JP5688829B2 - Differential Diabetes Prediction / Diagnosis Method and Diabetes Prediction / Diagnosis Kit - Google Patents

Description

  The present invention relates to a method for prior diagnosis and diagnosis of a disease, a method for evaluating a substance, and a method for screening a substance, and more specifically, the presence or absence of diabetes with the concentration of a marker substance in a body fluid of a subject as an index. Or a disease diagnosis method for determining the risk of future onset (preliminary diagnosis), an animal that has developed diabetes or an animal with a high risk of future onset, and the concentration of the marker substance in the body fluid of the animal As an index, a method for evaluating a substance for evaluating the effect of improving diabetes or reducing the risk of future onset of a test substance, and the effect of improving diabetes or reducing the risk of developing future using the evaluation method The present invention relates to a screening method for a substance for screening a substance having the same.

  The present invention relates to a method for diagnosing diabetes by measuring the concentration of a marker substance in blood and comparing the value with a healthy value, and a kit for use in diagnosing diabetes.

  In particular, the present invention relates to a means capable of differentially distinguishing between a marker substance found in healthy individuals and a marker substance found in affected individuals.

  In recent years, westernization of eating habits has progressed, and lifestyle-related diseases such as obesity, diabetes, and arteriosclerosis, which are considered to be caused by this, are increasing. These increased incidences are not genetic but mainly due to environmental factors. For example, abnormal lipid metabolism due to ingestion of a high-fat diet or a high-calorie diet causes increased blood lipids, development of insulin resistance, adipocyte hypertrophy, insulin secretion failure, and the like. As a result, diabetes, obesity, arteriosclerosis, etc. occur with high probability, leading to the progress of the disease state. Among these lifestyle-related diseases, diabetes often causes complications such as sensory paralysis, blindness, arteriosclerosis and the like, and is regarded as a problem particularly as a disease that causes a great obstacle to daily life.

  Diabetes is a complex disease caused by hyperglycemia due to insufficient action of insulin. Diabetes is classified into type 1 and type 2. In type 1 diabetes, pancreatic islets of Langerhans are inflamed, resulting in a decrease or depletion of insulin secretory ability, leading to hyperglycemia. On the other hand, type 2 diabetes causes hyperglycemia due to insufficient action of insulin due to other causes. This type 2 diabetes accounts for the majority of Japanese diabetes and is especially regarded as a problem. Although the pathogenesis of type 2 diabetes is still unclear, it is thought that the disease is caused mainly by environmental factors, and overeating and obesity are one of the major causes. For example, the amount of insulin secretion in the pancreas increases dramatically due to obesity, resulting in fatigue of the pancreas and conversely the decrease in insulin secretion, resulting in insufficient insulin action and hyperglycemia. Alternatively, insulin receptors decrease due to an increase in fat, resulting in insufficient insulin action and hyperglycemia. On the other hand, excess glucose born from insufficient action of insulin is stored as fat and obesity progresses, and diabetes is closely related to obesity in its onset mechanism. In addition, type 2 diabetes often has no subjective symptoms at the beginning of the onset, and at the time of discovery it is so advanced that it may be difficult to treat. In other words, it is expected that there will be a considerable number of reserves for type 2 diabetes.

  Currently, clinical laboratory items used for diagnosis of diabetes include urine sugar, fasting blood glucose, hemoglobin A1c (HbA1c), blood insulin level, blood / urine C-peptide level (CPR), and the like. In addition, an oral glucose tolerance test (OGTT) is also performed in which blood glucose concentration after ingesting glucose is monitored and its clearance ability is observed. As an example of a new clinical marker, blood retinoic acid has been proposed (Patent Document 1).

  However, the increase or decrease in insulin is not necessarily a clear indicator of diabetes. Thus, in addition to this, there have been no reports of diabetes markers useful as gene products, particularly markers that can be pre-diagnosed.

  In humans, the pathophysiology of other factors is intertwined, but it is unclear whether the factors are the same diagnostic indicators in pure model animals.

In addition, no means has yet been provided that can differentially distinguish between marker substances found in healthy individuals and marker substances found in affected individuals.
JP 2004-163379 A

  Prevention is particularly important for diseases with few subjective symptoms at the beginning of onset, such as type 2 diabetes. For this purpose, a so-called pre-diagnosis technique for detecting a stage before the onset of diabetes is necessary. If it does so, treatments, such as a diet restriction and exercise, can be performed before diabetes develops, and it becomes possible to prevent diabetes. However, it is difficult to detect diabetes at the stage before onset in conventional clinical examinations, and the current situation is that it is almost powerless in terms of prevention. On the other hand, there has already been proposed a method called a multi-marker system, which improves the diagnostic accuracy of clinical tests using a plurality of marker substances as indices. Therefore, it is conceivable to increase the diagnostic accuracy by applying a multi-marker system to the diagnosis of diabetes. However, in order to diagnose diabetes with a multi-marker system, a plurality of useful marker substances are required, but at present there are no such marker substances, and diabetes is detected by a multi-marker system. Has not been reported. Thus, there is a need for a method for diagnosing diabetes that is useful for the prevention and early detection of diabetes and can also be applied to a multi-marker system.

  Prevention is particularly important for diseases with few subjective symptoms at the beginning of onset, such as type 2 diabetes. For that purpose, in addition to the presence or absence of the onset of diabetes, a diagnostic technique for determining the risk of future onset of diabetes is required. By doing so, it is possible to perform treatments such as lifestyle correction, dietary restriction, exercise, etc. before the onset of diabetes, thereby preventing the onset of the disease. Furthermore, if there is a food material or the like that has an effect of reducing the risk of developing the disease, diabetes can be easily prevented by daily intake of food containing such a food material.

  However, no diagnostic technology has been established to determine the risk of developing diabetes in the future, and a method for evaluating the effect of reducing or reducing the onset risk of the substance, and the improvement or risk of onset of the disease. A method for screening a substance having an effect of reducing the above has not been developed.

  An object of the present invention is to provide a diagnostic method for determining the presence or absence of diabetes or the risk of future onset, and a method for evaluating the effect of improving diabetes or reducing the risk of future risk of the test substance, and It is an object of the present invention to provide a method for screening a substance having an effect of improving diabetes or reducing the risk of developing future using an evaluation method.

  It is another object of the present invention to provide a means capable of differentially distinguishing between a marker substance found in healthy persons and a marker substance found in affected persons.

  In order to search for a new marker substance of diabetes useful for pre-diagnosis and early detection for diabetes prevention, the present inventors comprehensively analyzed proteins in the blood of diabetic patients and healthy individuals using mass spectrometer spectra. In comparison, we searched for proteins that are gene products specific to diabetics. As a result, a plurality of proteins having a statistically significant difference between diabetic patients and healthy subjects were found. Furthermore, their proteins were identified and identified as transthyretin, apolipoprotein CII, apolipoprotein CIII, and serum albumin and their derivatives (eg, oxides, cysteinylates, glycosylated products, etc.). That is, the present inventors show that transthyretin and apolipoprotein CIII in blood are higher than those in healthy subjects in diabetic subjects and pre-diabetes groups, and that apolipoprotein CII and serum albumin are lower than those in healthy subjects. The present invention has been completed. That is, the gist of the present invention is as follows.

In order to solve the above problems, the present inventors also prepared body fluid samples of various animals from the stage before the onset of diabetes to the boundary between onset and non-onset and the stage of onset. The body fluid sample was comprehensively analyzed by proteomic analysis using a mass spectrometer. As a result, in addition to the presence / absence of diabetes, a plurality of proteins related to the risk of future onset (ie, enabling pre-diagnosis) were found. And the system which determines the presence or absence of diabetes or the future onset risk (namely, prior diagnosis) was constructed | assembled using the density | concentration of the said protein in the body fluid of a subject as a parameter | index. Furthermore, a system for evaluating the effect of improving diabetes or reducing the risk of future onset of the test substance was constructed using the concentration of the protein in the body fluid of the animal as an index. Furthermore, using this evaluation method, a system for screening a substance having an effect of improving diabetes or reducing the risk of developing future disease was constructed. The present invention has been completed. That is, the gist of the present invention is as follows.
(1) A prior diagnosis or whether a subject is diabetic, comprising a marker substance in a sample derived from a subject, a factor that specifically interacts with the marker substance, or a means for selectively recognizing the marker substance A system for diagnosing.
(2) The system according to item 1, wherein the marker substance is present in the body fluid of the subject.
(3) The system according to item 1, wherein the marker substance is present in the blood of the subject.
(4) The system according to item 1, wherein the marker substance is a gene product.
(5) The marker substance is selected from the group consisting of transthyretin, transthyretin derivative, apolipoprotein CII, apolipoprotein CII derivative, apolipoprotein CIII, apolipoprotein CIII derivative and serum albumin and proteins corresponding thereto. 2. A system according to item 1, comprising one or more substances.
(6) The system according to item 1, wherein the factor is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules, and complex molecules thereof.
(7) The system according to item 1, wherein the factor is a protein or the complex molecule.
(8) The system according to item 1, wherein the factor is an antibody.
(9) The system according to item 1, wherein the agent is labeled or labelable.
(10) The above means are mass spectrometer, nuclear magnetic resonance measuring apparatus, X-ray analyzer, SPR, chromatography, immunological means, biochemical means, electrophoresis instrument, chemical analyzer, fluorescent two-dimensional differential Item 2. The system according to Item 1, selected from the group consisting of electrophoresis, isotope labeling, tandem affinity purification, physical means, laser microdissection, and combinations thereof.
(11) The system according to item 1, further comprising a standard for the marker substance.
(12) The system according to item 1, further comprising means for purifying the sample.
(13) The system according to item 1, wherein the subject includes a mammal.
(14) The system according to item 1, wherein the subject includes a rodent.
(15) The system according to item 1, wherein the subject includes a human.
(16) The system according to item 1, wherein the factor or the means has an ability to quantify the marker substance.
(17) The system according to item 1, further comprising a quantification means for quantifying the marker substance.
(18) The system according to item 17, wherein the quantification unit includes a determination unit that compares the standard curve and the measurement result to determine whether the marker substance is within a normal value range.
(19) The system according to item 18, wherein the determination means is a computer.
(20) The system according to item 1, wherein the system is a composition containing the marker substance or the factor that specifically interacts with the marker substance.
(21) The marker substance includes at least one substance selected from the group consisting of transthyretin and a transthyretin derivative, and the transthyretin derivative includes S-cysteinyl transthyretin and S-cystein. Nyltransthyretin, glutathioneated transthyretin, SS bond forming transthyretin, oxidized transthyretin, formylated transthyretin, acetylated transthyretin, phosphorylated transthyretin, glycosylated transthyretin, The system according to item 1, wherein the system is selected from the group consisting of myristylated transthyretin and complex derivatives thereof.
(22) At least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of developing diabetes or a high risk of developing it in the future. The system according to item 21.
(23) The item, wherein at least one phenomenon selected from the group consisting of a decrease in transthyretin and an increase in the transthyretin derivative is an indicator of the degree of onset of diabetes or the risk of future onset The system according to 21.
(24) The transthyretin is encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the first 20 amino acids are excised Item 22. The system according to Item 21, wherein the system has been modified or has these modified sequences.
(25) The transthyretin derivative is a cysteine at position 30 in the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, respectively. The system according to item 21, wherein the cysteine of the corresponding cysteine is a cysteinylated derivative or the first 20 amino acids are excised.
(26) The system according to item 1, wherein the factor or the means has an ability to distinguish between a monomer and a tetramer of transthyretin.
(27) The system according to item 1, wherein the factor or the means has the ability to distinguish between transthyretin and S-cysteinyl transthyretin.
(28) The system according to item 1, wherein the factor or the means includes an antibody having an ability to distinguish between transthyretin and S-cysteinyl transthyretin.
(29) The factor or the means recognizes transthyretin and S-cysteinyl transthyretin, and the system has means for discriminating between transthyretin and S-cysteinyl transthyretin. The system according to item 1, further comprising:
(30) The factor or the means recognizes transthyretin and S-cysteinyl transthyretin, and the system distinguishes between the molecular weight of transthyretin and the molecular weight of S-cysteinyl transthyretin. The system of item 1, further comprising means and means for measuring a relative ratio of transthyretin to S-cysteinyl transthyretin.
(31) The system according to item 1, wherein the marker substance comprises apolipoprotein CII or apolipoprotein CII derivative, and the apolipoprotein CII derivative is a pro form.
(32) At least one phenomenon selected from the group consisting of a decrease in the apolipoprotein CII and a change in the apolipoprotein CII derivative is an indicator of developing diabetes or high risk of developing in the future. 32. The system according to item 31.
(33) The item, wherein at least one phenomenon selected from the group consisting of a decrease in the apolipoprotein CII and a change in the apolipoprotein CII derivative is an indicator of the degree of onset of diabetes or the risk of future onset 31. The system according to 31.
(34) The apolipoprotein CII is encoded by the nucleic acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7, or has the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 8, or a modified sequence thereof. 32. The system according to item 31.
(35) A system according to item 31, wherein the factor or the means has an ability to selectively identify apolipoprotein CII.
(36) A system according to item 31, wherein the factor or the means includes an antibody having an ability to selectively identify apolipoprotein CII.
(37) A system according to item 31, wherein the factor or the means has an ability to selectively identify apolipoprotein CII, and the system includes means for quantifying the apolipoprotein CII.
(38) The marker substance includes apolipoprotein CIII or apolipoprotein CIII derivative, the apolipoprotein CIII is apolipoprotein CIII0, and the apolipoprotein CIII derivative is selected from the group consisting of apolipoprotein CIII1 and apolipoprotein CIII2 The system according to item 1.
(39) that at least one phenomenon selected from the group consisting of an increase in apolipoprotein CIII, an increase in apolipoprotein CIII1 and an increase in apolipoprotein CIII2 is developing diabetes or having a high risk of developing in the future 39. The system according to item 38, which is an index.
(40) An indicator that at least one phenomenon selected from the group consisting of an increase in apolipoprotein CIII, an increase in apolipoprotein CIII1 and an increase in apolipoprotein CIII2 is a degree of developing diabetes or a high risk of developing in the future 39. The system according to item 38, wherein
(41) The apolipoprotein CIII is encoded by the nucleic acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, or has the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12, or a modified sequence thereof. 39. The system of item 38.
(42) The apolipoprotein CIII derivative is encoded by the nucleic acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, or in the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12, respectively. 39. A system according to item 38, which is a derivative having a chain.
(43) A system according to item 38, wherein the factor or the means has an ability to distinguish between apolipoprotein CIII and apolipoprotein CIII derivative.
(44) A system according to item 38, wherein the factor or the means has an ability to distinguish at least two of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2.
(45) A system according to item 38, wherein the factor or the means has the ability to distinguish all of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2.
(46) A system according to item 38, wherein the factor or the means comprises an antibody having an ability to distinguish between apolipoprotein CIII and apolipoprotein CIII derivative.
(47) A system according to item 38, wherein the factor or the means includes an antibody capable of distinguishing at least two of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2.
(48) A system according to item 38, wherein the factor or the means comprises a combination of antibodies capable of distinguishing all of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2.
(49) The system according to item 38, wherein the factor or the means recognizes apolipoprotein CIII and apolipoprotein CIII derivative, and the system further comprises means for discriminating between apolipoprotein CIII and apolipoprotein CIII derivative. .
(50) The factor or the means recognizes apolipoprotein CIII and apolipoprotein CIII derivative, and the system further comprises means for discriminating at least two of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2. 39. The system according to item 38.
(51) The factor or the means recognizes apolipoprotein CIII and apolipoprotein CIII derivative, and the system further comprises means for discriminating all of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2. The system described in.
(52) The system according to item 1, wherein the marker substance comprises serum albumin or a serum albumin derivative.
(53) Item 52, wherein at least one phenomenon selected from the group consisting of a decrease in serum albumin and a change in serum albumin derivative is an indicator of developing diabetes or a high risk of developing it in the future. The system described in.
(54) In item 52, wherein at least one phenomenon selected from the group consisting of a decrease in serum albumin and a change in serum albumin derivative is an indicator of the degree of developing diabetes or the risk of developing future The described system.
(55) The serum albumin is encoded by the nucleic acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 15, or has the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 16, or a modified sequence thereof. 52. The system according to 52.
(56) A system according to item 52, wherein the factor or the means has an ability to selectively distinguish serum albumin.
(57) A system according to item 52, wherein the factor or the means includes an antibody capable of selectively discriminating serum albumin.
(58) A system according to item 31, wherein the factor or the means has an ability to selectively distinguish serum albumin, and the system includes means for quantifying the serum albumin.
(59) The system according to item 1, which is a diagnostic agent.
(60) A method for pre-diagnosing or diagnosing whether a subject is diabetic, or for supporting the pre-diagnosis or diagnosis,
A) measuring a marker substance in a sample derived from the subject; and
B) determining from the measurement results whether the subject is diabetic or likely;
Including the method.
(61) The method according to item 60, which has the characteristics according to any one of items 2 to 59.
(62) Prior diagnosis or diagnosis of whether a subject is diabetic using a marker substance in a sample derived from a subject, a factor that specifically interacts with the marker substance, or a means for selectively recognizing the marker substance Use in the manufacture of a medicament for
(63) Use of item 62 which has the characteristic of any one of items 2-59.
(64) Prior diagnosis or diagnosis of whether a subject is diabetic using a marker substance in a sample derived from a subject, a factor that specifically interacts with the marker substance, or a means for selectively recognizing the marker substance Use to do.
(65) Use of item 64 which has the characteristic of any one of items 2-59.
(66)
Item 60. The method according to Item 60, wherein the concentration of a marker substance in blood is measured, the value is compared with a healthy value, and the marker substance is selected from the group of transthyretin, apolipoprotein CIII, and serum albumin. A method characterized in that it is one or more proteins.
(67)
70. The method for diagnosing diabetes according to item 66, wherein the apolipoprotein CIII is one or more proteins selected from the group consisting of apolipoprotein CIII0, apolipoprotein CIII1, and apolipoprotein CIII2.
(68)
The following steps (1) to (4):
(1) A step of preparing serum or plasma from the blood of a subject;
(2) a step of detecting diabetes by comparing the concentration of transthyretin in serum or plasma obtained in step (1) with a healthy value;
(3) If diabetes is not detected in step (2), the step of detecting diabetes by comparing the concentration of apolipoprotein CIII2 in serum or plasma with a healthy value; and
(4) If diabetes is not detected in step (3), the step of detecting diabetes by comparing the concentration value of apolipoprotein CIII1 in serum or plasma with a healthy value;
68. A method according to item 67, comprising:
(69)
The following steps (5) to (8):
(5) a step of preparing serum or plasma from the blood of the subject,
(6) a step of detecting diabetes by comparing the concentration of serum albumin in the serum or plasma obtained in step (5) with a healthy value,
(7) If diabetes is detected in step (6), the step of detecting diabetes by comparing the concentration of apolipoprotein CIII2 in serum or plasma with a healthy value;
(8) If diabetes is not detected in step (7), the step of detecting diabetes by comparing the concentration value of apolipoprotein CIII1 in serum or plasma with a healthy value;
68. The diagnostic method according to item 67, comprising:
(70)
Item 69. The method according to Item 68, wherein when diabetes is detected in the step (4), diabetes is detected by further comparing the concentration value of apolipoprotein CIII0 in serum or plasma with a healthy value. .
(71)
Item 70. The method according to Item 69, wherein when diabetes is detected in the step (8), diabetes is detected by comparing the concentration value of apolipoprotein CIII0 in serum or plasma with a healthy value. .
(72)
Item 60. The method according to Item 60, wherein the concentration of the marker substance in the blood is measured, the value is compared with a healthy value, and the marker substance is the following (a) to (e):
(A) a protein having a molecular weight of about 13800, supplemented by a cation exchanger at a pH of 7.0 or less;
(B) a protein having a molecular weight of about 8700, supplemented by a metal ion-immobilized carrier at a pH of 7.0 or lower;
(C) a protein having a molecular weight of about 9400, which is captured by a cation exchanger at a pH of 7.0 or less, or a protein having a molecular weight of about 9400, which is supplemented by a metal ion-immobilized support at pH 7.0;
(D) a protein having a molecular weight of about 9700, supplemented by a cation exchanger at a pH of 7.0 or less, or a protein having a molecular weight of about 9700, supplemented by a metal ion-immobilized support at pH 7.0. ;and
(E) a protein having a molecular weight of about 66000 supplemented by a cation exchanger at a pH of 7.0 or less, or a protein having a molecular weight of about 66000 supplemented by a metal ion-immobilized support at pH 7.0. ,
A method characterized in that it is one or more proteins selected from the group of
(73)
Item 60. The method according to Item 60, wherein the concentration of the marker substance in blood is measured, and the value is compared with a healthy value.
(A) a protein having a molecular weight of about 13800, wherein digestion with trypsin yields polypeptides having molecular weights of about 1270, about 1370, about 1390, about 1520, about 2450, about 2640, and about 3140; and / or
(B) a protein having a molecular weight of about 8700-9700, which upon digestion with trypsin yields polypeptides having a molecular weight of about 900, about 1200, about 1390, about 1710, about 1940, and about 2080;
A method characterized by being a protein of
(74)
Any of items 66 to 73, wherein serum or plasma is contacted with a carrier on which a substance having affinity for the marker substance is immobilized, the marker substance is captured, and the concentration of the marker substance is measured. The method described.
(75)
75. The method according to item 74, wherein the substance having affinity for the marker substance is an antibody specific for the marker substance.
(76)
75. The method for diagnosing diabetes according to item 74, wherein the substance having affinity for the marker substance has an ion exchange group.
(77)
77. The method according to any one of items 74 to 76, wherein the carrier has a planar portion, and the substance having affinity for the marker substance is immobilized on a part of the planar portion.
(78)
2. The system according to item 1, wherein the antibody is immobilized on a carrier.
(79)
Item 60. The method according to Item 60, wherein the following marker substances (a) to (n) in the body fluid of the subject:
(A) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 7040 when subjected to mass spectrometry;
(B) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8330 when subjected to mass spectrometry;
(C) A protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8530 when subjected to mass spectrometry.
(D) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 9060 when subjected to mass spectrometry;
(E) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 9260 when subjected to mass spectrometry;
(F) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 9450 when subjected to mass spectrometry;
(G) a protein that binds to a copper ion-binding metal chelate at pH 7.0 and a NaCl concentration of 0.5 M, and that when subjected to mass spectrometry, produces an ion peak with a mass / charge ratio of about 13700,
(H) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 76400 when subjected to mass spectrometry;
(I) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 79100 when subjected to mass spectrometry;
(J) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 3500 when subjected to mass spectrometry;
(K) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 3560 when subjected to mass spectrometry;
(L) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 4180 when subjected to mass spectrometry;
(M) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 12800 when subjected to mass spectrometry;
(N) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 65700 when subjected to mass spectrometry; and
Proteins or variants corresponding to the proteins (a) to (n)
A method comprising comparing the concentration of at least one protein selected from the group consisting of a healthy value and the presence or absence of diabetes or the risk of developing diabetes in the future.
(80)
80. A method according to item 79, wherein the body fluid is blood.
(81)
The body fluid or body fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, and the marker substance in the body fluid is captured on the carrier, and based on the amount of the captured marker substance Item 81. The disease diagnosis method according to Item 79 or 80, wherein the concentration of the marker substance in the body fluid is calculated.
(82)
84. The method according to item 81, wherein the carrier has a planar portion, and the substance having affinity for the marker substance is immobilized on a part of the planar portion.
(83)
83. The method according to item 81 or 82, wherein the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody.
(84) A method for evaluating a substance, characterized by evaluating an improvement effect of diabetes or a reduction effect of a future onset risk of a test substance,
a) ingesting a test substance in an animal that has developed diabetes or an animal at high risk of developing in the future; and
b) comparing the concentration of at least one of the marker substances in the body fluid of the animal with a reference value, and evaluating the effect of improving diabetes or reducing the risk of future onset of the test substance,
Including the method.
(85) The method of item 84 which has the characteristic of any one of items 2-59.
(86)
85. The method according to item 84, wherein the marker substance is the following marker substance (a) to (n):
(A) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 7040 when subjected to mass spectrometry;
(B) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8330 when subjected to mass spectrometry;
(C) A protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8530 when subjected to mass spectrometry.
(D) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 9060 when subjected to mass spectrometry;
(E) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 9260 when subjected to mass spectrometry;
(F) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 9450 when subjected to mass spectrometry;
(G) a protein that binds to a copper ion-binding metal chelate at pH 7.0 and a NaCl concentration of 0.5 M, and that when subjected to mass spectrometry, produces an ion peak with a mass / charge ratio of about 13700,
(H) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 76400 when subjected to mass spectrometry;
(I) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 79100 when subjected to mass spectrometry;
(J) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 3500 when subjected to mass spectrometry;
(K) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 3560 when subjected to mass spectrometry;
(L) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 4180 when subjected to mass spectrometry;
(M) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 12800 when subjected to mass spectrometry;
(N) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 65700 when subjected to mass spectrometry; and
Proteins or variants corresponding to the proteins (a) to (n)
A method wherein the protein is at least one protein selected from the group consisting of:
(87)
The above-mentioned reference value is obtained when an animal that has developed diabetes or has a high future risk of ingesting a known substance that does not have an effect of improving diabetes or a risk of reducing future risk of development. 89. The method according to item 86, which is the concentration of the marker substance in body fluid.
(88)
88. A method according to item 86 or 87, wherein the body fluid is blood.
(89)
The method according to any one of Items 86 to 88, wherein the test substance is a food material.
(90)
The bodily fluid or bodily fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, and the marker substance in the bodily fluid is captured on the carrier, and based on the amount of the captured marker substance 90. The method according to any one of items 86 to 89, wherein the concentration of the marker substance in the body fluid is calculated.
(91)
Item 91. The method according to Item 90, wherein the carrier has a flat portion, and the substance having affinity for the marker substance is immobilized on a part of the flat portion.
(92)
92. The method according to item 90 or 91, wherein the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody.
(93)
93. A substance screening method comprising: evaluating a test substance by the substance evaluation method according to any of items 84 to 92; and screening a substance having an effect of improving diabetes or a risk of reducing future risk of onset.
(94) A substance obtained by the method according to item 93.
(101) A pre-diagnosis whether a subject is diabetic or comprising a marker substance in a sample derived from a subject, a factor that specifically interacts with the marker substance, or a means for selectively recognizing the marker substance A system for diagnosis, wherein the means for recognizing has the ability to distinguish between transthyretin and a transthyretin derivative.
(102) The system according to item 101, wherein the factor is selected from the group consisting of a nucleic acid molecule, a polypeptide, a lipid, a sugar chain, a small organic molecule, and a complex molecule thereof.
(103) The system according to item 101, wherein the factor is a protein or the complex molecule.
(104) A system according to item 101, wherein the factor is an antibody.
(105) A system according to item 101, wherein the agent is labeled or labelable.
(106) The above means are mass spectrometer, nuclear magnetic resonance measuring apparatus, X-ray analyzer, SPR, chromatography, immunological means, biochemical means, electrophoresis instrument, chemical analyzer, fluorescent two-dimensional differential 102. The system of item 101, selected from the group consisting of electrophoresis, isotope labeling, tandem affinity purification, physical means, laser microdissection, and combinations thereof.
(107) The system according to item 101, further comprising a standard for the marker substance.
(108) The system according to item 101, further comprising means for purifying the sample.
(109) A system according to item 101, wherein the subject includes a mammal.
(110) The system according to item 101, wherein the subject includes a rodent.
(111) A system according to item 101, wherein the subject includes a human.
(112) The system according to item 101, wherein the factor or the means has an ability to quantify the marker substance.
(113) The system according to item 101, further comprising a quantification means for quantifying the marker substance.
(114) The system according to item 113, wherein the quantification unit includes a determination unit that compares the standard curve and the measurement result to determine whether the marker substance is within a normal value range.
(115) The system according to item 114, wherein the determination means is a computer.
(116) The system according to item 101, wherein the system is a composition containing the marker substance or the factor that specifically interacts with the marker substance.
(117) The transthyretin derivative includes S-cysteinyl transthyretin, glutathioneated transthyretin, SS bond-forming transthyretin, oxidized transthyretin, formylated transthyretin, acetylated transthyretin 102. The system of item 101, selected from the group consisting of: phosphorylated transthyretin, glycosylated transthyretin, myristylated transthyretin and complex derivatives thereof.
(118) A system according to item 101, wherein the transthyretin derivative is S-cysteinyl transthyretin.
(119) A system according to item 101, wherein the transthyretin derivative is S-cysteinyl transthyretin, and the means for identifying is an antibody.
(120) A system according to item 101, wherein the factor or the means reacts differentially with transthyretin and a transthyretin derivative.
(121) A system according to item 101, wherein the factor or the means reacts substantially only with either transthyretin or a transthyretin derivative.
(122) At least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of developing diabetes or a high risk of developing in the future. 102. The system according to item 101.
(123) The item, wherein at least one phenomenon selected from the group consisting of a decrease in transthyretin and an increase in the transthyretin derivative is an indicator of the degree of onset of diabetes or the risk of future onset 101. The system according to (101).
(124) The transthyretin is encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the first 20 amino acids are excised 101. The system according to item 101, wherein the system has been modified or has these modified sequences.
(125) The transthyretin derivative is a cysteine at position 30 in the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, respectively. 102. The system according to Item 101, wherein the cysteine of the corresponding cysteine is a cysteinylated derivative, or the first 20 amino acids are excised.
(126) A system according to item 101, wherein the factor or the means has the ability to distinguish between a monomer and a tetramer of transthyretin.
(127) A system according to item 101, wherein the factor or the means has the ability to distinguish between transthyretin and S-cysteinyl transthyretin.
(128) A system according to item 101, wherein the factor or the means includes an antibody having an ability to distinguish between transthyretin and S-cysteinyl transthyretin.
(129) The factor or the means recognizes transthyretin and S-cysteinyl transthyretin, and the system has means for discriminating between transthyretin and S-cysteinyl transthyretin. 102. The system according to item 101, further comprising:
(130) The factor or means recognizes transthyretin and S-cysteinyl transthyretin, and the system distinguishes between the molecular weight of transthyretin and the molecular weight of S-cysteinyl transthyretin. 102. The system of item 101, further comprising means and means for measuring a relative ratio of transthyretin to S-cysteinyl transthyretin.
(131) The system according to item 101, which is a diagnostic agent.
(132) A method for pre-diagnosing or diagnosing whether a subject is diabetic, or for supporting the pre-diagnosis or diagnosis,
A) measuring a marker substance in a sample derived from the subject; and
B) determining from the measurement results whether the subject is diabetic or likely;
Including the method.
(133) The method of item 32 which has the characteristic of any one of items 102-130.
(134) Prior diagnosis or diagnosis of whether a subject is diabetic using a marker substance in a sample derived from a subject, a factor that specifically interacts with the marker substance, or a means for selectively recognizing the marker substance Use in the manufacture of a medicament for the use wherein the means for recognizing has the ability to distinguish between transthyretin and a transthyretin derivative.
(135) Use of item 134 which has the characteristic of any one of items 102-130.
(136) Prior diagnosis or diagnosis of whether a subject is diabetic using a marker substance in a sample derived from a subject, a factor that specifically interacts with the marker substance, or a means for selectively recognizing the marker substance Use wherein the means for recognizing has the ability to distinguish between transthyretin and a transthyretin derivative.
(137) Use of item 134 which has the characteristic of any one of items 102-130.
(138) A method according to item 132, wherein the sample is blood.
(139) The sample is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, and the marker substance in a body fluid is captured on the carrier, and based on the amount of the captured marker substance 131. The disease diagnosis method according to Item 132, wherein the concentration of the marker substance in the body fluid is calculated.
(140) A method according to item 139, wherein the carrier has a planar portion, and the substance having affinity for the marker substance is immobilized on a part of the planar portion.
(141) The method according to item 139 or 140, wherein the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody.
(142) A method for evaluating a substance, characterized by evaluating an improvement effect of diabetes or a reduction effect of future onset risk of a test substance,
a) ingesting a test substance in an animal that has developed diabetes or an animal at high risk of developing in the future; and
b) comparing the concentration of at least one of the marker substances in the body fluid of the animal with a reference value, and evaluating the effect of improving diabetes or reducing the risk of future onset of the test substance,
Including the method.
(143) The method of item 84 which has the characteristic of any one of items 102-130.
(144) The above-mentioned reference value is obtained when an animal that has developed diabetes or an animal that has a high future risk of ingesting a known substance that does not have an effect of improving diabetes or a risk of reducing future risk of development. 142. The method according to item 142, which is the concentration of the marker substance in the body fluid of the animal.
(145) The method according to any one of items 142 to 144, wherein the body fluid is blood.
(146) The method according to any one of items 142 to 145, wherein the test substance is a food material.
(147) The amount of the marker substance captured by bringing the body fluid or body fluid component into contact with a carrier on which a substance having affinity for the marker substance is immobilized to capture the marker substance in the body fluid on the carrier. 150. The method according to any one of items 142 to 146, wherein the concentration of the marker substance in the body fluid is calculated based on the above.
(148) A method according to item 47, wherein the carrier has a planar portion, and the substance having affinity for the marker substance is immobilized on a part of the planar portion.
(149) The method according to item 146 or 147, wherein the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody.
(150) A substance characterized by evaluating a test substance by the substance evaluation method according to any of items 142 to 149, and screening for a substance having an effect of improving diabetes or reducing the risk of developing future disease Screening method.
(151) A substance obtained by the method according to item 150.

  Accordingly, it is understood that these and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description with reference to the accompanying drawings.

  Although transthyretin, apolipoprotein CIII, and serum albumin are known substances and have certain clinical significance, it has been revealed for the first time by the present invention that they can serve as marker substances for diabetes.

  According to the method and system or composition (diagnostic agent) for diabetes diagnosis and prior diagnosis of the present invention, diabetes diagnosis and prior diagnosis can be performed more reliably and accurately. In addition, diabetes can be diagnosed and pre-diagnosed by a multi-marker system by combining a plurality of marker substances.

  According to the kit for diagnosing diabetes of the present invention, diabetes can be diagnosed and pre-diagnosed more easily and with high accuracy.

  According to the method, composition (diagnostic agent) and system for diagnosis and pre-diagnosis of the disease of the present invention, it is possible to determine (that is, pre-diagnosis) the future risk of developing diabetes in addition to the presence of diabetes.

  According to the substance evaluation method of the present invention, in addition to the diabetes improvement effect of the test substance, the effect of reducing the future risk of developing diabetes can be evaluated.

  According to the method for screening a substance of the present invention, in addition to a substance having an effect of improving diabetes, a substance having an effect of reducing the future risk of developing diabetes can be screened.

  In particular, the present invention can differentially distinguish between a marker substance found in a healthy person and a marker substance found in an affected person, thereby enabling an efficient diagnosis.

It is a flowchart showing the procedure of the example which applied the diagnostic method of diabetes of this invention to the multimarker system. It is a flowchart showing the procedure of another example which applied the diagnostic method of diabetes of this invention to the multimarker system. FIG. 3 (a) is a graph in which peak intensities are plotted separately for diabetic patients and healthy individuals, and FIG. 3 (b) is the result of FIG. 3 (a). Is a graph showing a maximum value, a minimum value, a median value, and a cutoff value, and FIG. 3C is a graph showing an ROC curve. FIG. 4 (a) is a graph in which peak intensities are plotted separately for diabetic patients and healthy individuals, and FIG. 4 (b) is the result of FIG. 4 (a). Is a graph showing a maximum value, a minimum value, a median value, and a cutoff value, and FIG. 4C is a graph showing an ROC curve. It is a photograph showing the result of having used the serum of a diabetic patient for two-dimensional electrophoresis. Fig. 6 (a) is a graph in which peak intensities are plotted separately for diabetic patients and healthy subjects, and Fig. 6 (b) shows the results of Fig. 6 (a). Is a graph showing a maximum value, a minimum value, a median value, and a cutoff value, and FIG. 6C is a graph showing an ROC curve. It is a box diagram about the ion peak whose mass / charge ratio is 7043 (average value). It is a box diagram about an ion peak whose mass / charge ratio is 8325 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 8532 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 9062 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 9255 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 9445 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 13720 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 76404 (average value). It is a box diagram about an ion peak whose mass / charge ratio is 79085 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 3497 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 3559 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 4184 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 12786 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 65700 (average value). FIG. 21 shows healthy person-derived S-cysteinylated TTR. Measurement: M / Z = 13874.7480. FIG. 22 shows a healthy person-derived S-cysteinylated TTR after reduction treatment. Measurement: M / Z = 13759.7393. FIG. 23 shows diabetic patient-derived S-cysteinylated TTR. Measurement: M / Z = 13871.3320. FIG. 24 shows a diabetic patient-derived S-cysteinylated TTR after reduction treatment. Measurement: M / Z = 13755.6494. FIG. 25 shows the tetrameric structure of TTR and the amino acid sequence of the monomer. Thus, transthyretin normally has a tetrameric structure, and it is postulated that when it collapses, it becomes diabetic. FIG. 26 shows the three-dimensional structure (top) and secondary structure sequence (bottom) of the human TTRα-domain. FIG. 27 shows a gel photograph of a band identified as rat apolipoprotein CIII and analysis of the band by SELDI-TOF. FIG. 28 shows a gel photograph of a band identified as rat apolipoprotein CIII and analysis by SELDI-TOF of the band. FIG. 29 shows spots on two-dimensional electrophoresis that are considered to be spots of human apolipoprotein CIII (0 to 2). In FIG. 30, the result of the mass spectrometry of each spot of FIG. 29 is shown. FIG. 31 shows the SELDI-MS results for each spot. FIG. 32 shows the examination result of CM10. In FIG. 33, the examination result of Q10 is shown. FIG. 34 shows the optimum condition study results. From these results, it is clear that the obtained spot is a spot of human apolipoprotein CIII (0-2). FIG. 35 shows the relationship between ionic strength and HbA1c value of S-cysteinylated transthyretin (measured value: M / Z = 13,863, theoretical value: M / Z = 13880.53) in diabetic patients. FIG. 36 shows the relationship between the ionic strength and HbA1c value of S-cysteinylated transthyretin (measured value: M / Z = 13,874, theoretical value: M / Z = 13880.53) in diabetic patients. FIG. 37 shows the relationship between ionic strength and HbA1c value of S-cysteinylated transthyretin (measured value: M / Z = 13,884, theoretical value: M / Z = 13880.53) in diabetic patients. FIG. 38 shows measured data of M / Z: 13,863 in diabetic patients. FIG. 39 shows a diabetic rat serum 8.3K (Apo CII) box diagram. FIG. 40 shows the above time-series data. FIG. 41 shows the SELDI-MS results of the bands on the gel. FIG. 42 shows the confirmation of the obtained band by Western blot. FIG. 43 shows a state of being enhanced by the anti-ApoC2 antibody in the chromatographic fractionation. FIG. 44 shows that apolipoprotein CII in humans is also a pre-diagnostic marker. Here, the mass spectrometry result of a healthy person's apolipoprotein CII is shown. FIG. 45 shows that apolipoprotein CII in humans is also a pre-diagnostic marker. Here, the mass spectrometry result of apolipoprotein CII of a diabetic patient is shown. FIG. 46 shows that apolipoprotein CII in humans with another fraction is also a pre-diagnostic marker. Here, the mass spectrometry result of a healthy person's apolipoprotein CII is shown. FIG. 47 shows that apolipoprotein CII in humans with another fraction is also a pre-diagnostic marker. Here, the mass spectrometry result of apolipoprotein CII of a diabetic patient is shown.

Brief description of sequence listing

SEQ ID NO: 1-2 transthyretin human (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NO: 3-4 transthyretin rat (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NOs: 5-6 apolipoprotein CII human (nucleic acid sequence and amino acid sequence, respectively),
SEQ ID NOs: 7-8 Apolipoprotein CII rat (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NOs: 9-10 Apolipoprotein CIII human (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NOs: 11-12 Apolipoprotein CIII rat (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NOs: 13-14 Serum albumin human (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NOs: 15-16 Serum albumin rat (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NOs: 17-18 Serum albumin mouse (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NOs: 19-20 Serum albumin dogs (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NOs: 21-22 Serum albumin rabbit (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NO: 23-24 Serum albumin monkey (nucleic acid sequence and amino acid sequence, respectively)
SEQ ID NO: 25: Amino acid sequence of a cysteine residue of human transthyretin SEQ ID NO: 26: Amino acid sequence of a cysteine residue of mouse transthyretin SEQ ID NO: 27: Amino acid sequence of a cysteine residue of rat transthyretin 28: Amino acid sequence of cysteine residues of transthyretin of Gallus Gallus

  The present invention will be described below. Throughout this specification, singular articles (eg, “a”, “an”, “the”, etc. in English, corresponding articles in other languages, adjectives, etc.) It should be understood that it also includes the plural concept. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described in, for example, Maniatis, T. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associate ES and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Sambrook, J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer & Expression Analysis”, Yodosha, 1997, etc., which are related in this specification (may be all) Is incorporated by reference.

  Examples of the technology related to the protein chip include a technology available from Cyphergen.

(Explanation of terms)
The terms used in this specification will be explained below.

(Representative gene products identified as marker substances)
In the present specification, the “marker substance” refers to a substance that serves as an indicator for tracking whether or not the patient is suffering from or at risk of a certain condition (for example, a disease such as diabetes). Examples of such marker substances include genes, gene products, metabolites, and enzymes.

  As used herein, “gene product” refers to a protein or mRNA encoded by a gene. Herein, it has been found that gene products not directly related to sugar metabolism (ie, proteins not related to sugar metabolism such as insulin) can be used as an indicator of diabetes.

  In this specification, “transthyretin (TTR)”, also called prealbumin, is known as a protein that forms a tetramer composed of homogeneous subunits, and binds to retinol, which is a vitamin A transport protein in blood. It is known to form a protein complex with protein (RBP) and bind to thyroxine (T4). In rats, it is known as the main T4 transport protein.

  Transthyretin is described in Raz, A. et al. And the primary structure was identified by Kanda et al. (Raz, A. & Goodman DS, (1969), J. Biol. Chem. 224, 3230-3237; Kanda, Y. et al. (1974), J. Biol. Chem., 247, 6796-6805). To date, it is known that the abnormality is associated with Alzheimer's dementia and familial amyloidosis polyneuropathy.

A typical nucleotide sequence of transthyretin is
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment sequence thereof;
(B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment thereof;
(C) a variant polypeptide or fragment thereof in which one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 A polynucleotide encoding a variant polypeptide having biological activity;
(D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof;
(E) a polynucleotide encoding a species homologue of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment thereof;
(F) a polynucleotide that hybridizes to any one of the polynucleotides of (a) to (e) under a stringent condition and encodes a polypeptide having biological activity; or (g) (a) to It may be a polynucleotide that consists of a base sequence that is at least 70% identical to any one polynucleotide of (e) or its complementary sequence, and encodes a polypeptide having biological activity.

As the amino acid sequence of transthyretin,
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment thereof;
(B) In the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion, and have biological activity A polypeptide;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (e) at least 70% identity to any one polypeptide of (a)-(d) A polypeptide having an amino acid sequence that is and having biological activity,
It can be.

  Representative sequences of transthyretin are shown in SEQ ID NO: 1 or SEQ ID NO: 3 (nucleic acid sequence) and SEQ ID NO: 2 or SEQ ID NO: 4 (amino acid sequence).

  Here, the biological activity of transthyretin is known as, for example, a protein that forms a tetramer, and forms a protein complex with retinol-binding protein (RBP) that is a vitamin A transport protein in blood. And the ability to bind to thyroxine (T4).

  Transthyretin is a complex protein consisting of four subunits with a molecular weight of about 14,000 and is synthesized in the liver. The clinical significance of transthyretin in the blood is considered to reflect nutritional status and hepatic protein synthesis ability, and is known to show high values in the recovery phase of nephrotic syndrome and acute hepatitis. In the present specification, transthyretin refers to both a tetrameric complex protein and a subunit alone without any particular distinction.

  Transthyretin and its derivatives are known not only in humans, rats but also in other animals (for example, mammals) and homologs thereof (referred to herein as “corresponding” genes or proteins). Therefore, in the present specification, transthyretin and derivatives thereof generally refer to transthyretin and derivatives thereof which exist in general organisms in addition to humans and rats unless otherwise specified.

  As used herein, “transthyretin derivative” refers to any derivative of transthyretin, and particularly refers to a metabolite in vivo such as post-translational modification. Typical transthyretin derivative modifications are shown below with mass variation values:

.

  In this specification, typical transthyretin derivatives include cysteinylation (cysteinyl), glutathioneation, SS bond formation, oxidation (eg, oxidation of methionine side chain), formylation, acetylation, phosphorylation. , Sugar chain addition, myristylation, and the like, but are not limited thereto.

  In the present invention, in diabetic patients or high-risk subjects, the amount of transthyretin is reduced and, instead, certain derivatives of transthyretin (eg, cysteinyl transthyretin, glutathioneated transthyretin). S-S bond-formed transthyretin, oxidized transthyretin (eg, transthyretin with methionine side chain oxidized), formylated transthyretin, acetylated transthyretin, phosphorylated transthyretin, glycosylation It was revealed that transthyretin and myristylated transthyretin) increased. Therefore, it is possible to diagnose or pre-diagnose a subject with diabetes or a high risk thereof using the decrease in transthyretin or the increase in transthyretin derivatives as an index.

  In the present specification, “apolipoprotein” or “apolipid protein” refers to a protein that binds to a lipid to form a lipid protein, and is roughly classified into A, B, C, D, and E. It is the protein component of the lipoprotein complex, which is a typical component such as human plasma milk fat particles (chylomicron), HDL, LDL, and VLDL. Apolipoprotein C-II (may be abbreviated as APOC2) is an apolipoprotein present in VLDL, HDL and chylomicron. It is an activator of lipoprotein lipase. This protein deficiency causes hyperchylomicronemia and hypertriglyceridemia. Apolipoprotein C-III (sometimes abbreviated as APOC3) is an apolipoprotein present in VLDL, HDL, and chylomicron, and is known to suppress many lipases. For a review of apolipoprotein C and others, see Bondarenko, P. et al. V. et al. J. Lipid Res. 1999; 40: 543-555.

A representative nucleotide sequence of apolipoprotein CII is:
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7 or a fragment sequence thereof;
(B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8 or a fragment thereof;
(C) In the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 8, one or more amino acids are a variant polypeptide or a fragment thereof having one mutation selected from the group consisting of substitution, addition and deletion A polynucleotide encoding a variant polypeptide having biological activity;
(D) a polynucleotide which is a splice variant or allelic variant of the base sequence described in SEQ ID NO: 5 or SEQ ID NO: 7 or a fragment thereof;
(E) a polynucleotide encoding a species homologue of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8 or a fragment thereof;
(F) a polynucleotide that hybridizes to any one of the polynucleotides of (a) to (e) under a stringent condition and encodes a polypeptide having biological activity; or (g) (a) to It may be a polynucleotide that consists of a base sequence that is at least 70% identical to any one polynucleotide of (e) or its complementary sequence, and encodes a polypeptide having biological activity.

As an amino acid sequence of apolipoprotein CII,
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8 or a fragment thereof;
(B) In the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8, one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion, and have biological activity A polypeptide;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8; or (e) at least 70% identity to any one polypeptide of (a)-(d) A polypeptide having an amino acid sequence that is and having biological activity,
It can be.

  Representative sequences of apolipoprotein CII are shown in SEQ ID NO: 5 or SEQ ID NO: 7 (nucleic acid sequence) and SEQ ID NO: 6 or SEQ ID NO: 8 (amino acid sequence).

  Examples of the biological activity of apolipoprotein CII include the ability to form VLDL, HDL and chylomicron.

  Apolipoprotein CII is known not only in humans and rats but also in other animals (for example, mammals) and homologs thereof (referred to herein as “corresponding” genes or proteins). Accordingly, in the present specification, the apolipoprotein CII usually refers to the apolipoprotein CII that exists not only in humans, rats but also in general organisms unless otherwise specified.

  Apolipoprotein is produced as a pro form. When making a more detailed determination, it is preferable to distinguish the pro form from the mature form.

A representative nucleotide sequence of apolipoprotein CIII is:
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 11 or a fragment sequence thereof;
(B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12 or a fragment thereof;
(C) In the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, one or more amino acids are a variant polypeptide having one mutation selected from the group consisting of substitution, addition and deletion, or a fragment thereof. A polynucleotide encoding a variant polypeptide having biological activity;
(D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 11 or a fragment thereof;
(E) a polynucleotide encoding a species homologue of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12 or a fragment thereof;
(F) a polynucleotide that hybridizes to any one of the polynucleotides of (a) to (e) under a stringent condition and encodes a polypeptide having biological activity; or (g) (a) to It may be a polynucleotide that consists of a base sequence that is at least 70% identical to any one polynucleotide of (e) or its complementary sequence, and encodes a polypeptide having biological activity.

As an amino acid sequence of apolipoprotein CIII,
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12 or a fragment thereof;
(B) In the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12, one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion, and have biological activity A polypeptide;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 11;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12; or (e) at least 70% identity to any one polypeptide of (a)-(d) A polypeptide having an amino acid sequence that is and having biological activity,
It can be.

  Representative sequences of apolipoprotein CIII are shown in SEQ ID NO: 9 or SEQ ID NO: 11 (nucleic acid sequence) and SEQ ID NO: 10 or SEQ ID NO: 12 (amino acid sequence).

  Examples of the biological activity of apolipoprotein CIII include the ability to form VLDL, HDL, and chylomicron.

  Apolipoprotein CIII is known not only in humans, rats, but also in other animals (for example, mammals) and homologs thereof (referred to herein as “corresponding” genes or proteins). Therefore, in the present specification, the apolipoprotein CIII usually refers to apolipoprotein CIII which exists in general organisms, in addition to humans and rats, unless otherwise specified.

  Apolipoprotein CIII is known to exist in at least three types, CIII0, CIII1, and CIII2, depending on the degree of sugar chain binding.

  Thus, apolipoprotein CIII is a general term for three types of proteins, and is classified into apolipoprotein CIII0, apolipoprotein CIII1, and apolipoprotein CIII2. And in the diagnostic method of diabetes of this invention, the density | concentration of at least 1 sort (s) of these 3 types of apolipoprotein CIII is measured. According to the method for diagnosing diabetes of the present invention, the concentration of apolipoprotein CIII is subdivided and measured, so that the accuracy is high.

  Apolipoprotein CIII is one of 10 or more apolipoproteins present in the blood and is synthesized in the liver. The clinical significance of apolipoprotein CIII in blood is known to be high in obstructive jaundice and antilipidemia. Apolipoprotein CIII is further classified into three types, apolipoprotein CIII0, apolipoprotein CIII1, and apolipoprotein CIII2, depending on the presence or absence of sugar chains and the difference in structure. Apolipoprotein CIII0 has no sugar chain, apolipoprotein CIII1 has a sugar chain added to apolipoprotein CIII0, and apolipoprotein CIII2 has a sugar chain added more complexly to apolipoprotein CIII1 It is.

  Bondarenko, P.A. V. et al. J. Lipid Res. According to 1999; 40: 543-555, apolipoprotein CIII0, apolipoprotein CIII1, and apolipoprotein CIII2 have the following structures.

  To identify these, use specific factors such as identifiable antibodies, or treat them with enzymes that selectively cleave these specific sugar chains to determine whether the molecular weight decreases, etc. Can be confirmed.

In the present specification, “serum albumin” refers to albumin contained in serum, and is the most abundant in serum protein (about 4 g per 100 ml) and occupies 60% of the total protein. In humans, it has a molecular weight of 64000-68000 and an isoelectric point of pH 4.7-4.9. It has several roles, such as maintaining blood osmotic pressure, binding and transporting various substances (ions, pigments, some water-soluble vitamins, drugs, etc.), and providing a source of amino acids to tissues It is. Serum albumin is a kind of albumin and is the most abundant protein in serum. Serum albumin is a protein that does not have a sugar chain with a molecular weight of about 69000 (calculated from the primary amino acid structure is 66439) and is synthesized in the liver. The clinical significance of serum albumin in blood is known to show a low value reflecting the deterioration of nutritional status and the degree of liver damage.

The typical nucleotide sequence of serum albumin is
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 15 or a fragment sequence thereof;
(B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 16 or a fragment thereof;
(C) In the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 16, one or more amino acids are a variant polypeptide or a fragment thereof having one mutation selected from the group consisting of substitution, addition and deletion A polynucleotide encoding a variant polypeptide having biological activity;
(D) a polynucleotide which is a splice variant or allelic variant of the base sequence described in SEQ ID NO: 13 or SEQ ID NO: 15 or a fragment thereof;
(E) a polynucleotide encoding a species homologue of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 16 or a fragment thereof;
(F) a polynucleotide that hybridizes to any one of the polynucleotides of (a) to (e) under a stringent condition and encodes a polypeptide having biological activity; or (g) (a) to It may be a polynucleotide that consists of a base sequence that is at least 70% identical to any one polynucleotide of (e) or its complementary sequence, and encodes a polypeptide having biological activity.

As the amino acid sequence of serum albumin,
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 16 or a fragment thereof;
(B) In the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 16, one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion, and have biological activity A polypeptide;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 15;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 16; or (e) at least 70% identity to a polypeptide of any one of (a)-(d) A polypeptide having an amino acid sequence that is and having biological activity,
It can be.

  Representative sequences of serum albumin are shown in SEQ ID NO: 13 or SEQ ID NO: 15 (nucleic acid sequence) and SEQ ID NO: 14 or SEQ ID NO: 16 (amino acid sequence).

  The biological activity of serum albumin includes, for example, maintaining blood osmotic pressure, binding and transporting various substances (ions, dyes, some water-soluble vitamins, drugs, etc.), supplying amino acids to tissues The ability to become a source can be mentioned.

  Serum albumin is known not only in humans and rats but also in other animals (for example, mammals) and homologs thereof (referred to herein as “corresponding” genes or proteins). Accordingly, in the present specification, serum albumin usually refers to serum albumin present in general organisms, in addition to humans and rats, unless otherwise specified. Other animal sequences of serum albumin can include mice (SEQ ID NO: 17-18), dogs (SEQ ID NO: 19-20), rabbits (SEQ ID NO: 21-22), monkeys (SEQ ID NO: 23-24). .

(Diagnosis / pre-diagnosis (prevention) factors)
In the present invention, diagnosis or pre-diagnosis can be realized by using a factor or means specific to the marker substance.

  As used herein, an “agent” can be any substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.) that can achieve the intended purpose. Good. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto. As a factor specific for a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity, Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.

  Therefore, in the present specification, a “factor that specifically interacts” with a biological agent such as a polynucleotide or a polypeptide means an affinity for the biological agent such as the polynucleotide or the polypeptide, The affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides is typically equivalent or higher, preferably significantly (eg, statistically significant) ) Includes the expensive. Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.

  As used herein, a first substance or factor “specifically interacts” with a second substance or factor means that the first substance or factor has a second Interacting with a higher affinity than a substance or factor other than a substance or factor (especially another substance or factor present in a sample containing a second substance or factor). Specific interactions for a substance or factor include, for example, when both nucleic acid and protein are involved, such as hybridization in nucleic acids, antigen-antibody reactions in proteins, ligand-receptor reactions, enzyme-substrate reactions, etc. Examples include, but are not limited to, protein-lipid interactions, nucleic acid-lipid interactions, and the like, such as reactions with transcription factor binding sites. Thus, when both a substance or factor is a nucleic acid, the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor. Further, for example, when both substances or factors are proteins, the fact that the first substance or factor “specifically interacts” with the second substance or factor includes, for example, interaction by antigen-antibody reaction, receptor- Examples include, but are not limited to, interaction by a ligand reaction and enzyme-substrate interaction. When two substances or factors include proteins and nucleic acids, the first substance or factor “specifically interacts” with the second substance or factor means that the transcription factor and the transcription factor Interaction between the binding region of the nucleic acid molecule to be included.

  As used herein, “antibody” refers to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotype antibodies, and fragments thereof such as F (ab ′) 2 and Fab fragments, and other recombinants. Conjugates produced by In addition, such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like.

  In this specification, “means” refers to anything that can be an arbitrary tool that achieves a certain purpose. In particular, in this specification, “means for selectively recognizing” refers to an object as another. Means that can be recognized differently. Such ability is also referred to herein as “ability to distinguish one subject (eg, transthyretin) from another (eg, a transsilentin derivative such as cysteinyl transthyretin). is there. As used in this specification, as a means for selectively recognizing both, it is only necessary to be able to recognize both differentially and not to recognize the other. There is no need. However, it is preferable that the means is preferably one that recognizes only one and not the other. This is because the results can be interpreted efficiently.

  As used herein, “antigen” refers to any substrate that can be specifically bound by an antibody molecule. As used herein, “immunogen” refers to an antigen capable of initiating lymphocyte activation that produces an antigen-specific immune response.

  It will be understood that antibodies of any specificity may be used as long as false positives are reduced. Therefore, the antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.

  As used herein, “compound” means any identifiable chemical or molecule, including small molecules, peptides, proteins, sugars, nucleotides, or nucleic acids, including: Without limitation, such compounds can be natural or synthetic.

  In the present specification, the “small organic molecule” means an organic molecule having a relatively small molecular weight. Usually, a low molecular weight organic material has a molecular weight of about 1000 or less, but may have a higher molecular weight. The organic small molecule can be synthesized usually by using a method known in the art or a combination thereof. Such small organic molecules may be produced by living organisms. Examples of small organic molecules include hormones, ligands, signal transmitters, small organic molecules, molecules synthesized by combinatorial chemistry, and small molecules that can be used as pharmaceuticals (for example, small molecule ligands). It is not limited.

  As used herein, “ligand” refers to a substance that specifically binds to a certain protein. Examples of the ligand include lectins, antigens, antibodies, hormones, neurotransmitters, and the like that specifically bind to various receptor protein molecules present on the cell membrane.

  As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also refer to a complex assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is included. When specifically mentioned, the “polypeptide” of the present invention may refer to a marker substance.

    In the present specification, the “amino acid” may be natural or non-natural. “Derivative amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art.

  As used herein, “natural amino acid” means an L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to in this specification are in L form.

  As used herein, “unnatural amino acid” means an amino acid that is not normally found naturally in proteins. Examples of non-natural amino acids include the above-mentioned D-type amino acids, norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homoarginine D-form or L-form and D-phenylalanine Is mentioned.

  As used herein, “amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by the generally accepted single letter code.

  As used herein, “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. “Oligonucleotide derivatives” or “polynucleotide derivatives” refer to oligonucleotides or polynucleotides that include derivatives of nucleotides or that have unusual linkages between nucleotides, and are used interchangeably. Specific examples of such oligonucleotides include, for example, 2′-O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides. Derivative converted to N3′-P5 ′ phosphoramidate bond, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide is C− Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives wherein uracil in oligonucleotide is substituted with C-5 thiazole uracil, cytosine in oligonucleotide is C-5 propynylcytosine Substituted oligonucleotide derivatives, oligonucleotide derivatives in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA is replaced with 2'-O-propylribose Examples thereof include oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-). 98 (1994)).

  In the present specification, the “nucleotide” may be natural or non-natural. “Nucleotide derivative” or “nucleotide analog” refers to a substance that is different from a naturally occurring nucleotide but has a function similar to that of the original nucleotide. Such nucleotide derivatives and nucleotide analogs are well known in the art. Examples of such nucleotide derivatives and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, peptide-type nucleic acids (PNA). Not.

  As used herein, “complex molecule” refers to a molecule formed by linking a plurality of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules. Examples of such complex molecules include, but are not limited to, glycolipids and glycopeptides. In the present specification, a nucleic acid molecule encoding a polypeptide having the amino acid of SEQ ID NO: 2, or a variant or fragment thereof, as long as it has biological activity involved in diagnosis, and the like. Can also be used. A complex molecule containing such a nucleic acid molecule can also be used.

  As used herein, “nucleic acid” is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. Particular nucleic acid sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a gene that affects its expression is called a regulatory gene. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein” “polypeptide”, “oligopeptide” and “peptide”.

  As used herein, “homology” of genes refers to the degree of identity of two or more gene sequences with respect to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous.

  In the present specification, comparison of similarity, homology and identity of amino acid and base sequences is calculated using BLAST, which is a tool for sequence analysis, using default parameters. The identity search can be performed using, for example, NCBI BLAST 2.2.9 (issued 2004.5.12). In the present specification, the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.

  As used herein, a “corresponding” amino acid or nucleic acid has or has the same action as a predetermined amino acid or nucleotide in a reference polypeptide or polynucleotide in a polypeptide molecule or polynucleotide molecule. In particular, in the case of an enzyme molecule, it means an amino acid that is present at the same position in the active site and contributes similarly to the catalytic activity. For example, an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. Corresponding amino acids are, for example, cysteinylated, glutathione, SS bond formation, oxidation (eg, oxidation of methionine side chain), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc. It can be a specific amino acid. Alternatively, the corresponding amino acid can be an amino acid responsible for dimerization. Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain.

  As used herein, a “corresponding” gene (eg, a polypeptide molecule or a polynucleotide molecule) has, or is predicted to have, in a certain species, the same effect as a given gene in a species as a reference for comparison. Gene (for example, a polypeptide molecule or a polynucleotide molecule), and when there are a plurality of genes having such an action, the genes having the same evolutionary origin. Thus, a gene corresponding to a gene can be an ortholog of that gene. Therefore, mouse, rat apolipoprotein CII, apolipoprotein CIII, transthyretin and serum albumin can respectively find corresponding apolipoprotein CII, apolipoprotein CIII, transthyretin and serum albumin in humans. Such corresponding genes can be identified using techniques well known in the art. Thus, for example, the corresponding gene in an animal (for example, mouse) is the query sequence of the sequence of the gene that serves as a reference for the corresponding gene (for example, apolipoprotein CII, apolipoprotein CIII, transthyretin, serum albumin). And can be found by searching a sequence database of the animal (eg, human, rat).

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg 11 etc.) are also suitable as lower limits obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, it is understood that such a fragment falls within the scope of the present invention as long as the full-length fragment functions as a marker, as long as the fragment itself also functions as a marker.

  As used herein, “contacting” means bringing a compound into physical proximity, either directly or indirectly, to a polypeptide or polynucleotide of the present invention. . The polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like. Contact includes placing the compound in, for example, a beaker, microtiter plate, cell culture flask or microarray (eg, gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof.

    As used herein, “polynucleotide that hybridizes under stringent conditions” refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A PR Applical Approach, Second Edition, Oxford Universe. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. Accordingly, a polypeptide (for example, transthyretin) used in the present invention is a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule encoding a polypeptide particularly described in the present invention. The polypeptide encoded by is also encompassed.

  In the present specification, the “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, as a polynucleotide capable of hybridizing, a polynucleotide having at least 60% or more homology with a DNA base sequence encoding a polypeptide having the amino acid sequence represented by SEQ ID NOs: 2, 4, 6, etc., A polynucleotide having a homology of 80% or more is preferable, and a polynucleotide having a homology of 95% or more is more preferable.

Factors that affect the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one of ordinary skill in the art to apply these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
Tm (° C.) = 81.5 + 16.6 (log [Na +]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na +] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is (guanine + cytosine in the hybrid) ) Percentage of base. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch.

  As used herein, a “purified” biological agent (such as a nucleic acid or protein) refers to a product from which at least part of the factors naturally associated with the biological agent has been removed. Thus, typically the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.

  The term “purified” as used herein is preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight, Means the presence of the same type of biological agent.

  In the present specification, “expression” of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form. Preferably, a gene, polynucleotide or the like is transcribed and translated into a polypeptide form. However, transcription and production of mRNA can also be an aspect of expression. More preferably, such polypeptide forms may be post-translationally processed (derivatives as referred to herein).

  As used herein, “detection” or “quantification” of polypeptide expression can be accomplished using any suitable method, including, for example, measuring mRNA and immunological methods. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR. Examples of the immunological measurement method include an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a Western blot method, and an immunohistochemical staining method. Examples of the quantitative method include an ELISA method and an RIA method.

  In the present specification, the “expression level” refers to an amount at which a polypeptide or mRNA is expressed in a target cell or the like. Such expression level is evaluated by any appropriate method including immunoassay methods such as ELISA method, RIA method, fluorescent antibody method, Western blot method, and immunohistochemical staining method using the antibody of the present invention. The amount of expression of the polypeptide of the present invention at the protein level, or the polypeptide used in the present invention evaluated by any suitable method including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. The expression level of the peptide at the mRNA level can be mentioned. “Change in expression level” means expression at the protein level or mRNA level of the polypeptide used in the present invention evaluated by any appropriate method including the above immunological measurement method or molecular biological measurement method. Means that the amount increases or decreases.

  The term “binding” as used herein means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. A physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates.

  The term “modulate” or “modify” as used herein means an increase or decrease or maintenance in the amount, quality or effect of a particular activity, transcript or protein.

  As used herein, “decrease” or “suppression” or synonyms for activity, expression products (eg, proteins, transcripts (RNA, etc.)) or a synonym is a decrease in the quantity, quality or effect of a particular activity, transcript or protein. Or activity to decrease.

  As used herein, “increase” or “activation” of an activity, an expression product (eg, a protein, transcript (such as RNA)) or a synonym thereof refers to a quantity, quality or effect of a particular activity, transcript or protein. An activity that increases or increases.

  As used herein, the term “probe” refers to a substance that serves as a search means used in biological experiments such as screening in vitro and / or in vivo. For example, a nucleic acid molecule containing a specific base sequence or a specific nucleic acid molecule Examples include, but are not limited to, peptides containing amino acid sequences. In this specification, the probe is used as a marker detection means.

  Examples of nucleic acid molecules that are usually used as probes include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is homologous or complementary to the nucleic acid sequence of the target gene. Such a nucleic acid sequence is preferably at least 12 contiguous nucleotides long, at least 9 contiguous nucleotides, more preferably at least 10 contiguous nucleotides, and even more preferably at least 11 contiguous nucleotides. At least 13 contiguous nucleotide lengths, at least 14 contiguous nucleotide lengths, at least 15 contiguous nucleotide lengths, at least 20 contiguous nucleotide lengths, at least 25 contiguous nucleotide lengths, at least 30 It can be at least a nucleic acid sequence of at least 40 contiguous nucleotides, at least 50 contiguous nucleotides long, of contiguous nucleotides. Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above. Is included.

  As used herein, “search” refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods. Say. As an electronic search, BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-). 2448 (1988)), the Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and the Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:44:44). -453 (1970)) and the like. Biological searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization, etc. It is not limited to. In the present specification, it is intended that the gene used in the present invention should include a corresponding gene identified by such an electronic search or biological search.

  In the present specification, the “primer” refers to a substance necessary for initiation of a reaction of a polymer compound to be synthesized in a polymer synthase reaction. In the synthesis reaction of a nucleic acid molecule, a nucleic acid molecule (for example, DNA or RNA) complementary to a partial sequence of a polymer compound to be synthesized can be used. In the present specification, the primer can be used as a marker detection means.

  Examples of nucleic acid molecules that are usually used as primers include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the target gene. Such a nucleic acid sequence is preferably at least 12 contiguous nucleotides long, at least 9 contiguous nucleotides, more preferably at least 10 contiguous nucleotides, and even more preferably at least 11 contiguous nucleotides. At least 13 contiguous nucleotides, at least 14 contiguous nucleotides, at least 15 contiguous nucleotides, at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 At least 19 contiguous nucleotides, at least 19 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 40 Nucleotides long that connection, at least 50 contiguous nucleotides in length, may be a nucleic acid sequence. Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above. Is included. A sequence suitable as a primer may vary depending on the nature of the sequence intended for synthesis (amplification), but those skilled in the art can appropriately design a primer according to the intended sequence. Such primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar).

  As used herein, “biological activity” refers to an activity that a certain factor (eg, polypeptide or protein) may have in vivo, and includes an activity that exhibits various functions. For example, when an agent is a ligand, the biological activity includes the activity of the ligand binding to the corresponding receptor.

(Detection of transthyretin, transthyretin derivative, apolipoprotein CII, apolipoprotein CII derivative, apolipoprotein CIII, apolipoprotein CIII derivative and serum albumin and their corresponding proteins)
When detecting transthyretin, transthyretin derivatives, apolipoprotein CII, apolipoprotein CII derivatives, apolipoprotein CIII, apolipoprotein CIII derivatives and serum albumin and serum albumin contained in body fluids and the like and their corresponding proteins, Retin, transthyretin derivative, apolipoprotein CII, apolipoprotein CII derivative, apolipoprotein CIII, apolipoprotein CIII derivative and serum albumin and antibodies that specifically recognize these proteins (hereinafter referred to as transthyretin antibody, transthyretin) Retin derivative antibody, apolipoprotein CII antibody, apolipoprotein CII derivative antibody, apolipoprotein CIII antibody, apoli Protein CIII derivatives referred such as antibodies and serum albumin antibodies as well as antibodies of the proteins corresponding to these) to produce. Such an antibody can be prepared using a conventionally known technique. The antibody may be a monoclonal antibody or a polyclonal antibody. For other marker substances (for example, transthyretin, transthyretin derivative, apolipoprotein CII, apolipoprotein CII derivative, apolipoprotein CIII, apolipoprotein CIII derivative and serum albumin and their corresponding proteins) Can be produced.

  As an example, a method for preparing a transthyretin monoclonal antibody is described below. Transthyretin monoclonal antibody is a hybridoma prepared by cell fusion between an antibody-producing cell obtained from an animal immunized with an antigen and a myeloma cell, and an antibody that specifically inhibits the activity of transthyretin is produced from the resulting hybridoma. Can be prepared by selecting a clone to be selected.

  Examples of the transthyretin protein used as an antigen for animal immunization include all or part of the amino acid sequence of the transthyretin protein prepared by recombinant DNA method or chemical synthesis. For example, a peptide consisting of the 21st to 147th amino acid sequences in the amino acid sequence of the transthyretin protein shown in SEQ ID NO: 2 (that is, mature type) can be used as the antigen. In addition, as a transthyretin monoclonal antibody for specifically detecting the transthyretin protein present on the cell surface, any 10 or more peptides in the amino acid sequence of the transthyretin protein shown in SEQ ID NO: 2 can be used as the antigen. It is preferable to use as. Any factor in the molecular pathway of other transthyretins (eg, transthyretin, transthyretin derivatives, apolipoprotein CII, apolipoprotein CII derivative, apolipoprotein CIII, apolipoprotein CIII derivative and serum albumin and their corresponding proteins) Etc.), the antigen can be designed in the same manner.

  The obtained transthyretin for antigen is bound to a carrier protein (for example, thyroglobulin), and then an adjuvant is added. Examples of adjuvants include Freund's complete adjuvant and Freund's incomplete adjuvant, and any of these may be mixed.

  The antigen obtained as described above is administered to mammals such as mammals such as mice, rats, horses, monkeys, rabbits, goats and sheep. Any method can be used for immunization as long as it is an existing method, but it is mainly carried out by intravenous injection, subcutaneous injection, intraperitoneal injection or the like. Immunization intervals are not particularly limited, and immunization is performed at intervals of several days to several weeks, preferably at intervals of 4 to 21 days.

  Antibody-producing cells are collected 2-3 days after the last immunization. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells. Generally, spleen cells are used. For example, 100 μg of antigen is used per mouse at a time.

  In order to confirm the immune response level of the immunized animal and to select the target hybridoma from the cells after cell fusion treatment, the antibody titer in the blood of the immunized animal or the antibody titer in the culture supernatant of the antibody producing cell Measure. Examples of antibody detection methods include known techniques such as EIA (enzyme immunoassay), RIA (radioimmunoassay), ELISA (enzyme linked immunosorbent assay) and the like.

  As myeloma (myeloma) cells to be fused with antibody-producing cells, cell lines derived from various animals such as mice, rats, humans and generally available to those skilled in the art are used. As a cell line to be used, a cell line having drug resistance and having the property that it cannot survive in a selective medium (for example, HAT medium) in an unfused state but can survive only in a fused state is used. An 8-azaguanine resistant strain is generally used, and this cell line is deficient in hypoxanthine-guanine-phosphoribosyltransferase and cannot grow in hypoxanthine / aminopterin / thymidine (HAT) medium.

  Myeloma cells are known in various known cell lines such as P3 (P3x63Ag8.653) (J. Immunol. (1979) 123: 1548-1550), P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81: 1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol. (1976) 6: 511-519), MPC-. 11 (Margulies, DH et al., Cell (1976) 8: 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276: 269-270), FO (de St Groth, SF et al., J. Immunol. Methods (1980) 35: 1-21), S194 (Travebridge, IS, J. Exp. Med. (1978) 148: 313-323). , R210 . Galfre, G.et al, Nature (1979) 277: 131-133) or the like is preferably used.

  Antibody-producing cells are obtained from spleen cells, lymph node cells, and the like. That is, spleen, lymph nodes, etc. are removed or collected from the various animals, and these tissues are crushed. The obtained crushed material is suspended in a medium or buffer solution such as PBS, DMEM, RPMI 1640, etc., filtered through a stainless mesh, etc., and then centrifuged to prepare the desired antibody-producing cells.

  Next, the myeloma cell and the antibody-producing cell are fused. Cell fusion is performed by mixing myeloma cells and antibody-producing cells at a mixing ratio of 1: 1 to 1:10 in an animal cell culture medium such as MEM, DMEM, and RPME-1640 medium in the presence of a fusion promoter. It is performed by contacting at 37 ° C. for 1 to 15 minutes. In order to promote cell fusion, a fusion promoter or fusion virus such as polyethylene glycol, polyvinyl alcohol or Sendai virus having an average molecular weight of 1,000 to 6,000 can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

  The target hybridoma is selected from the cells after cell fusion treatment. Examples of the method include a method utilizing selective growth of cells in a selective medium. That is, after the cell suspension is diluted with an appropriate medium, it is plated on a microtiter plate, a selective medium (such as HAT medium) is added to each well, and then the selective medium is appropriately replaced and cultured. As a result, growing cells can be obtained as hybridomas.

  Hybridoma screening is performed by limiting dilution, fluorescence excitation cell sorter, or the like, and finally a monoclonal antibody-producing hybridoma is obtained. Examples of a method for collecting a monoclonal antibody from the obtained hybridoma include a normal cell culture method and ascites formation method. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10 to 20% fetal bovine serum, MEM medium, or serum-free medium (eg, 37 ° C., 5% CO 2 concentration). ) For 2 to 14 days, and antibodies are obtained from the culture supernatant. In the ascites formation method, a hybridoma is administered into the abdominal cavity of an animal of the same kind as a mammal derived from myeloma cells, and the hybridoma is proliferated in large quantities. Then, ascites or serum is collected after 1 to 4 weeks.

  When antibody purification is required in the above antibody collection method, purification is performed by appropriately selecting a known method such as ammonium sulfate salting-out method, ion exchange chromatography, affinity chromatography, or a combination thereof. To do.

  Therefore, an antigen binds to an antibody or binds to a specific receptor such as B lymphocyte, T lymphocyte, etc., and causes an immune reaction such as antibody production and / or cytotoxicity (eg, protein, lipid, sugar). But are not limited to these). Here, the binding property with an antibody or a lymphocyte receptor is referred to as “antigenicity”. Properties that induce an immune response, such as antibody production, are referred to as “immunogenicity”. The substance used as the antigen includes, for example, at least one target substance (for example, protein). The substance to be contained is preferably full length, but may be a partial sequence as long as it contains at least one epitope capable of inducing immunity. As used herein, “epitope” or “antigenic determinant” refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by those skilled in the art using such well known techniques once the primary sequence of the nucleic acid or amino acid is provided.

  Epitopes can be used even if their exact location and structure are not known. Thus, an epitope is required for a set of amino acid residues involved in recognition by a particular immunoglobulin, or in the case of T cells, for recognition by T cell receptor proteins and / or major histocompatibility complex (MHC) receptors. A set of amino acid residues is included. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site”. In the immune system field, in vivo or in vitro, an epitope is a molecular feature (eg, primary peptide structure, secondary peptide structure or tertiary peptide structure and charge) and is recognized by immunoglobulins, T cell receptors or HLA molecules. Form a site. An epitope comprising a peptide can comprise more than two amino acids in a spatial conformation unique to the epitope. In general, an epitope consists of at least 5 such amino acids and typically consists of at least 6, 7, 8, 9, or 10 such amino acids. Longer epitope lengths are generally preferred because they are more similar to the antigenicity of the original peptide, but may not necessarily be so in view of conformation. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance spectroscopy. Furthermore, identification of epitopes in a given protein is readily accomplished using techniques well known in the art. See, for example, Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998 (a general method for rapidly synthesizing peptides to determine the location of immunogenic epitopes in a given antigen); US Pat. No. 4,708,871 (identifying epitopes of antigen and See Procedure for Chemical Synthesis); and Geysen et al. (1986) Molecular Immunology 23: 709 (a technique for identifying peptides with high affinity for a given antibody). Antibodies that recognize the same epitope can be identified in a simple immunoassay. Thus, methods for determining epitopes comprising peptides are well known in the art, and once such epitopes are provided with the primary sequence of a nucleic acid or amino acid, one skilled in the art will use such well known techniques. Can be determined.

  Thus, for use as an epitope comprising a peptide, a sequence of at least 3 amino acids in length is required, preferably this sequence is at least 4 amino acids, more preferably at least 5 amino acids, at least 6 amino acids, at least 7 A sequence of amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids in length may be required. Epitopes may be linear or conformational.

  In recent years, methods for acquiring a desired antibody more rapidly have been known. For example, a method described in JP-A-2006-149383 can be used. This method comprises antibodies that specifically bind to streptavidin from a population of DT40 cells (see WO2004 / 011644) that induces somatic recombination at an immunoglobulin locus and produces various immunoglobulin molecules. This is done by sorting the molecules.

  The procedure consists of a diversified library (eg, a library composed of chicken-derived DT40 cells capable of presenting various antibody molecules on the cell surface) and at least one kind that specifically binds to the ligand of interest. This is a method of selecting a protein. This method includes the following steps: (1) Various proteins present in the library and a ligand of interest (anything can be used as long as it binds to a protein, but is not limited to, for example, A step of contacting a protein, nucleic acid, lipid, carbohydrate, low molecular weight compound, etc.), incubating a mixture of the protein group and the target ligand, and recovering a complex of at least one protein and the target ligand (2) step (1) A step of contacting the target ligand with at least several proteins selected in step), incubating a mixture of the protein and the target ligand, and confirming the binding between the protein and the target ligand, (3) selecting in step (1) Contacting at least some of the produced proteins with one or two specific control ligands, A step of incubating a mixture of the proteins to determine whether the protein and the control ligand are bound, and (4) the binding to the target ligand is confirmed in step (1), and the control in step (c) Selecting a protein that did not bind to the ligand. Selection of a protein that binds to the target ligand can be performed based on ordinary knowledge in the art. For example, the target ligand is bound to an appropriate carrier, the bound target ligand is brought into contact with the protein present in the diversified library, incubated under appropriate conditions, and the resulting carrier-target ligand-protein complex is then obtained. A protein that is recovered by centrifugation or the like and that binds to the target ligand can be selected.

(Modified)
Certain amino acids can be replaced with other amino acids in a protein structure such as, for example, a sugar chain binding region, a cysteinylation region, a cationic region, or a binding site of a substrate molecule, without an apparent reduction or loss of interaction binding capacity . It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is well known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, an equivalent protein in enzymatic activity). It is. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.

  The hydrophilicity index is also useful for modifying the amino acid sequence of the present invention. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present invention, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine; However, it is not limited to these.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). .

  As used herein, “conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. The addition of amino acids means that one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids are added to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such a number retains the target function (for example, marker etc.) in the variant having the substitution, addition or deletion. You can do as much as you can. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

  In the present specification, the “derivative” may also exist for the “variant” as described above.

(Diagnosis method)
As used herein, “diagnosis” refers to identifying various parameters related to a disease, disorder, or condition in a subject and determining the current state or future of such a disease, disorder, or condition. By using the method, apparatus, and system of the present invention, the state in the body can be examined, and such information is used to formulate a disease, disorder, condition, treatment to be administered or prevention in a subject. Alternatively, various parameters such as methods can be selected. In the present specification, in a narrow sense, “diagnosis” refers to diagnosing the current state, but in a broad sense includes “preliminary diagnosis”.

  In particular, in the present specification, “preliminary diagnosis” refers to detecting the stage before the onset of diabetes when referring to diabetes, and is associated with diabetes for the purpose of determining the risk of future onset and preventing diabetes. This includes determining whether or not there is a risk of doing so. By using the method, apparatus, and system of the present invention, the state of the body can be examined in advance, and such information can be used to treat a disease, disorder, condition, treatment to be administered or prevention in a subject. Various parameters such as formulation or method can be selected.

  The diagnostic method of the present invention is industrially useful because, in principle, the diagnostic method can be used from the body and can be carried out away from the hands of medical personnel such as doctors. In this specification, in order to clarify that it can be performed away from the hands of medical personnel such as doctors, it may be specifically referred to as “advanced diagnosis or assistance”.

  As used herein, “treatment” refers to preventing a disease or disorder from deteriorating, preferably maintaining the status quo, more preferably reducing, when a certain disease or disorder becomes such a condition. Preferably, it means elimination.

  In the method for diagnosing diabetes of the present invention, the concentration of at least one of these three proteins in blood is measured as a marker substance for diabetes. And the diagnosis of diabetes is performed by comparing the measured value with a healthy value. Here, “diagnosis of diabetes” is not only determining whether or not the patient is suffering from diabetes, but also determining whether or not there is a risk of suffering from diabetes for the purpose of preventing diabetes, And monitoring for recurrence. In the method for diagnosing diabetes according to the present invention, the concentrations of only a part of the three types of marker substances may be measured, or the concentrations of all three types may be measured. In particular, when measuring the concentration of all marker substances, a multimarker system can be assembled to diagnose diabetes from various directions, and the accuracy of diagnosis is high. In addition, since all of these three types of proteins are also present in the blood of healthy subjects, by monitoring the fluctuations in the concentration, it is possible to detect signs that the healthy subjects will develop diabetes.

  The disease diagnosis method of the present invention comprises comparing at least one concentration of the marker substance (for example, (a) to (n)) in a body fluid of a subject with a healthy value, The risk of onset is determined. In the disease diagnosis method of the present invention, blood glucose is not directly used as an index, but another marker substance is used as an index, so that it is possible to capture a state before the blood glucose level rises. As a result, in addition to the presence or absence of diabetes, the risk of future onset of diabetes can be determined. “Preliminary diagnosis of diabetes” and “determining the risk of future development of diabetes” are used interchangeably, and may have diabetes in the future (risk) at the time when diabetes does not occur Determining whether or not there is or the degree of possibility (risk).

  Here, values such as “about 7040”, “about 8330”, and “about 8530” of the mass / charge ratio (hereinafter sometimes abbreviated as “M / Z”) in each marker substance are used in mass spectrometry. This is a value that takes into account the error range of the measured value, and has a width of approximately ± 0.2%. That is, about 7040 represents about 7040 ± 0.2%, about 8330 represents about 8330 ± 0.2%, and about 8530 represents about 8530 ± 0.2%. The other mass / charge ratios have a width of approximately ± 0.2% in a similar manner. In addition, these marker substances are mainly proteins present in blood. When the subject has developed diabetes or when the risk of developing diabetes is high, the marker substances (a), (b), (c), (d), (e), ( The concentrations of f), (g), (h) and (i) are higher, and the concentrations of the marker substances (j), (k), (l), (m) and (n) are lower. Show.

  In a preferred embodiment of the disease diagnosis method of the present invention, a carrier on which a substance having affinity for a marker substance is immobilized is used. Then, the body fluid or body fluid component is brought into contact with the carrier, and the marker substance contained in the body fluid or body fluid component is captured on the carrier via a substance having affinity for the marker substance, and the amount of the captured marker substance is increased. Based on this, the concentration of the marker substance in the body fluid is calculated. According to the disease diagnosis method of the present invention, since the marker substance captured on the carrier is the measurement target, the influence of the contaminant substance contained in the measurement sample can be reduced, and the sensitivity and precision can be increased. The concentration of the marker substance can be measured. Examples of the body fluid component include serum or plasma when the body fluid is blood.

  In a preferred embodiment of the disease diagnosis method of the present invention, a carrier having a planar portion is used, and the substance having affinity for the marker substance is immobilized on a part of the planar portion. With this configuration, a substance having affinity for the marker substance can be spot-fixed at a plurality of locations on the carrier. As a result, it is possible to simultaneously process a plurality of measurement samples with one carrier, and simultaneously measure the concentrations of a plurality of marker substances with one carrier, and work efficiency is improved. Furthermore, by reducing the area of each spot, the concentration of the marker substance can be measured even from a very small amount of measurement sample. An example of the carrier having a planar portion is a substrate such as a chip.

  In a preferred embodiment of the disease diagnosis method of the present invention, an ion exchanger, a metal chelate or an antibody is used as a substance having an affinity for a marker substance, and the measurement sample is passed through the ion exchanger, metal chelate or antibody. The marker substance is captured on the carrier. When the substance is an ion exchanger or a metal chelate, various substances are easily available, and a carrier for capturing the marker substance can be easily prepared. Further, when the substance is an antibody, the marker substance can be captured more specifically. Examples of the method for measuring the amount of the captured marker substance include mass spectrometry and immunoassay (in the case of an antibody).

(system)
In this specification, “system” refers to an arbitrary system for diagnosis, and generally includes one or a plurality of components, and when there are a plurality of components, these components act and relate to each other. It means a system that satisfies the three conditions of exhibiting harmonious behavior and function as a whole. The system can be in any form, such as a device, composition, diagnostic agent. Accordingly, the system is, for example, an in-vitro drug including a large-scale system including a measuring device, a system including a chromatography, a kit using an immune reaction, a composition including an antibody (that is, a monoclonal antibody as a marker substance). It is understood to include diagnostic agents).

(screening)
In the present invention, it is also intended that a drug by computer modeling is provided based on the disclosure of the present invention.

  As used herein, “screening” refers to selecting a target such as a target organism or substance having a specific property from a large number of populations by a specific operation / evaluation method. For screening, an agent (eg, antibody), polypeptide or nucleic acid molecule of the invention can be used. For the screening, a system using an actual substance such as in vitro or in vivo may be used, or a library generated using an in silico (computer system) system may be used. In the present invention, it is understood that compounds obtained by screening having a desired activity are also encompassed within the scope of the present invention. The present invention also contemplates providing a drug by computer modeling based on the disclosure of the present invention.

  In one embodiment, the invention screens for candidate or test compounds that bind to or modulate the activity of the protein of the invention or polypeptide of the invention, or biologically active portion thereof. An assay is provided. Test compounds of the invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, including: biological libraries; spatially Accessible parallel solid phase or solution phase library; synthetic library method requiring deconvolution; “one bead one compound” library method; and synthetic library method using affinity chromatography selection. The biological library approach is limited to peptide libraries, but the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam (1997) Anticancer Drug Des. 12). 145).

  Examples of methods for the synthesis of molecular libraries can be found in the art, for example: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994) J. MoI. Med. Chem 37: 2678; Cho et al. (1993) Science 2611: 1303; Carrell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2059; Carrell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2061; and Gallop et al. (1994) J. MoI. Med. Chem 37: 1233.

  The library of compounds can be in solution (eg, Houghten (1992) BioTechniques 13: 412-421), or on beads (Lam (1991) Nature 354: 82-84), on chip (Fodor (1993) Nature 364: 555-556), bacteria (Ladner US Pat. No. 5,223,409), spores (Ladner, supra), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-1869) or phage (Scott and Smith (1990) Science 249: 386-390; Devlin (1990) Science 249: 404-406; Cwilla et al. (1990) Proc. Natl. Acad. ci.U.S.A.87: 6378~6382; Felici (1991) J Mol Biol 222: 301~310; Ladner above) can be shown in.

  In another embodiment, the present invention provides a quantitative structure-activity relationship by computer as a tool for screening factors that are equally effective as the active ingredient (eg, polypeptide or nucleic acid) of the present invention. Compounds obtained using QSAR) modeling techniques are also encompassed by the present invention. Here, the computer technology includes substrate templates, pharmacophores created by several computers, and creation of a homology model of the active site of the present invention. In general, from data obtained in vitro, a method for modeling the normal characteristic groups of an interacting substance for a substance is described by the CATALYST ™ pharmacophore method (Ekins et al., Pharmacogenetics, 9: 477-489, 1999). Ekins et al., J. Pharmacol. & Exp. Ther., 288: 21-29, 1999; Ekins et al., J. Pharmacol. & Exp. Ther., 290: 429-438, 1999; , J. Pharmacol. & Exp. Ther., 291: 424-433, 1999) and comparative molecular field analysis (CoMFA). (. Jones et al, Drug Metabolism & Disposition, 24: 1~6,1996) are shown using, for example. In the present invention, computer modeling may be performed using molecular modeling software such as CATALYSTTM version 4 (Molecular Simulations, Inc., San Diego, CA).

  Fitting a compound to the active site can be done using any of a variety of computer modeling techniques known in the art. Visual inspection and manual manipulation of compounds to the active site are described in QUANTA (Molecular Simulations, Burlington, MA, 1992), SYBYL (Molecular Modeling Software, Tripos Associates, Inc., St. AM, St. Louis, MO, 1992). et al., J. Am. Chem. Soc., 106: 765-784, 1984), CHARMM (Brooks et al., J. Comp. Chem., 4: 187-217, 1983), etc. Can be done using. In addition, energy can be minimized using standard force fields such as CHARMM, AMBER, and the like. Other more specialized computer modeling are GRID (Goodford et al., J. Med. Chem., 28: 849-857, 1985), MCSS (Miranker and Karplus, Function and Genetics, 11: 29-34, 1991), AUTODOCK (Goodsell and Olsen, Proteins: Structure, Function and Genetics, 8: 195-202, 1990), DOCK (Kuntz et al., J. Mol. Biol., 161: 269-288, (1982). ) Etc. Further structural compounds include LUDI (Bohm, J. Comp. Aid. Molec. Design, 6: 61-78, 1992), LEGEND (Nishibata and Itai), such as blank active sites, active sites in known low molecular weight compounds, and the like. , Tetrahedron, 47: 8985, 1991), LeapFrog (Tripos Associates, St. Louis, Mo.), etc. Such modeling is well known and commonly used in the art, and those skilled in the art can appropriately design compounds that fall within the scope of the present invention according to the disclosure of the present specification.

(Administration / Infusion / Medicine)
The composition containing the substance obtained by the screening method of the present invention can be provided in any preparation form as long as the composition is suitable for transfer to an organism. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent. Examples of administration routes include oral administration, parenteral administration, and direct administration to the affected area.

  As used herein, the term “kit” refers to a unit provided with a portion (eg, antibody, label, etc.) to be provided, usually divided into two or more compartments. This kit form is preferred when it is intended to provide a composition that should not be provided in a mixed form but is preferably used in a mixed state immediately prior to use. Such a kit preferably comprises a provided part (eg, instructions or instructions describing how the reagent should be processed. In this specification the kit is a reagent kit. When used as a kit, the kit usually includes instructions describing how to use the antibody.

  In the present specification, the “instruction” describes an explanation for a person who administers a method of administering the medicine of the present invention, such as a doctor or a patient. This instruction manual describes a word for instructing how to use the diagnostic agent of the present invention or administering a medicine or the like. In addition, the instruction sheet may include a word for instructing administration (for example, by injection) to skeletal muscle as an administration site. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the US) in which the present invention is implemented, and is approved by the regulatory authority It is clearly stated that it has been received. The instruction is a so-called package insert and is usually provided as a paper medium, but is not limited thereto. For example, a form such as an electronic medium (for example, a homepage or e-mail provided on the Internet) is used. But it can be provided.

  As used herein, “subject” refers to an organism to which the treatment of the present invention is applied, and is also referred to as a “patient”. The patient or subject may preferably be a human.

  As used herein, “in vivo” or “in vivo” refers to the inside of a living body. In a particular context, “in vivo” refers to the location where a target substance is to be placed.

  As used herein, “in vitro” refers to a state in which a part of a living body is removed or released “in vitro” (eg, in a test tube) for various research purposes. A term that contrasts with in vivo.

  In this specification, “ex vivo” refers to a series of operations ex vivo when a target cell for gene transfer is extracted from a subject, a therapeutic gene or factor is introduced in vitro, and then returned to the same subject again. .

  The polypeptides, nucleic acids, medicaments used in the present invention, and compositions prepared with such polypeptides or nucleic acids can be provided in any dosage form as long as they are in a form suitable for transfer to an organism. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent. Administration methods include oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, topical administration to affected areas, skin administration, etc.) Examples include direct administration to the affected area. Formulations for such administration can be provided in any dosage form. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent. The compositions and medicaments of the present invention may be in the form of an orally acceptable aqueous solution that is pyrogen-free when administered systemically. The preparation of such pharmaceutically acceptable protein solutions is within the skill of the artisan, provided that considerable attention is paid to pH, isotonicity, stability, and the like.

  The solvent used for pharmaceutical formulation in the present invention may have either aqueous or non-aqueous properties. In addition, the vehicle can be used to modify or maintain the pH, osmolarity, viscosity, clarity, color, sterility, stability, isotonicity, disintegration rate, or odor of the formulation. Material may be included. Similarly, the compositions of the present invention may include other formulation materials to modify or maintain the release rate of the active ingredient or to promote absorption or permeation of the active ingredient.

  The formulation procedure of the preparation of the present invention is known in the art, and is described in, for example, the Japanese Pharmacopeia, the US Pharmacopeia, the pharmacopoeia of other countries, and the like. Thus, one skilled in the art can determine the amount to be administered without undue experimentation as described herein.

(Preferred embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(Diagnostic system)
In one aspect, the present invention includes a marker substance in a sample derived from a subject, a factor that specifically interacts with the marker substance, or a means for selectively recognizing the marker substance. Provide a system for pre-diagnosis or diagnosis if there is.

  In certain specific aspects, the present invention provides a system or composition for pre-diagnosing whether a subject is diabetic, comprising a marker substance in a sample from the subject.

  In certain specific aspects, the present invention provides a system or composition for pre-diagnosing whether a subject is diabetic comprising an agent that specifically interacts with a marker substance in a sample from the subject. To do.

  In one specific aspect, the present invention provides a system for pre-diagnosing whether a subject is diabetic, comprising means for selectively recognizing a marker substance in a sample from the subject.

  In certain specific aspects, the present invention provides a system or composition for diagnosing whether a subject is diabetic, comprising a marker substance in a sample from the subject.

  In one specific aspect, the present invention provides a system or composition for diagnosing whether a subject is diabetic, comprising an agent that specifically interacts with a marker substance in a sample from the subject. .

  In one specific aspect, the present invention provides a system for diagnosing whether a subject is diabetic, comprising means for selectively recognizing a marker substance in a sample derived from the subject.

  As long as the marker substance can be identified, these compositions or systems selectively select the marker substance in a sample from any subject, a factor that specifically interacts with the marker substance, or the marker substance. It can be understood that means for recognizing can be used. Thus, it is understood that any equivalent factors or means known in the art can be used, not just the factors or means specifically described herein.

  In one embodiment, the marker substance used is characterized in that it is present in the body fluid of the subject, preferably blood. Although it is not desired to be bound by theory, if it is a body fluid, the post-treatment after removal is simple, and a large amount of diagnosis or diagnosis support is possible. Although not wishing to be bound by theory, blood is preferred because it significantly reflects the behavior of the marker substance of the present invention.

  In one embodiment, the marker substance used in the present invention is characterized by being a gene product. In particular, the gene product is preferably one that has not been previously known to be directly related to sugar metabolism. This is because it has not been known that a marker that is not directly related to glucose metabolism can be diagnosed or pre-diagnosed as a marker substance for diabetes. Because it becomes possible. In addition, the marker substance identified in the present invention has been shown to be a marker in a model animal, and can vary depending on a number of etiologies, such as a marker that has been empirically found in humans. In many cases, it is unclear whether or not it is caused only by the disease in the case of diabetes. However, the marker substance of the present invention does not have such an ambiguity. This is because the marker substance of the present invention has been found as a result of exhaustive analysis using a protein chip and has also been confirmed in model animals.

  In a specific embodiment, the marker substance used in the present invention is transthyretin, transthyretin derivative, apolipoprotein CII, apolipoprotein CII derivative, apolipoprotein CIII, apolipoprotein CIII derivative and serum albumin and corresponding to these One or more substances selected from the group consisting of proteins. Preferably, two or more, three or more or more marker substances (in particular, a plurality of marker substances selected from different groups when each paired with a derivative is regarded as one group) Is advantageously included. This is because a more accurate diagnosis can be performed as a multi-marker system, and a definitive diagnosis is also possible.

  In one embodiment, the factor used in the present invention is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof, preferably the factor is a protein or complex. A molecule (eg, glycoprotein, lipid protein, etc.). Preferably, the factor is an antibody (eg, a polyclonal antibody or a monoclonal antibody). Such factors are preferably labeled or labelable. This is because it is easy to diagnose.

  In this specification, the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others. Examples of such a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like. When both the nucleic acid fragment and the complementary oligonucleotide are labeled by the fluorescence method, the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. The difference in the maximum fluorescence emission wavelength is preferably 10 nm or more. As the fluorescent substance, any substance can be used as long as it can bind to the base portion of the nucleic acid. However, cyanine dyes (eg, CyDye ™ series Cy3, Cy5, etc.), rhodamine 6G reagent, N-acetoxy-N 2 -acetylamino Fluorene (AAF), AAIF (iodine derivative of AAF) or the like is preferably used. Examples of the fluorescent substance having a difference in fluorescence emission maximum wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, a combination of rhodamine 6G reagent and fluorescein, and the like. In the present invention, by using such a label, the target object can be modified so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can appropriately carry out such methods depending on the label and the target object.

  In a preferred embodiment of the present invention, the means used are mass spectrometer, nuclear magnetic resonance analyzer, X-ray analyzer, SPR, chromatography (eg, HPLC, thin layer chromatography, gas chromatography), immunology (E.g., Western blotting, ELISA, RIA), biochemical means (e.g., pI electrophoresis, Southern blotting, two-dimensional electrophoresis), electrophoresis instrument, chemical analysis instrument, fluorescence two-dimensional differential electrophoresis ( 2DE-DIGE), isotope labeling method (ICAT), tandem affinity purification method (TAP method), physical means, laser microdissection, and combinations thereof.

  In a preferred embodiment of the invention, the system of the invention further comprises a marker substance standard. Such a standard is used to confirm whether a marker substance detection means (such as a factor that specifically interacts with the marker substance or a means for selectively recognizing the marker substance) functions normally. It is preferable to use it.

  In a preferred embodiment, the present invention may further comprise means for purifying the sample of interest. Examples of such purification means include chromatography. Since purification can increase the accuracy of the diagnosis, it can be used in preferred embodiments, but this is not essential.

  In the present invention, the subject includes a mammal, and in one embodiment, the subject includes a rodent. Such rodents (for example, rats, mice, etc.) are preferable because model animals, particularly diabetic model animals, have been prepared. In preferred embodiments, the subject includes a human.

  In one embodiment, the factor or means used in the present invention has the ability to quantify the marker substance of the present invention. Such quantification may be a means or factor that can draw a calibration curve properly when a standard curve is drawn. Preferable examples include antibodies, mass spectrometry, and chromatographic analysis. Therefore, in one embodiment, the system of the present invention further comprises a quantification means for quantifying the marker substance.

  In one embodiment, the quantitative unit includes a determination unit that compares a standard curve with a measurement result to determine whether the marker substance is within a normal value range. Such determination means can be realized using a computer.

  In one embodiment, the system of the present invention is a composition comprising a marker substance or said agent that interacts specifically with a marker substance.

(Transthyretin related)
In one aspect, the marker substance of interest in the system of the present invention includes at least one substance selected from the group consisting of transthyretin and transthyretin derivative, and the transthyretin derivative comprises S-cysteine. Nyltransthyretin, glutathioneated transthyretin, SS bond forming transthyretin, oxidized (eg, oxidation of methionine side chain) transthyretin, formylated transthyretin, acetylated transthyretin, phosphorylated transthy Examples include retin, glycosylated transthyretin, and myristylated transthyretin. In particular, S-cysteinyl transthyretin is preferable. Further, the present invention can determine the degree of deterioration or risk of diabetes by examining the quantitative ratio between transthyretin and transthyretin derivatives (particularly metabolites appearing in the redox pathway). It should be said that the point which found is remarkable.

  Accordingly, in one embodiment, at least one phenomenon selected from the group consisting of a decrease in transthyretin and an increase in transthyretin derivative is an indication that diabetes is developing or that there is a high risk of developing in the future. It is.

  Preferably, at least one phenomenon selected from the group consisting of a decrease in transthyretin and an increase in a transthyretin derivative may be an indicator of the degree of developing diabetes or a high risk of developing in the future. It will be understood that such indicators can be determined by those skilled in the art based on the description herein.

  In a specific embodiment, the transthyretin of interest in the present invention is encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Or have these modified sequences.

  In another embodiment, the transthyretin derivative of interest in the present invention is the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Each of the cysteine at position 30 (position 10 in the mature form) or the corresponding cysteine is cysteinylated, or may have these modified sequences.

  In one embodiment, the factor or means used in the present invention has the ability to distinguish between transthyretin monomers and tetramers.

  In another embodiment, the factor or means used in the present invention has the ability to distinguish between transthyretin and S-cysteinyl transthyretin (eg, an antibody). For example, in the case of an antibody, a factor or means having such a capability is to prepare a library of antibodies, and from the library, either transthyretin or S-cysteinyl transthyretin is used. One can be made by selecting one that reacts specifically (preferably selectively) on one side, and such techniques can be achieved using techniques well known in the art. Similarly to antibodies other than antibodies, this can be achieved using techniques well known in the art.

  In a preferred embodiment, the factor or means in the present invention recognizes transthyretin and S-cysteinyl transthyretin, and the system of the present invention comprises transthyretin and S-cysteinyl transthyretin. Means for identifying. For example, transthyretins are identified by providing a combination of identification means such as antibody + electrophoresis and molecular weight, etc., but electrophoresis or mass spectrometry is used to distinguish a derivative from the others. It is understood that identification can be achieved. Such a technique can be achieved using techniques well known in the art.

  In a preferred embodiment, the factor or means in the present invention recognizes transthyretin and S-cysteinyl transthyretin, and the system of the present invention uses the molecular weight of transthyretin and S-cysteinyl transthyretin. It further comprises means for discriminating the molecular weight and means for measuring the relative ratio of transthyretin to S-cysteinyl transthyretin. By providing such a system, the present invention can determine the degree of deterioration of diabetes and the onset probability.

(Apolipoprotein CII)
In a preferred aspect, the marker substance used in the system of the present invention includes apolipoprotein CII or apolipoprotein CII derivative, and examples of the apolipoprotein CII derivative include proapolipoprotein CII.

  In one embodiment, at least one phenomenon selected from the group consisting of apolipoprotein CII decrease and apolipoprotein CII derivative variation is an indicator of developing diabetes or an increased risk of developing in the future obtain.

  In one embodiment, at least one phenomenon selected from the group consisting of apolipoprotein CII decrease and apolipoprotein CII derivative variation may be an indicator of the degree of developing diabetes or an increased risk of developing in the future. .

  In a specific embodiment, the apolipoprotein CII of interest in the present invention is encoded by the nucleic acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7, or the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 8, Alternatively, these modified sequences are included.

  In another embodiment, the proapolipoprotein CII that is the subject of the present invention may be one in which the lead sequence is bound in the above sequence.

  In one embodiment, the factor or means used in the present invention has the ability to selectively identify apolipoprotein CII (eg, an antibody). For example, in the case of an antibody, a factor or means having such a capability is to produce a library of antibodies, and specifically (preferably, selectively) apolipoprotein CII from the library. It can be made by selecting what reacts, and such techniques can be achieved using techniques well known in the art. Similarly to antibodies other than antibodies, this can be achieved using techniques well known in the art.

  In a preferred embodiment, the factor or means in the present invention has the ability to selectively identify apolipoprotein CII and the system comprises means for quantifying the apolipoprotein CII. By providing such a system, the present invention can determine the degree of deterioration of diabetes and the onset probability.

  In a particularly preferred aspect, the present invention comprises a marker substance in a sample derived from a subject, a factor that specifically interacts with the marker substance, or a means for selectively recognizing the marker substance, and the subject is diabetic A system for pre-diagnosing or diagnosing whether the means for recognizing has the ability to distinguish between transthyretin and a transthyretin derivative. Here, as an embodiment of the marker substance, the interacting factor, the means for selectively recognizing and the like, the above (diagnostic system) as long as it is ensured that it has the ability to distinguish between transthyretin and a transthyretin derivative. It is understood that any of the forms illustrated in FIG.

  In a preferred embodiment, the transthyretin used in the system of the invention is S-cysteinyl transthyretin.

  In a preferred embodiment, the selectively recognizing means used in the system of the invention is an antibody. This antibody may have any property as long as it can differentially recognize transthyretin and S-cysteinyl transthyretin. Preferably, it is an antibody that reacts with only one of transthyretin or S-cysteinyl transthyretin and does not substantially react with the other, more preferably it reacts only with the former. A set of both antibodies and antibodies that react only with the latter may be provided.

  In this specification, “substantially does not react” means that it is below the detection limit in a commonly used assay system (for example, ELISA or the like). Thus, such “substantially unreactive” conditions vary depending on the assay system used, but typically assume detection limits in an ELISA assay system using biotin-streptavidin. Those skilled in the art can set concentrations and limit values that do not prevent detection in an actual assay, which can be said to be substantially unresponsive.

(Apolipoprotein CIII)
In a preferred aspect, the marker substance used in the system of the present invention comprises apolipoprotein CIII or apolipoprotein CIII derivative, the apolipoprotein CIII is apolipoprotein CIII0, and the apolipoprotein CIII derivative comprises apolipoprotein CIII1 and apolipoprotein CIII1. Selected from the group consisting of CIII2. In addition, the present invention is remarkable in that it has been found that the degree of aggravation or risk of diabetes can be accurately determined by examining the quantitative ratio of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2. Should be.

  Thus, in one embodiment, at least one phenomenon selected from the group consisting of an increase in apolipoprotein CIII, an increase in apolipoprotein CIII1 and an increase in apolipoprotein CIII2 is developing diabetes or risk of developing in the future Is an indicator of high.

  Preferably, at least one phenomenon selected from the group consisting of an increase in apolipoprotein CIII, an increase in apolipoprotein CIII1 and an increase in apolipoprotein CIII2 is an indicator of the degree of developing diabetes or the risk of developing in the future. possible. It will be understood that such indicators can be determined by those skilled in the art based on the description herein.

  In a specific embodiment, the apolipoprotein CIII of interest in the present invention is encoded by the nucleic acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, or the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12. Or have these modified sequences.

  In another embodiment, the apolipoprotein CIII derivative of interest in the present invention is encoded by the nucleic acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, or in the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12. These are derivatives having sugar chains at positions 73 and 74 of SEQ ID NOs: 10 and 12, respectively, or threonine corresponding thereto.

  In one embodiment, the factor or means used in the present invention has the ability to distinguish between apolipoprotein CIII and apolipoprotein CIII derivatives.

  In another embodiment, the factor or means used in the present invention has the ability to distinguish at least two of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2. If the factor or means having such ability is, for example, an antibody, a library of antibodies is prepared, and at least two of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2 are further selected from the library. Can be made by selecting one that reacts specifically, preferably selectively, and such techniques can be accomplished using techniques well known in the art. Similarly to antibodies other than antibodies, this can be achieved using techniques well known in the art.

  In a preferred embodiment, the factor or means in the present invention has the ability to distinguish all of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2 (eg, antibody). For example, in the case of an antibody, a factor or means having such a capability is to create a library of antibodies, and further distinguish all of apolipoprotein CIII0, apolipoprotein CIII1, and apolipoprotein CIII2 from the library. Can be made by selecting those that react specifically (preferably, selectively), and such techniques can be achieved using techniques well known in the art. Similarly to antibodies other than antibodies, this can be achieved using techniques well known in the art.

  Accordingly, in one embodiment, the factor or said means comprises an antibody having the ability to distinguish between apolipoprotein CIII and apolipoprotein CIII derivative, preferably at least of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2. It includes antibodies that have the ability to distinguish between the two, and combinations of antibodies that have the ability to distinguish all of apolipoprotein CIII0, apolipoprotein CIII1, and apolipoprotein CIII2.

  In a preferred embodiment, the factor or means in the present invention recognizes apolipoprotein CIII and apolipoprotein CIII derivative, and the system further comprises means for discriminating between apolipoprotein CIII and apolipoprotein CIII derivative. For example, apolipoprotein CIIIs can be identified by providing a combination of identification means such as antibody + electrophoresis and molecular weight, etc., but use electrophoresis or mass spectrometry to distinguish derivatives from others It is understood that identification can be achieved. Such a technique can be achieved using techniques well known in the art.

  In a preferred embodiment, the factor or means in the present invention recognizes apolipoprotein CIII and apolipoprotein CIII derivative and the system of the present invention distinguishes at least two of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2. And a means for performing. For example, apolipoprotein CIIIs can be identified by providing a combination of identification means such as antibody + electrophoresis and molecular weight, etc., but use electrophoresis or mass spectrometry to distinguish derivatives from others It is understood that identification can be achieved. Such a technique can be achieved using techniques well known in the art.

  In a preferred embodiment, the factor or means in the present invention recognizes apolipoprotein CIII and apolipoprotein CIII derivative, and the system further comprises means for discriminating all of apolipoprotein CIII0, apolipoprotein CIII1 and apolipoprotein CIII2. . By providing such a system, the present invention can determine the degree of deterioration of diabetes and the onset probability.

(Serum albumin)
In another aspect, the marker substance used in the system of the present invention includes serum albumin or a serum albumin derivative, and the serum albumin derivative includes oxidized serum albumin, fractionated serum albumin and the like.

  Thus, in one embodiment, at least one phenomenon selected from the group consisting of a decrease in serum albumin and a variation in serum albumin derivatives is an indicator of developing diabetes or a high risk of developing in the future. . Albumin is measured as an index of various diseases, but it has not been known so far as an index of diabetes. In particular, it is worth noting that it has been found for the first time to increase the accuracy of diagnosis when combined with transthyretin, apolipoprotein CII, apolipoprotein III and the like.

  Preferably, at least one phenomenon selected from the group consisting of a decrease in serum albumin and a change in serum albumin derivative is an indicator of the degree of developing diabetes or a high risk of developing in the future. It will be understood that such indicators can be determined by those skilled in the art based on the description herein.

  In a specific embodiment, the subject serum albumin is encoded by the nucleic acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 15, or the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 16, or Have these modified sequences. Alternatively, it may have a serum albumin sequence as described herein or otherwise known.

  In one embodiment, the factor or means used in the present invention has the ability to selectively distinguish serum albumin (eg, an antibody). For example, in the case of an antibody, a factor or means having such a capability is to prepare a library of antibodies, and further react (preferably, selectively) with serum albumin from the library. Can be made by selecting what to do, and such techniques can be achieved using techniques well known in the art. Similarly to antibodies other than antibodies, this can be achieved using techniques well known in the art.

  In a preferred embodiment, the factor or means in the present invention has the ability to selectively discriminate serum albumin and the system comprises means for quantifying the serum albumin. For example, serum albumins are identified by providing a combination of identification means such as antibody + electrophoresis and molecular weight, etc., but electrophoresis or mass spectrometry is used to distinguish serum albumin itself from others It is understood that identification can be achieved. Such a technique can be achieved using techniques well known in the art.

  In one aspect, the system of the present invention can preferably be used as a diagnostic agent.

(Diagnosis method)
In one aspect, the invention provides a method for prediagnosis or diagnosis of whether a subject is diabetic or to support the prediagnosis or diagnosis, comprising: A) in a sample from the subject Providing a method comprising: measuring a marker substance; and B) determining from the measurement results whether the subject is diabetic or likely. Here, any means may be used for obtaining the sample. Usually, when a person in charge other than the doctor is engaged in the measurement, it may have been acquired by the doctor in some form. The step of determining whether or not there is a possibility of diabetes from the measurement result can be carried out by determining whether or not it is abnormal compared to each normal substance compared to the normal value.

  In the method of the present invention, the marker substance or the like to be used is any one of those described in the above sections (System), (Transthyretin), (Apolipoprotein CIII), (Apolipoprotein CII) and (Serum albumin). It is understood that a plurality of features may be provided as long as they do not contradict each other.

(use)
In one aspect, the present invention relates to a marker substance in a sample derived from a subject, a factor that specifically interacts with the marker substance, or a means for selectively recognizing the marker substance, wherein the subject is diabetic. Whether in the manufacture of a medicament for pre-diagnosis or diagnosis is provided. Here, any means may be used for obtaining the sample. Usually, when a person in charge other than the doctor is engaged in the measurement, it may have been acquired by the doctor in some form. The step of determining whether or not there is a possibility of diabetes from the measurement result can be carried out by determining whether or not it is abnormal compared to each normal substance compared to the normal value.

  In the method of the present invention, the marker substance or the like to be used is any one of those described in the above sections (System), (Transthyretin), (Apolipoprotein CIII), (Apolipoprotein CII) and (Serum albumin). It is understood that a plurality of features may be provided as long as they do not contradict each other.

  In another aspect, the present invention relates to a marker substance in a sample derived from a subject, a factor that specifically interacts with the marker substance, or a means for selectively recognizing the marker substance, wherein the subject is diabetic. Provide pre-diagnosis or use for diagnosis. Here, any means may be used for obtaining the sample. Usually, when a person in charge other than the doctor is engaged in the measurement, it may have been acquired by the doctor in some form. The step of determining whether or not there is a possibility of diabetes from the measurement result can be carried out by determining whether or not it is abnormal compared to each normal substance compared to the normal value.

  In the method of the present invention, the marker substance or the like to be used is any one of those described in the above sections (System), (Transthyretin), (Apolipoprotein CIII), (Apolipoprotein CII) and (Serum albumin). It is understood that a plurality of features may be provided as long as they do not contradict each other.

(Further explanation)
In the method for diagnosing diabetes according to the present invention, as a method for measuring the concentration of a marker substance, a method generally used for protein quantification may be used as it is as long as the method can specifically measure the concentration of the marker substance. it can. For example, various immunoassays, mass spectrometry (MS), chromatography, electrophoresis and the like can be used.

  According to the immunoassay, the concentration of the marker substance can be accurately measured even with a sample having a lot of contaminants. Examples of immunoassays include classical methods such as precipitation, agglutination, and hemolysis, which directly or indirectly measure antigen-antibody conjugates, and enzyme immunoassays (EIA) with enhanced detection sensitivity in combination with labeling methods. , Radioimmunoassay (RIA), fluorescent immunoassay (FIA) and the like. The antibody specific for the marker substance used in these immunoassays may be monoclonal or polyclonal.

  As the ionization method for measuring the concentration of the marker substance by mass spectrometry, either matrix-assisted laser desorption / ionization (MALDI) or electrospray ionization (ESI) can be applied. However, MALDI is preferred because it produces less multivalent ions. In particular, according to MALDI-TOF-MS combined with a time-of-flight mass spectrometer (TOF), the concentration of the marker substance can be measured more accurately. Furthermore, according to MS / MS using two mass spectrometers, the concentration of the marker substance can be measured more accurately.

  When measuring the concentration of the marker substance by electrophoresis, for example, the test material is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the target marker substance, and the gel is prepared with an appropriate dye or fluorescent substance. It is only necessary to stain and measure the intensity and fluorescence intensity of the band corresponding to the target marker substance. When the separation of the marker substance is insufficient by SDS-PAGE alone, two-dimensional electrophoresis combined with isoelectric focusing (IEF) can also be used. Furthermore, instead of detecting directly from the gel, Western blotting can also be performed to measure the amount of marker substance on the membrane.

  When measuring the concentration of the marker substance by chromatography, for example, a method by liquid high performance chromatography (HPLC) can be used. That is, the concentration of the marker substance in the sample can be measured by subjecting the sample to HPLC to separate the target marker substance and measuring the peak area of the chromatogram.

  The diagnostic method of diabetes of the present invention comprises (a) a protein having a molecular weight of about 13800, supplemented by a cation exchanger at a pH of 7.0 or less, and (b) metal ion immobilization at a pH of 7.0 or less. A protein with a molecular weight of about 8700, supplemented by a carrier, (c) a protein with a molecular weight of about 9400, trapped by a cation exchanger at a pH of 7.0 or less, or metal ion immobilization at pH 7.0 A protein having a molecular weight of about 9400, supplemented by a fluorinated carrier, (d) a protein having a molecular weight of about 9700, supplemented by a cation exchanger at a pH of 7.0 or less, or a metal ion at pH 7.0 A protein with a molecular weight of about 9700 supplemented by an immobilization carrier, (e) a protein with a molecular weight of about 66000 supplemented by a cation exchanger at a pH of 7.0 or less, or H7.0 in supplemented to the metal ion-immobilized carrier, the protein molecular weight of about 66,000, and a marker substance, also includes a method of comparing the amount of the blood and healthy value. The physical properties of (a), (b), (c), (d), and (e) are the same as those of transthyretin subunit, apolipoprotein CIII0, apolipoprotein CIII1, apolipoprotein CIII2, and serum albumin, respectively. To do.

  The method for diagnosing diabetes of the present invention comprises: (a) digesting with trypsin yields polypeptides having molecular weights of about 1270, about 1370, about 1390, about 1520, about 2450, about 2640, and about 3140; Protein and / or (b) digestion with trypsin yields a polypeptide having a molecular weight of about 900, about 1200, about 1390, about 1710, about 1940, and about 2080 with a molecular weight of about 8700-9700 And a method of comparing the amount in the blood with a healthy value. When peptide mass fingerprinting is performed using the ProFound database, (a) is identified as transthyretin. When peptide mass fingerprinting is performed using the MS-Fit database, (b) is identified as apolipoprotein CIII.

  One of the preferred embodiments of the method for diagnosing diabetes of the present invention is to capture a marker substance on a carrier and measure the concentration of the captured marker substance. That is, a substance having affinity for the marker substance is immobilized on the carrier, and the marker substance is captured on the carrier via the substance having the affinity. According to this embodiment, it is possible to reduce the influence of contaminants contained in the sample, and to measure the concentration of the marker substance with higher sensitivity and higher accuracy. Examples of “affinity” include chemical actions such as ion binding and hydrophobic interaction, as well as bioaffinity such as antigen and antibody, enzyme and substrate, and hormone and receptor.

  In this embodiment, when an immunoassay is used as a method for measuring a marker substance, it is preferable to use a carrier on which an antibody is immobilized. In this way, an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed. For example, two types of antibodies specific to a marker substance and having different epitopes are prepared, one is immobilized on a carrier as a primary antibody, and the other is enzyme-labeled as a secondary antibody to construct a sandwich EIA system. . In addition, immunoassay systems based on binding inhibition methods and competitive methods can be constructed. Further, when a substrate is used as a carrier, immunoassay using an antibody chip is possible. According to the antibody chip, the concentration of a plurality of marker substances can be measured simultaneously, and rapid measurement is possible.

  On the other hand, when mass spectrometry is used as a method for measuring a marker substance in the present embodiment, the marker substance can be captured on a carrier by ionic bonds or hydrophobic interactions in addition to antibodies. Ion binding and hydrophobic interactions are not as specific as bioaffinity, such as antigens and antibodies, and substances other than marker substances are captured, but mass spectrometry quantifies them using mass spectrometer spectra that reflect molecular weight. ,No problem. In particular, using a protein chip using a substrate as a carrier, surface-enhanced laser desorption / ionization-time-of-flight mass spectrometry (hereinafter referred to as "SELDI-TOF"). (Referred to as “−MS”), the concentration of the marker substance can be measured more accurately. As the types of substrates that can be used, cation exchange substrates, anion exchange substrates, normal phase substrates, reverse phase substrates, metal ion substrates, antibody substrates, etc. can be used, but cation exchange substrates, particularly weak cation exchanges. A substrate and a metal ion substrate are preferably used.

  When capturing the marker substance on the carrier by ionic bonding, the ion exchanger is immobilized on the carrier. In this case, both an anion exchanger and a cation exchanger can be used as the ion exchanger, and further, a strong anion exchanger, a weak anion exchanger, a strong cation exchanger, and a weak cation exchanger. Any of these can be used. For example, examples of weak anion exchangers include those having weak anion exchange groups such as dimethylaminoethyl (DE) and diethylaminoethyl (DEAE). Examples of strong anion exchangers include quaternary ammonium (trimethylaminomethyl) (QA), quaternary aminoethyl (diethyl, mono-2-hydroxybutylaminoethyl) (QAE), and quaternary ammonium (trimethylammonium. And those having a strong anion exchange group such as (QMA). Examples of weak cation exchangers include those having weak cation exchange groups such as carboxymethyl (CM). Further, examples of the strong cation exchanger include those having a strong cation exchange group such as sulfopropyl (SP). On the other hand, when a marker substance is captured on a carrier by hydrophobic interaction, a substance having a hydrophobic group is immobilized on the carrier. Examples of hydrophobic groups include C4-C20 alkyl groups, phenyl groups, and the like. Furthermore, the marker substance can be captured on a carrier on which metal ions such as Cu 2+, Zn 2+, Ni 2+, Ca 2+, Co 2+ and Mg 2+ are immobilized.

As examples of the carrier used in the present embodiment, known ones such as beads, microtiter plates, and resins can be used. In particular, beads and microtiter plates are conventionally used in immunoassays, and the measurement system can be easily constructed. on the other hand,
A carrier having a planar portion such as a substrate can also be used. In this case, it is preferable to immobilize a substance having affinity for the marker substance on a part of the planar portion. An example is a carrier in which a chip is used as a substrate and an antibody specific to a marker substance is immobilized in spots on a plurality of locations on the surface.

  In the method for diagnosing diabetes of the present invention, it is preferable to use blood collected from a subject as a specimen and serum or plasma prepared from the blood as a test material. Serum or plasma can be prepared from blood by a known method such as centrifugation.

  One preferred embodiment of the method for diagnosing diabetes according to the present invention includes diabetes diagnosis using a multi-marker system. An example in which the method for diagnosing diabetes of the present invention is applied to a multimarker system will be described with reference to FIGS. FIG. 1 is a flowchart showing the procedure of the method for diagnosing diabetes of the present invention using a multi-marker system. According to the method of the flowchart of FIG. 1, firstly, a primary determination is made using the concentration of transthyretin in blood as an index. When the concentration of transthyretin is higher than the normal value, it is determined as diabetes. On the other hand, when the concentration of transthyretin is below a healthy value, secondary determination is performed using the concentration of apolipoprotein CIII2 in blood as an index. And when the density | concentration of apolipoprotein CIII2 is higher than a healthy value, it determines with diabetes. On the other hand, when the concentration of apolipoprotein CIII2 is below a healthy value, a third determination is made using the concentration of apolipoprotein CIII1 in blood as an index. And when the density | concentration of apolipoprotein CIII1 is below a healthy value, it determines with it being normal (it is not diabetes). On the other hand, when the concentration of apolipoprotein CIII1 is higher than the healthy value, the fourth determination is made using the concentration of apolipoprotein CIII0 in blood as an index. And when the density | concentration of apolipoprotein CIII0 is higher than a healthy value, it determines with diabetes. On the other hand, when the concentration of apolipoprotein CIII0 is below a healthy value, it is determined as normal (not diabetic).

  FIG. 2 is a flowchart showing the procedure of the method for diagnosing diabetes of the present invention using another multimarker system. According to the method of the flowchart of FIG. 2, firstly, a primary determination is made using the serum albumin concentration in the blood as an index. And when the density | concentration of serum albumin is more than a healthy value, it determines with normal (it is not diabetes). On the other hand, when the concentration of serum albumin is lower than the normal value, secondary determination is performed using the concentration of apolipoprotein CIII2 in blood as an index. And when the density | concentration of apolipoprotein CIII2 is higher than a healthy value, it determines with diabetes. On the other hand, when the concentration of apolipoprotein CIII2 is below a healthy value, a third determination is made using the concentration of apolipoprotein CIII1 in blood as an index. And when the density | concentration of apolipoprotein CIII1 is below a healthy value, it determines with it being normal (it is not diabetes). On the other hand, when the concentration of apolipoprotein CIII1 is higher than the healthy value, the fourth determination is made using the concentration of apolipoprotein CIII0 in blood as an index. And when the density | concentration of apolipoprotein CIII0 is higher than a healthy value, it determines with diabetes. On the other hand, when the concentration of apolipoprotein CIII0 is below a healthy value, it is determined as normal (not diabetic).

  It is possible to construct a multi-marker system by combining transthyretin, apolipoprotein CIII, and serum albumin used as diabetes markers in the present invention with clinical markers such as HbA1c and CPR that are conventionally known. It is.

  According to the method for diagnosing diabetes using the multi-marker system as described above, since it can be determined from many aspects, it is possible to diagnose diabetes with considerably high accuracy. Furthermore, it is also suitable for detection of a stage before the onset of diabetes, that is, diagnosis of a pre-diabetes, which is difficult with a conventional method for diagnosing diabetes. Further, according to the multi-marker system, not only the detection of diabetes but also the detection of diabetes reserves and the monitoring of the improvement state of diabetes can be performed with high accuracy.

  The diabetes diagnosis kit of the present invention comprises an antibody specific for a marker substance such as transthyretin. The antibody contained in the kit may be a single reagent or may be immobilized in advance on a carrier. In the case of a single reagent, the shape may be a solution or a lyophilized product. In addition, a plurality of antibodies may be included. For example, a labeled antibody used in an immunoassay may be included as a secondary antibody. The diabetes diagnosis kit of the present invention may contain other reagents. For example, if the kit is for EIA, a carrier such as beads, a blocking solution, a buffer solution such as PBS, a chromogenic substrate, etc. It may be included.

The method for diagnosing a disease according to the present invention compares at least one concentration of the following marker substances (a) to (n) in a body fluid of a subject with a healthy value, and determines the presence or absence of diabetes or the risk of future onset. It is.
(A) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 7040 when subjected to mass spectrometry;
(B) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8330 when subjected to mass spectrometry;
(C) A protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8530 when subjected to mass spectrometry.
(D) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 9060 when subjected to mass spectrometry;
(E) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 9260 when subjected to mass spectrometry;
(F) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 9450 when subjected to mass spectrometry;
(G) a protein that binds to a copper ion-binding metal chelate at pH 7.0 and a NaCl concentration of 0.5 M, and that when subjected to mass spectrometry, produces an ion peak with a mass / charge ratio of about 13700,
(H) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 76400 when subjected to mass spectrometry;
(I) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 79100 when subjected to mass spectrometry;
(J) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 3500 when subjected to mass spectrometry;
(K) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 3560 when subjected to mass spectrometry;
(L) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 4180 when subjected to mass spectrometry;
(M) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 12800 when subjected to mass spectrometry;
(N) A protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 65700 when subjected to mass spectrometry.

  All of these marker substances are mainly proteins present in blood. When the subject has developed diabetes or when the risk of developing diabetes is high, the marker substances (a), (b), (c), (d), (e), ( The concentrations of f), (g), (h) and (i) are higher, and the concentrations of the marker substances (j), (k), (l), (m) and (n) are lower. Show. Hereinafter, the group consisting of the marker substances (a), (b), (c), (d), (e), (f), (g), (h) and (i) is referred to as “Group 1”, the marker substance A group composed of (j), (k), (l), (m) and (n) may be referred to as “group 2”.

  The healthy value used in the disease diagnosis method of the present invention is, for example, collecting concentration data in the body fluid of the marker substances (a) to (n) in a healthy person who is definitely diagnosed as not developing diabetes, It can be set based on the density value. When determining the future risk of developing diabetes, a healthy value can be set based on the concentration value in the healthy person. It is also possible to set a plurality of healthy values in stages and quantitatively determine the presence or absence of diabetes or the risk of future onset.

  Blood is preferably used as the body fluid used in the disease diagnosis method of the present invention. In particular, it is preferable to use serum or plasma (body fluid component) prepared from blood collected from a subject as a measurement sample. Serum or plasma can be prepared from blood by a known method such as centrifugation.

(Evaluation method)
In one aspect, the present invention provides a method for evaluating a substance, in which the test substance is ingested by an animal that has developed diabetes or an animal that has a high risk of developing in the future, and is a marker substance in the body fluid of the animal. At least one concentration of (for example, 14 types of (a) to (n)) is compared with a reference value to evaluate the effect of improving diabetes or reducing the future risk of developing the test substance. In the method for evaluating a substance of the present invention, blood glucose is not directly used as an index, but another marker substance is used as an index, so that it is possible to capture a state before an increase in blood glucose level in an animal. As a result, in addition to the diabetes improving effect of the test substance, the effect of reducing the future risk of developing diabetes can be evaluated. In addition, “animal” includes humans in addition to animals such as rats.

  With this configuration, it is possible to more accurately evaluate the diabetes improvement effect or the future risk reduction effect of the test substance.

  With this configuration, it is possible to easily collect a body fluid as a measurement sample, and it is possible to more easily and quickly evaluate a diabetic improvement effect or a future risk reduction effect of a test substance.

  With this configuration, for the purpose of developing functional foods, it is possible to evaluate the effect of improving diabetes or reducing the risk of developing the future.

  Similar to the disease diagnosis method of the present invention described above, also in the substance evaluation method of the present invention, the body fluid or body fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, and the body fluid The marker substance is captured on a carrier, and the concentration of the marker substance in a body fluid is calculated based on the amount of the captured marker substance (Claim 10), the carrier has a planar portion, It is recommended that the substance having affinity for the marker substance is immobilized on a part of the planar portion, and the substance having affinity for the marker substance is an ion exchanger, metal chelate or antibody. Is done.

  In the method for evaluating a substance of the present invention, an animal that develops diabetes or an animal that has a high risk of developing in the future is allowed to ingest a test substance, and the concentration of at least one of the marker substances (a) to (n) in the animal is determined. Compared to the reference value, the test substance evaluates the effect of improving diabetes or reducing the risk of future onset. When the test substance has an effect of improving diabetes or reducing the risk of developing the future, the concentration of the marker substance belonging to group 1 in the body fluid shows a lower value, and the concentration of the marker substance belonging to group 2 is more Indicates a high price.

  In a preferred embodiment, when the animal having diabetes or having a high future risk is ingested with a known substance having no effect of improving diabetes or reducing the risk of future development as the reference value, The concentration of the marker substance in the body fluid of the animal is used. That is, when an animal that has developed diabetes or an animal that has a high risk of developing in the future is ingested with a known substance that has no effect of improving diabetes or of reducing the risk of developing future disease, the marker substance in the body fluid The concentration of is an “abnormal value”. Then, the value (measured value) in the animal ingested with the test substance is compared with the reference value (abnormal value), and the measured value is significantly different from the reference value and is on the normal side (normal side) In other words, it can be evaluated that the test substance has an effect of improving diabetes or reducing the risk of developing the disease in the future. Specifically, when the marker substance belonging to group 1 is used as an index, when the measured value is significantly lower than the reference value, when the marker substance belonging to group 2 is used as an index, the measured value is the reference. When the value is significantly higher than the value, it can be evaluated that the test substance has an effect of improving diabetes or an effect of reducing the risk of developing the future.

  Furthermore, there may be a plurality of reference values. For example, in addition to the above abnormal values, values (normal values, negative controls) in animals that have not developed diabetes or that have a low risk of developing diabetes can be added to the reference values. Specifically, (1) a group in which an animal that has not developed diabetes or a low risk of developing diabetes is ingested with a normal diet or a test substance (a group that exhibits normal values), and (2) that has developed diabetes. A group (a group showing an abnormal value) of ingesting a known substance that does not have an effect of improving diabetes or a risk of reducing future development risk to an animal or a high risk of developing diabetes, and (3) Diabetes mellitus Three groups are set, which are a group in which a test substance is ingested by an onset animal or an animal at high risk of developing diabetes, and the animals are bred. And the said marker substance in the bodily fluid of each animal is measured, and each measured value is compared. At this time, there is a significant difference between (1) and (2), there is a significant difference between (3) and (2), and (3) is closer to the normal side ((1) than (2)) If the test substance is maintained on the normal side, it can be evaluated that the test substance has an effect of improving diabetes or reducing the risk of developing the future. That is, if the test substance has an effect of improving diabetes or reducing the risk of developing the future, the blood glucose level is maintained at a normal value in (3), and the marker substance concentration is close to the normal value (1) Take.

  Furthermore, as a reference value, (4) in an animal of a group in which an animal that develops diabetes or an animal that has a high risk of developing in the future receives a known substance having an effect of improving diabetes or reducing the risk of developing future disease A value (positive control) can also be added. Specifically, in addition to (1) to (3) above, the group (4) above is set and animals are raised. At this time, there is a significant difference between (1) and (2), there is a significant difference between (3) and (2), and (3) is normal ((1) and (2) compared to (2). 4), it can be evaluated that the test substance has an effect of improving diabetes or an effect of reducing the risk of future onset. That is, such a test substance exhibits the same behavior as the above-mentioned known substance adopted in (4) and can be said to be a substance having the same action.

  The “animal that develops diabetes or an animal that has a high risk of developing diabetes” described above can be realized, for example, by using an animal that necessarily genetically develops diabetes. More specifically, for example, OLETF (Otsuka Long-Evans Tokushima Fatty) rats supplied from Tokushima Laboratory, Otsuka Pharmaceutical Co., Ltd. can be used. The OLETF rat is a model rat that spontaneously develops type 2 diabetes with obesity. In males, almost all cases are diagnosed as diabetes by OGTT at 25 weeks of age. Similarly, “an animal that does not develop diabetes or an animal that has a low risk of developing diabetes” can be realized, for example, by using a model animal that does not genetically develop diabetes at all. More specifically, for example, a LETO (Long-Evans Tokushima Otsuka) rat supplied from Tokushima Laboratory, Otsuka Pharmaceutical Co., Ltd. can be used. The LETO rat is a control rat that does not develop diabetes at all, and is genetically related to the OLETF rat.

  The animal used in the method for evaluating a substance of the present invention is not particularly limited, and for example, a mouse, rat, rabbit, pig or the like can be used. In particular, rats and mice are easily used for the evaluation method of the present invention because they are easy to breed. There is no limitation in particular as an animal breeding method, For example, it is sufficient to feed freely for about 3 to 20 days. Furthermore, humans can also be used as animals. When humans are used, substances are evaluated based on the results of clinical trials.

  As an animal body fluid used in the method for evaluating a substance of this aspect, blood is preferably used. In particular, serum or plasma (body fluid component) prepared from blood is preferably used as a measurement sample. Serum or plasma can be prepared from blood by a known method such as centrifugation.

  Examples of the test substance in the substance evaluation method of the present invention include food materials and drug substances. In particular, when food materials are to be evaluated, it can be used for the development of functional foods.

  In order to easily perform the method for evaluating a substance of the present invention, an evaluation kit can be constructed by collecting necessary reagents. Examples of the evaluation kit include those containing a carrier on which a substance having affinity for a marker substance is immobilized. In particular, according to the evaluation kit including a carrier on which a weak cation exchanger such as CM, a metal chelate such as copper ion, or a substrate on which an antibody against a marker substance is immobilized, SELDI-TOF-MS or antibody chip Can be easily performed. The kit may contain other reagents such as standard substances and various pretreatment buffers.

  The substance screening method of the present invention evaluates a test substance by the substance evaluation method of the present invention, and screens for a substance having an effect of improving diabetes or reducing the risk of developing the future. In the method for screening a substance of the present invention, the same embodiment as the above-described embodiment of the method for evaluating a substance of the present invention can be employed. Furthermore, a screening kit having the same configuration as the above-described evaluation kit can be constructed.

  The present invention is also a substance screening method characterized in that a test substance is evaluated by the substance evaluation method described in the present invention, and a substance having an effect of improving diabetes or a risk of reducing future risk of development is screened. is there.

  The present invention relates to a method for screening a substance, and compares at least one concentration of a marker substance (for example, 14 species (a) to (n)) in a body fluid of an animal with a reference value, thereby improving the effect of improving diabetes or A substance having an effect of reducing the onset risk is screened. In the method for screening a substance of the present invention, blood glucose is not directly used as an index, but another marker substance is used as an index, so that it is possible to capture a state before an increase in blood glucose level in an animal. As a result, in addition to a substance having an effect of improving diabetes, a substance having an effect of reducing the risk of developing future diabetes can be screened. In particular, when the test substance is a food material, it is possible to screen for a food material useful for developing a functional food having an effect of improving diabetes or a functional food having an effect of reducing the risk of developing the future.

  The present invention also provides a substance obtained by such a screening method.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
[Example 1]

1. Search for Diabetes Marker Candidate Proteins Using Protein Chip Serum samples for 20 diabetic patients and 10 healthy individuals were collected. Each serum sample was pretreated by adding a denaturation buffer (9 M urea, 2% CHAPS, 50 mM Tris-HCl (pH 9.0)) to remove some contaminating proteins. Next, each pretreated serum sample was adsorbed on a strong anion exchange resin, and then eluted in order with eluents having different pHs. Fraction 1 (through), fraction 2 (eluted at pH 7.0), fraction Six fractions were obtained: fraction 3 (eluted at pH 5.0), fraction 4 (eluted at pH 4.0), fraction 5 (eluted at pH 3.0), and fraction 6 (eluted with an organic solvent). Each fraction was subjected to SELDI-TOF-MS using a protein chip to comprehensively search for proteins specific to the serum of diabetic patients. Four types of protein chips (metal ions (Cu2 +), weak cation exchanger (CM), strong anion exchanger (Q), and reverse phase (C16 alkyl chain)) (all manufactured by Cyphergen) were examined. Two types of energy-absorbing substances (EAM), α-cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid (SPA) were examined. As a result, a large number of peaks were detected depending on combinations of protein chip type, fraction type, chip washing condition (pH), EAM type and the like. From these peaks, peaks having significantly different intensities between diabetic patients and healthy subjects were searched.
2. Search for candidate peaks (1)
Using a protein chip on which a weak cation exchanger is immobilized and the washing pH is 7.0 for fraction 4, the mass / charge ratio (m / z) is 13867, 14049, 13885, 14087, and 13661. Five peaks were detected. Further, when a protein chip on which a weak cation exchanger was immobilized was used and the washing pH was set to 4.0 for fraction 5, a peak having an m / z of 13858 was detected. From these six peaks, it was suggested that a candidate protein (hereinafter referred to as “candidate protein (1)”) exists in the vicinity of a molecular weight range of 13800 to 14100.

  For each peak, the peak intensity was plotted separately for diabetic patients and healthy individuals, an ROC curve was created, and a cut-off value was set. As an example, the example about the peak whose m / z is 13867 is shown to Fig.3 (a)-(c). FIG. 3A is a graph in which the peak intensity is plotted separately for diabetic patients and healthy individuals, and the horizontal line is the cutoff value. FIG. 3B is a graph showing the result of FIG. 3A as a maximum value, a minimum value, a median value, and a cutoff value. FIG. 3C is an ROC curve, and shows that the closer the ROC area is to 1 (the closer the curve is to the upper left), the higher the accuracy of the measurement system. Similar graphs (not shown) were prepared for the other five peaks. Table 2 below summarizes the P value, ROC area, chip used, chip cleaning conditions, fractions, and used EAM for each peak.

3. Purification of Candidate Protein (1) Candidate protein (1) was purified from the serum of diabetic patients by the following procedure. First, 75 μL of a denaturing buffer (9 M urea, 2% CHAPS, 50 mM Tris-HCl (pH 9.0)) was added to 50 μL of the serum of a diabetic patient and treated at 4 ° C. for 20 minutes. Further, after adding and diluting 1.5 mL of washing / binding buffer (50 mM phosphate buffer (pH 7.0) containing 100 mM NaCl), Q Ceramic HyperD F Spin Column ( BioSepla). The mixture was stirred at 4 ° C. for 30 minutes, and then washed twice with 500 μL of washing / binding buffer. Next, fractionation was performed by sequentially eluting with 100 μL of 5 types of 50 mM phosphate buffer (pH 7.0) containing 125 mM, 150 mM, 175 mM, 200 mM, or 250 mM NaCl. When each fraction was analyzed by SELDI-TOF-MS, a peak having a molecular weight of about 13800 to 14100 similar to that of the candidate protein (1) was found in the fraction eluted with a buffer containing 175 mM, 200 mM, and 250 mM NaCl. was detected. All albumin was eluted in the washing step before elution, and the candidate protein (1) and albumin were completely separated under these conditions.
4). Identification of candidate protein (1) Fractions with NaCl concentrations of 175 mM, 200 mM, and 250 mM were mixed (90 μL × 3). The mixed fractions were concentrated by centrifugation with VivaSpin 5000 Cut Off (Sartorius), and further 10-fold volume of 50 mM phosphate buffer (pH 7.0) was added to concentrate to a final volume of 50 μL, and the buffer was exchanged. Next, the concentrated sample was subjected to SDS-PAGE having a gel concentration of 16%, and the gel was stained with Coomassie Brilliant Blue (CBB). As a result, a target band was detected at a molecular weight of about 13800. Next, 0.02 μg / μL of a trypsin solution (dissolved in 25 mM ammonium bicarbonate (pH 8.0)) was allowed to act on the target band, and digested in the gel. When the MALDI-MS / MS analysis was performed on the digested sample, at least 7 peaks were detected, and their molecular weights were “126.960”, “136.300”, “1394.270”, “1522.140”. ”,“ 245.790 ”,“ 2646.030 ”, and“ 3141.310 ”. Based on these data, a known protein was searched using the ProFound database, and peptide mass fingerprinting was performed. As a result, the target protein was identified as a subunit of transthyretin with a probability of 99% or more. The conventionally known transthyretin subunit has a molecular weight of 13890 and an isoelectric point of 5.3, whereas the isolated protein has a molecular weight of about 13800 and an expected isoelectric point of 5.3. The physicochemical properties of both were almost the same.

It is known that transthyretin is a tetramer consisting of four identical subunits, but according to the results of this example, transthyretin is not in the form of a tetramer but a subunit alone. Detected in
[Example 2]

1. Search for candidate peaks (2)
Similar to Example 1, another candidate peak was searched. As a result, when a protein chip on which a weak cation exchanger was immobilized was used and the washing pH was set to 4.0 for fraction 6, two peaks with m / z of 9279, 9705 were detected. In addition, when a protein chip with a weak cation exchanger immobilized thereon was used and the washing pH was set to 4.0 for fraction 5, two peaks having m / z of 9285 and 9415 were detected. In addition, when a protein chip on which a weak cation exchanger was immobilized was used and the washing pH was set to 7.0 for fraction 6, three peaks at m / z of 9289, 9638, and 9712 were detected. In addition, when a protein chip on which metal ions (Cu2 +) are immobilized is used and the washing pH is set to 7.0 for fraction 6, four peaks at m / z of 8690, 9289, 9638, and 9712 are detected. It was. From these 11 peaks, it was suggested that a candidate protein (hereinafter referred to as “candidate protein (2)”) exists in the vicinity of a molecular weight range of 8600 to 9800.

  In the same manner as in Example 1, for each peak, the peak intensity was plotted separately for diabetic patients and healthy individuals, an ROC curve was created, and a cut-off value was set. As an example, FIGS. 4A to 4C show an example of a peak having an m / z of 8690. FIG. FIG. 4A is a graph in which peak intensities are plotted separately for diabetic patients and healthy individuals, and FIG. 4B shows the results of FIG. 4A with the maximum value, minimum value, median value, and cutoff. FIG. 4C is a ROC curve. Similar graphs (not shown) were prepared for the other 10 peaks. Table 3 below summarizes the P value, ROC area, chip used, chip cleaning conditions, fractions, and used EAM for each peak.

2. Purification of candidate protein (2) Candidate protein (2) was purified from the serum of diabetic patients by the following procedure. First, 75 μL of a denaturation buffer was added to 50 μL of serum of a diabetic patient and treated at 4 ° C. for 20 minutes. Furthermore, 1.5 mL of washing / binding buffer (50 mM phosphate buffer (pH 6.0)) was added and diluted, and then applied to Q Ceramic HyperDF Spin Column equilibrated with the washing / binding buffer. The mixture was stirred at 4 ° C. for 30 minutes, and then washed twice with 500 μL of washing / binding buffer. Next, fractionation was performed by sequentially eluting with 100 μL of three types of 50 mM phosphate buffer (pH 6.0) containing 50 mM, 150 mM, or 250 mM NaCl. When each fraction was analyzed by SELDI-TOF-MS, peaks with molecular weights of about 8690, 9415, and 9712 similar to those of the candidate protein (2) were detected. All albumin was eluted in the washing step before elution, and the candidate protein (2) and albumin were completely separated under these conditions.
3. Identification of Candidate Protein (2) 0.02 μg / μL trypsin solution (dissolved in 25 mM ammonium bicarbonate (pH 8.0)) was allowed to act on the target band purified by SDS-PAGE and digested in gel. When MALDI-MS / MS analysis was performed on the digested sample, at least 5 peaks were detected, and their molecular weights were calculated as “898”, “1197”, “1717”, “1939”, “2076”. It was. Based on these data, a known protein was searched using the MS-Fit database, and peptide mass fingerprinting was performed. Was identified. The molecular weights of apolipoprotein CIII0, apolipoprotein CIII1, and apolipoprotein CIII2 that are conventionally known are 8765, 9421, and 9713, respectively, and the isoelectric points are 4.95, 4.80, and 4.65, respectively. The three separated proteins had molecular weights of 8690, 9415, and 9712, and the expected isoelectric points were all 4.5 to 5.0, and the physicochemical properties of the two proteins were almost the same.
4. Analysis of diabetic patient serum by two-dimensional electrophoresis The serum of diabetic patients was subjected to two-dimensional electrophoresis of IEF in the first dimension (horizontal direction) and SDS-PAGE in the second dimension (vertical direction). Three bands A, B and C were detected in the vicinity of 6.5 to 14.4 kDa and isoelectric point (pI) 5 (FIG. 5). These bands were not detected in the serum of healthy subjects. When these bands A, B, and C were analyzed by SELDI-TOF-MS, the molecular weights of A, B, and C coincided with the molecular weights of apolipoprotein CIII2, apolipoprotein CIII1, and apolipoprotein CIII0, respectively. This result coincided with the search result of the candidate protein (2) using the protein chip.
[Example 3]

Search for candidate peaks (3)
In the same manner as in Example 1, another candidate peak was searched. As a result, when a protein chip on which a weak cation exchanger was immobilized was used and the washing pH was set to 7.0 for fraction 3, two peaks with m / z of 66418 and 66449 were detected. Further, when a protein chip on which a weak cation exchanger was immobilized was used and the washing pH was set to 4.0 for fraction 5, a peak having an m / z of 66449 was detected. In addition, when a protein chip having a weak cation exchanger immobilized thereon was used and the washing pH was set to 7.0 for fraction 6, a peak having an m / z of 66572 was detected. When a protein chip on which metal ions (Cu2 +) were immobilized was used and the washing pH was set to 7.0 for fraction 4, a peak having an m / z of 66216 was detected. Further, when a protein chip on which metal ions (Cu2 +) were immobilized was used and the washing pH was set to 7.0 for fraction 6, two peaks with m / z of 66572, 66582 were detected. When a protein chip on which metal ions (Cu2 +) were immobilized was used and the washing pH was set to 7.0 for fraction 5, a peak with an m / z of 66596 was detected. These peaks differed from candidate protein (1) and candidate protein (2), and showed low values in diabetic patients. From these 8 peaks, it was suggested that a candidate protein (hereinafter referred to as “candidate protein (3)”) exists in the vicinity of a molecular weight range of 66000 to 67000. Since this molecular weight value is very close to the known human serum albumin molecular weight (66439) value, and a peak almost identical to the molecular weight of human serum albumin whose m / z is 66449 was detected, the candidate protein ( 3) was identified as serum albumin.

  In the same manner as in Examples 1 and 2, for each peak, the peak intensity was plotted separately for diabetic patients and healthy individuals, an ROC curve was created, and a cut-off value was set. As an example, the example about the peak whose m / z is 66216 is shown to Fig.6 (a)-(c). FIG. 6A is a graph in which peak intensities are plotted separately for diabetic patients and healthy individuals, and FIG. 6B shows the results of FIG. 6A with the maximum value, minimum value, median value, and cutoff. FIG. 6C is an ROC curve. In addition, since this diabetic patient shows a lower value in this peak, the ROC curve in FIG. 6C is inclined to the lower right and indicates that the accuracy is higher toward the lower right. Similar graphs (not shown) were prepared for the other seven peaks. Table 4 below shows a table summarizing the P value, ROC area, chip used, chip cleaning conditions, fractions, and used EAM for each peak.

[Example 4]

  A diabetes detection kit (1) having the following constitution was constructed. This kit includes a substrate on which an antibody is immobilized. The kit captures a marker substance on the substrate and measures transthyretin, apolipoprotein CIII, and serum albumin in a sandwich EIA system. Detection is performed with fluorescently labeled streptavidin.

[Example 5]

  A diabetes detection kit (2) having the following constitution was constructed. This kit is for measuring transthyretin, apolipoprotein CIII, and serum albumin by sandwich EIA using a microtiter plate.

[Example 6]

1. Animal Experiments Using Rats OLETF rats were prepared as model rats with spontaneous development of type 2 diabetes, and LETO rats were prepared as rats that did not develop type 2 diabetes of the same strain (genetically related) as the model rats. Both the OLETF rat and the LETO rat were provided by Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd. A group of LETO rats (first group) and a group of OLETF rats (second group) were set and each rat was raised. CRF-1 (Oriental Bioscience) was used as the feed, and each group was allowed to eat freely. Each group started the test at 5 weeks of age and was reared until 50 or 62 weeks. OGTT was performed at each age of 5 (at the start of breeding), 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, and 49, and blood was sampled. Serum was prepared from each of these blood samples. After the termination of the breeding, 4 rats from the first group and 7 mice from the second group were selected from rats in which no abnormality was observed. The evaluation of OGTT in these rats was evaluated according to a conventional method in three stages: normal (◯), boundary (Δ), and diabetes (●). The results of OGTT are shown in Table 1. In the table, 1-1 to 1-4 are the results of the first group of rats (4 animals in total), and 2-1 to 2-7 are the results of each of the rats in the second group (7 animals in total). The integer of is the age of the rat. That is, the first group of rats did not develop diabetes, but the second group of rats gradually developed diabetes from around 21 weeks of age. Next, 21 samples specifically shown in Table 1 were subjected to the following analysis.

  To 20 μL of each serum sample, 30 μL of denaturation buffer (9 M urea, 2% CHAPS, 50 mM Tris-HCl (pH 9.0)) was added for pretreatment to denature the protein. Next, each pretreated serum sample was applied to a strong anion exchange resin column (Q Ceramic Hyper D, Biosepra). Next, a buffer solution of pH 9.0 (50 mM Tris-HCl (pH 9.0), 0.1% (w / v) 1-o-N-octyl-β-D-glucopyranoside (hereinafter referred to as “OGP”) )), PH 7.0 buffer (50 mM HEPES-NaOH (pH 7.0), 0.1% (w / v) OGP), pH 5.0 buffer (100 mM sodium acetate (pH 5.0), 0 0.1% (w / v) OGP), pH 4.0 buffer (100 mM sodium acetate (pH 4.0), 0.1% (w / v) OGP), pH 3.0 buffer (50 mM sodium citrate) (PH 3.0), 0.1% (w / v) OGP), and organic solvent (mixed solution consisting of 33.3% isopropyl alcohol, 16.7% acetonitrile, 0.1% trifluoroacetic acid) at 200 μL each. Elution in order Fraction 1 (eluted at pH 9.0, pass through), fraction 2 (eluted at pH 7.0), fraction 3 (eluted at pH 5.0), fraction 4 (eluted at pH 4.0), fraction Six crude fractions of 5 (eluted at pH 3.0) and fraction 6 (organic solvent) were obtained.

10 μL of each of the obtained fractions was diluted 10-fold with a pH 4.0 protein chip binding buffer (100 mM sodium acetate) and then added to the cation exchange chip CM10 (Cyphergen). Similarly, 10 μL of each obtained fraction was diluted 10-fold with a pH 7.0 protein chip binding buffer (100 mM phosphoric acid, 0.5 M NaCl), and then added to a copper-modified chip IMAC30 (Cyphergen). Each protein chip was washed 3 times with each binding buffer, then once with deionized water and dried. Next, sinapinic acid (SPA) or α-cyano-4-hydroxycinnamic acid (CHCA) which is an energy absorbing molecule is added, and SELDI-TOF- is used using a protein chip leader Model PBS IIc (Cyphergen). MS was performed. In addition, the measurement molecular weight range (M / Z) was performed in the range of 3000-200000. Moreover, the measurement was performed in duplicate and the average value of M / Z was calculated. Data analysis was performed using Protein Chip Software, Ciphergen Express Data Manager, and Biomarker Patterns Software (all from Ciphergen). Specifically, after baseline correction, molecular weight calibration, and spectrum normalization, single marker analysis and multiflow analysis combining several markers were performed. As a result, a large number of peaks were detected depending on the combination of the type of crude fraction, the type of protein chip, the washing conditions of the chip, and the like. From these peaks, 14 peaks having a significant difference between the samples of the first group and the second group were selected.
2. In the specific fraction 5 of the marker substance (a), when the cation exchange chip CM10 was used, an ion peak having a mass / charge ratio of 7043 (average value) was detected. FIG. 7 shows a box diagram when the peak intensity of this peak at 21 weeks of age is plotted for each group. In the figure, the upper and lower edges of the bag are the maximum and minimum values, respectively, the upper and lower sides of the box are the third quartile (75th percentile) and the first quartile (25th percentile), respectively, and the line in the box is the center Value (the same applies to the following figures). That is, this peak showed a low value in the first group and a high value in the second group. Based on the above, the protein (marker substance (a)) that produces a peak with a mass / charge ratio of about 7040 when subjected to SELDI-TOF-MS is specific to rats that have developed diabetes or have a high risk of developing in the future. It was found that this substance can be a marker for the disease. Thereby, when the marker substance (a) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (a) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (a) in the blood of the animal ingested the test substance as an index, the evaluation of the diabetes improving effect or the future risk reduction effect of the test substance, and such It has been shown that screening of substances can be performed. For example, when a serum sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 7040 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.
3. In the specific fraction 6 of the marker substance (b), when the cation exchange chip CM10 was used, an ion peak having a mass / charge ratio of 8325 (average value) was detected. FIG. 8 shows a box diagram when the peak intensity of this peak at 21 weeks of age is plotted for each group. That is, this peak showed a low value in the first group and a high value in the second group. Based on the above, a protein (marker substance (b)) that produces a peak with a mass / charge ratio of about 8330 when subjected to SELDI-TOF-MS is specific to a rat that has developed diabetes or has a high risk of developing in the future. It was found that this substance can be a marker for the disease. Thereby, when the marker substance (b) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (b) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (b) in the blood of the animal ingested the test substance as an indicator, the evaluation of the diabetes improving effect or the future risk reduction effect of the test substance, and such It has been shown that screening of substances can be performed. For example, when a similar animal experiment is performed using a desired test substance to prepare a serum sample, and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 8330 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.
4). In the specific fraction 6 of the marker substance (c), when the cation exchange chip CM10 was used, an ion peak having a mass / charge ratio of 8532 (average value) was detected. FIG. 9 shows a box diagram when the peak intensity of this peak at the age of 21 weeks is plotted for each group. That is, this peak showed a low value in the first group and a high value in the second group. From the above, the protein (marker substance (c)) that produces a peak with a mass / charge ratio of about 8530 when subjected to SELDI-TOF-MS is specific to rats that are developing diabetes or have high risk of developing in the future. It was found that this substance can be a marker for the disease. Thereby, when the marker substance (c) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (c) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (c) in the blood of the animal ingested the test substance as an index, the evaluation of the diabetes improving effect or the future risk reduction effect of the test substance, and such It has been shown that screening of substances can be performed. For example, when a similar animal experiment is performed using a desired test substance to prepare a serum sample, and a SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 8530 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.
5. In the specific fraction 1 of the marker substance (d), when the copper-modified chip IMAC30 was used, an ion peak having a mass / charge ratio of 9062 (average value) was detected. FIG. 10 shows a box diagram when the peak intensity of this peak at the age of 21 weeks is plotted for each group. That is, this peak showed a low value in the first group and a high value in the second group. From the above, a protein (marker substance (d)) that produces a mass / charge ratio of about 9060 peak when subjected to SELDI-TOF-MS is specific to a rat that has developed diabetes or has a high risk of developing in the future. The substance was found to be a marker for the disease. Thereby, when the marker substance (d) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (d) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (d) in the blood of the animal ingested the test substance as an indicator, the evaluation of the diabetes improving effect or the future risk reduction effect of the test substance, and such It has been shown that screening of substances can be performed. For example, when a similar animal experiment is performed using a desired test substance to prepare a serum sample and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 9060 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.
6). In the specific fraction 6 of the marker substance (e), when the cation exchange chip CM10 was used, an ion peak having a mass / charge ratio of 9255 (average value) was detected. FIG. 5 shows a box diagram when the peak intensity of this peak at 21 weeks of age is plotted for each group. That is, this peak showed a low value in the first group and a high value in the second group. From the above, a protein (marker substance (e)) that produces a peak with a mass / charge ratio of about 9260 when subjected to SELDI-TOF-MS is specific to a rat that has developed diabetes or has a high risk of developing in the future. It was found that this substance can be a marker for the disease. Thereby, when the marker substance (e) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (e) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (e) in the blood of the animal ingested the test substance as an indicator, the evaluation of the diabetes improving effect or the future risk reduction effect of the test substance, and such It has been shown that screening of substances can be performed. For example, when a similar animal experiment is performed using a desired test substance to prepare a serum sample and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 9260 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.
7). In the specific fraction 6 of the marker substance (f), when the cation exchange chip CM10 was used, an ion peak having a mass / charge ratio of 9445 (average value) was detected. FIG. 12 shows a box diagram when the peak intensity of this peak at the age of 21 weeks is plotted for each group. That is, this peak showed a low value in the first group and a high value in the second group. 2 or more, a protein (marker substance (f)) that produces a peak with a mass / charge ratio of about 9450 when subjected to SELDI-TOF-MS is specific to a rat that has developed diabetes or has a high risk of developing in the future It was found that the substance can be a marker of the disease. Thereby, when the marker substance (f) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (f) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (f) in the blood of the animal ingested the test substance as an index, evaluation of the effect of improving the diabetes of the test substance or reducing the risk of developing the future, and such It has been shown that screening of substances can be performed. For example, when a serum sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 9450 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.
8). In the specific fraction 4 of the marker substance (g), when the copper modified chip IMAC30 was used, an ion peak having a mass / charge ratio of 13720 (average value) was detected. FIG. 13 shows a box diagram when the peak intensity of this peak at the age of 21 weeks is plotted for each group. That is, this peak showed a low value in the first group and a high value in the second group. From 2 or more, a protein (marker substance (g)) that produces a mass / charge ratio of about 13700 peak when subjected to SELDI-TOF-MS is specific to a rat that has developed diabetes or a high risk of developing in the future. It was found that this substance can be a marker for the disease. Thereby, when the marker substance (g) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (g) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (g) in the blood of the animal ingested the test substance as an index, the evaluation of the effect of improving the diabetes of the test substance or reducing the risk of future onset, and such It has been shown that screening of substances can be performed. For example, when a serum sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 13700 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.
9. In the specific fraction 3 of the marker substance (h), when the cation exchange chip CM10 was used, an ion peak having a mass / charge ratio of 76404 (average value) was detected. FIG. 14 shows a box diagram when the peak intensity of this peak at 21 weeks of age is plotted for each group. That is, this peak showed a low value in the first group and a high value in the second group. 2 or more, a protein (marker substance (h)) that produces a peak with a mass / charge ratio of about 76400 when subjected to SELDI-TOF-MS is specific to a rat that has developed diabetes or has a high risk of developing it It was found that the substance can be a marker of the disease. Thereby, when the marker substance (h) is also present in human blood, the presence or absence of diabetes or the risk of developing it in the future can be determined using the concentration of the marker substance (h) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (h) in the blood of the animal ingested the test substance as an index, the test substance evaluates the effect of improving diabetes or reducing the risk of future onset, and such It has been shown that screening of substances can be performed. For example, when a similar animal experiment is performed using a desired test substance to prepare a serum sample, and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 76400 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.

10. In the specific fraction 4 of the marker substance (i), when the cation exchange chip CM10 was used, an ion peak having a mass / charge ratio of 79085 (average value) was detected. FIG. 15 shows a box diagram when the peak intensity of this peak at the age of 21 weeks is plotted for each group. That is, this peak showed a low value in the first group and a high value in the second group. From the above, a protein (marker substance (i)) that produces a peak with a mass / charge ratio of about 79100 when subjected to SELDI-TOF-MS is specific to a rat that has developed diabetes or has a high risk of developing in the future. It was found that this substance can be a marker for the disease. Thereby, when the marker substance (i) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (i) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (i) in the blood of the animal ingested the test substance as an indicator, the evaluation of the diabetes improving effect or the future risk reduction effect of the test substance, and such It has been shown that screening of substances can be performed. For example, when a serum sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak having a mass / charge ratio of about 79100 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.

11. In the specific fraction 6 of the marker substance (j), when the cation exchange chip CM10 was used, an ion peak having a mass / charge ratio of 3497 (average value) was detected. FIG. 16 shows a box diagram when the peak intensity of this peak at the age of 21 weeks is plotted for each group. That is, this peak showed a high value in the first group and a low value in the second group. From 2 or more, a protein (marker substance (j)) that produces a peak with a mass / charge ratio of about 3500 when subjected to SELDI-TOF-MS is specific to rats that are developing diabetes or have a high risk of developing in the future It was found that the substance can be a marker of the disease. Thereby, when the marker substance (j) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (j) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (i) in the blood of the animal ingested the test substance as an indicator, the evaluation of the diabetes improving effect or the future risk reduction effect of the test substance, and such It has been shown that screening of substances can be performed. For example, when a serum sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 3500 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.

12 In the specific fraction 1 of the marker substance (k), when the copper modified chip IMAC30 was used, an ion peak having a mass / charge ratio of 3559 (average value) was detected. FIG. 17 shows a box diagram when the peak intensity of this peak at the age of 21 weeks is plotted for each group. That is, this peak showed a high value in the first group and a low value in the second group. From the above, a protein (marker substance (k)) that produces a peak with a mass / charge ratio of about 3560 when subjected to SELDI-TOF-MS is specific to a rat that has developed diabetes or has a high risk of developing in the future. It was found that this substance can be a marker for the disease. Thereby, when the marker substance (k) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (k) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (k) in the blood of the animal ingested the test substance as an index, the test substance evaluates the effect of improving diabetes or reducing the future risk of developing, and such It has been shown that screening of substances can be performed. For example, when a similar animal experiment is performed using a desired test substance to prepare a serum sample and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 3560 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.

13. In the specific fraction 4 of the marker substance (l), when the copper-modified chip IMAC30 was used, an ion peak having a mass / charge ratio of 4184 (average value) was detected. FIG. 18 shows a box diagram when the peak intensity of this peak at 21 weeks of age is plotted for each group. That is, this peak showed a high value in the first group and a low value in the second group. Based on the above, the protein (marker substance (l)) that produces a peak with a mass / charge ratio of about 4180 when subjected to SELDI-TOF-MS is specific to rats that have developed diabetes or have a high risk of developing in the future. It was found that this substance can be a marker for the disease. Thereby, when the marker substance (l) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (l) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (l) in the blood of the animal ingested the test substance as an index, an evaluation of the diabetes improving effect or the future risk reduction effect of the test substance, and such It has been shown that screening of substances can be performed. For example, when a similar animal experiment is performed using a desired test substance to prepare a serum sample and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 4180 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.

14 In the specific fraction 5 of the marker substance (m), when the cation exchange chip CM10 was used, an ion peak having a mass / charge ratio of 12786 (average value) was detected. FIG. 19 shows a box diagram when the peak intensity of this peak at the age of 21 weeks is plotted for each group. That is, this peak showed a high value in the first group and a low value in the second group. Based on the above, a protein (marker substance (m)) that produces a peak with a mass / charge ratio of about 12800 when subjected to SELDI-TOF-MS is specific to a rat that has developed diabetes or has a high risk of developing in the future. It was found that this substance can be a marker for the disease. Thereby, when the marker substance (m) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (m) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (m) in the blood of the animal ingested the test substance as an index, the evaluation of the effect of improving diabetes or reducing the risk of future onset of the test substance, and such It has been shown that screening of substances can be performed. For example, when a similar animal experiment is performed using a desired test substance to prepare a serum sample, and a SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 12800 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.

15. In the specific fraction 4 of the marker substance (n), when the cation exchange chip CM10 was used, an ion peak having a mass / charge ratio of 65700 (average value) was detected. FIG. 80 shows a box diagram when the peak intensity of this peak at the age of 21 weeks is plotted for each group. That is, this peak showed a high value in the first group and a low value in the second group. Based on the above, a protein (marker substance (n)) that produces a peak with a mass / charge ratio of about 65700 when subjected to SELDI-TOF-MS is specific to a rat that has developed diabetes or has a high risk of developing in the future. It was found that this substance can be a marker for the disease. Thereby, when the marker substance (n) is also present in human blood, it is possible to determine the onset of diabetes or the risk of future onset using the concentration of the marker substance (n) in the blood as an index. It has been shown. Furthermore, using the concentration of the marker substance (n) in the blood of the animal ingested as the test substance as an index, the evaluation of the effect of improving the diabetes of the test substance or reducing the risk of future onset, and such It has been shown that screening of substances can be performed. For example, when a similar animal experiment is performed using a desired test substance to prepare a serum sample and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 65700 is generated. When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving diabetes or reducing the risk of developing the future.
[Example 7]

  In the above Examples, a scheme for identifying a marker substance predicted to be S-cysteinyl transthyretin from the molecular weight is described below.

  The purification and identification scheme of S-cysteinylated TTR derived from serum of diabetic patients and healthy controls is described.

(A. Purification method by ion exchange)
500 μL of serum of a diabetic patient and a healthy person is diluted 5 times with 50 mM Tris-HCl (pH 7.0), centrifuged at 20 kG, 4 ° C., 10 min, and then 1 mL of Amersham HiTrapQ HP as a strong anion exchange column is added. Fractionation was performed. First, 5 CV washing was performed with 50 mM Tris-HCl (pH 7.0), and then 5 CV washing was performed with 50 mM Tris-HCl (pH 7.0) and 160 mM NaCl. Elution was performed at 2 CV using 50 mM Na-Acetate (pH 4.0). The elution sample was subjected to acetone precipitation with 5 volumes of acetone, and the resulting precipitate was added to 200 μL of a 62.5 mM Tris-HCl (pH 6.8), 1% SDS, 20% glycerol, 0.005% BPB mixed solution. Dissolved and used as a sample for SDS-PAGE.

(B. Purification method by SDS-PAGE)
A. 20 μL of the sample prepared as described above was used, and fractionation was performed at a constant voltage (100 V, 2.5 hr) using DRC Perfect NT Gel polypeptide analysis. As the running buffer, 100 mM Tris, 100 mM Tricine, 0.1% SDS was used. After completion of the electrophoresis, the cells were fixed with 10% AcOH in 40% MeOH, washed with ultrapure water, and then stained with CBB.

(C. Identification method by Western blotting)
B. After performing SDS-PAGE as described above, staining was not performed with CBB, but blotting was performed on a PVDF membrane as described below, and detection was performed by BIO-RAD Amplified Alkaline Phosphatate Goat Anti-Rabbit Immun-Blot Assay Kit.

(1) Preparation of reagent and anode buffer
60 mM Tris, 40 mMCAPS, 0.1% SDS, pH 9.6
・ Cathode buffer
60 mM Tris, 40 mM CAPS, 15% MeOH, pH 9.6
・ Tris-bufferedsaline (TBS)
20 mM Tris-HCl, 500 mM NaCl, pH 7.5
・ Cleaning solution (TTBS)
20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5
・ 5% non-fat dry milk in blocking buffer TBS ・ Primary antibody solution Anti-human Transthyretin antibody (rabbit IgG 200μg / mL) diluted 1000 times with TTBS (final concentration 0.2μg / mL)

・ Secondary antibody solution
Biotinylated Goat Anti Rabbit IgG (H + L) diluted 3000 times with TTBS / Streptavidin-biotinylated alkaline phosphatase complex
Dissolve 10 μL of Streptavidin in 30 mL of TTBS, add 10 μL of biotinylated alkaline phosphatase, and incubate for 2 hours at room temperature.
・ AP coloring solution
Dilute 25x stock 25 times with ultrapure water to make AP color buffer. Add 200 µL of AP coloring reagent A and 200 µL of AP coloring reagent B to 20 mL of AP coloring buffer solution to make an AP coloring solution.

(2) A filter paper wetted with an anode buffer solution on an anode plate, a PVDF membrane (7 × 6 cm) wetted with methanol for 5 minutes, and a gel placed thereon to wet the cathode buffer solution. And a cathode plate was placed thereon, and a current was applied at a constant current of 105 mA for 30 minutes.
(3) After energization, the PVDF membrane was immersed in ultrapure water and shaken for 5 minutes.
(4) The PVDF membrane was air-dried and then blocked with a blocking buffer. Blocking was performed at room temperature under shaking for 1 hour.
(5) After blocking, the substrate was immersed in TTBS and shaken at room temperature for 5 minutes × 3 times to wash the blocking buffer.
(6) It was immersed in a primary antibody solution and shaken at room temperature for 2 hours.
(7) After the primary antibody reaction, it was immersed in TTBS and washed with shaking for 5 minutes × 3 times at room temperature.
(8) It was immersed in a secondary antibody solution and shaken at room temperature for 2 hours.
(9) During the operation of (8), a streptavidin-biotinylated alkaline phosphatase complex was prepared.
(10) After the secondary antibody reaction, it was immersed in TTBS and washed by shaking for 5 minutes × 3 times at room temperature.
(11) It was immersed in a streptavidin-biotinylated alkaline phosphatase complex solution and shaken at room temperature for 2 hours.
(12) After the streptavidin-biotinylated alkaline phosphatase complex reaction, it was immersed in TTBS and washed by shaking at room temperature for 5 minutes × 3 times.
(13) It was immersed in an AP coloring solution and confirmed to be human TTR.

(D. Extraction from gel and MALDI-TOF-MS analysis)
Extraction from the gel was performed using a mixed solution of equal amounts of methanol and 100 mM ammonium bicarbonate, and mixed at a ratio of ultrapure water: 100 mM ammonium bicarbonate: MeCN = 2: 5: 3 (15 μL) It was performed using.

  The obtained extract (2 μL) was placed on a metal plate, and 0.4 μL of saturated CHCA was used as a matrix, and measurement was performed with an Applied Biosystems 4700 Proteomics Analyzer.

(result)
The results are shown in FIGS.

(Confirmation of TTR monomer by Western blotting)
By the above method, it was confirmed that a protein around 13.8K was a TTR monomer using a TTR antibody. It was also found that each derivative was included in the monomer fraction.

  The MS spectrum of the monomer fraction is as follows.

Theoretical value: S-cysteinylated TTR M / Z = 13880.53
After reduction TTR M / Z = 13761.41
FIG. 21 shows healthy person-derived S-cysteinylated TTR. Measurement: M / Z = 13874.7480.

  FIG. 22 shows a healthy person-derived S-cysteinylated TTR after reduction treatment. Measurement: M / Z = 13759.7393.

  FIG. 23 shows diabetic patient-derived S-cysteinylated TTR. Measurement: M / Z = 13871.3320.

  FIG. 24 shows a diabetic patient-derived S-cysteinylated TTR after reduction treatment. Measurement: M / Z = 13755.6494.

  FIG. 25 shows the tetrameric structure of TTR and the amino acid sequence of the monomer. Thus, transthyretin normally has a tetrameric structure, and it is postulated that when it collapses, it becomes diabetic.

FIG. 26 shows the three-dimensional structure and secondary structure arrangement of human TTRα-domain. Derivatives can be analyzed by mass spectrometry using typical post-translational modifications and their mass changes in proteins and peptides shown in Table 1.
[Example 8]

  In the above examples, a scheme for identifying the marker substance predicted to be apolipoprotein CIII from the molecular weight is described below.

  The purification identification scheme of Rat Apolipoprotein CIII is described below.

(A. Purification method by ion exchange)
750 μL of Rat Serum was diluted 5 times with 50 mM Tris-HCl (pH 6.0) and filtered using a Millipore Millex-HV (0.45 μm) filter unit as a sample. The obtained sample was fractionated using 1 mL of Amersham HiTrapQ HP which is a strong anion exchange column. First, 5 CV washing was performed with 50 mM Tris-HCl (pH 6.0), then 12 CV washing was performed with 50 mM Tris-HCl (pH 6.0) and 200 mM NaCl, and then 5 CV was washed with 50 mM Tris-HCl (pH 6.0). Washing was performed. Elution was performed at 2 CV using 50 mM Na-Acetate (pH 3.0). The elution sample was subjected to acetone precipitation with 10 times the amount of acetone, and the resulting precipitate was added to 100 μL of 62.5 mM Tris-HCl (pH 6.8), 1% SDS, 20% glycerol, 0.005% BPB mixed solution. Dissolved and used as a sample for SDS-PAGE.

(B. Purification method by SDS-PAGE)
A. 20 μL of the sample prepared as described above was used, and fractionation was performed at a constant voltage (100 V, 2.5 hr) using DRC Perfect NT Gel polypeptide analysis. As the running buffer, 100 mM Tris, 100 mM Tricine, 0.1% SDS was used. After completion of the electrophoresis, the cells were fixed with 10% AcOH in 40% MeOH, washed with ultrapure water, and then stained with CBB.

(C. Extraction from gel and SELDI-TOF-MS analysis)
Extraction from the gel was performed using a mixed solution of equal amounts of methanol and 100 mM ammonium bicarbonate, and mixed at a ratio of ultrapure water: 100 mM ammonium bicarbonate: MeCN = 2: 5: 3 (15 μL) It was performed using.

  15 μL of the obtained extract was placed on a CIPHERGEN H50 ProteinChipArray, washed twice with 5 μL of 0.1% TFA, and measured by placing 0.5 μL of 25% SPA twice on the matrix.

(D. Identification method by in-gel digestion)
The sample whose molecular weight was measured using SELDI-TOF-MS was subjected to in-gel digestion and identification. (See protocol below)
(1) Preparation of reaction solution Adjustment of reduction solution <Essential adjustment>
Dissolve DTT (1.5 mg) in 100 mM ammonium bicarbonate (1 mL).
Adjustment of alkylation solution
Dissolve iodoacetamide (10 mg) in 100 mM ammonium bicarbonate (1 mL).
Adjustment of trypsin solution
-Trypsin (20 μg) is dissolved in 50 mM acetic acid (0.1 mL) to make a trypsin stock solution. To a trypsin stock solution (25 μL), add ultrapure water (200 μL), 100 mM ammonium bicarbonate (500 μL), and MeCN (300 μL) to make a trypsin solution.
(2) Cut out the band and put it in a 500 μL tube.
(3) Add 100 μL of an equal volume mixed solution of methanol and 100 mM ammonium bicarbonate and decolorize.
(4) Add 100 μL of the reducing solution and incubate for 30 minutes.
(5) Add 100 μL of alkylation solution and incubate for 30 minutes.
(6) Add 15 μL of trypsin solution and incubate at 37 ° C. overnight.
(7) 1/10 times the amount of 1% TFA is added to stop the reaction.

1 μL of the digested sample was placed on a metal plate, and 0.4 μL of saturated CHCA was used as a matrix, and measurement was performed with an Applied Biosystems 4700 Proteomics Analyzer.

(About the data obtained)
A database search of Mascot (http://www.matrixscience.com/search_form_select.html) was performed to identify rat apolipoprotein CIII. The results are shown in FIGS. 27 and 28.

  FIG. 27 shows a gel photograph of the identified band and analysis by SELDI-TOF of the band.

  FIG. 28 shows a gel photograph of the identified band and analysis by SELDI-TOF of the band.

  The presence of similar markers in humans was confirmed as follows.

  The selection of the purified resin and the optimum adsorption conditions for the resin were investigated.

Used ProteinChips Q10 and CM10
PH pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 7.0
Use Buffer
pH3.0: 50mM Na-Citrate Buffer
pH3.5 ~ 5.5: 50mM Na-Acetate Buffer
pH7.0: 50mM HEPES Buffer
FIG. 29 shows spots on two-dimensional electrophoresis that are considered to be spots of human apolipoprotein CIII (0 to 2).

  FIG. 30 shows the results of mass spectrometry of each spot.

  FIG. 31 shows the SELDI-MS results for each spot.

FIG. 32 to FIG. 34 show the results of the optimum adsorption condition study. FIG. 32 shows the examination result of CM10, and FIG. 33 shows the examination result of Q10. FIG. 34 shows the optimum condition study results. From these results, it is clear that the obtained spot is a spot of human apolipoprotein CIII (0-2).
[Example 9]

(S-cysteinylated transthyretin is also correlated in severity assessment by glycated hemoglobin (HbA1c) value)
It was shown in this example that the S-cysteinylated transthyretin identified as described above shows a correlation also in the severity evaluation by glycated hemoglobin (HbA1c) value. HbA1c is currently used as the most reliable diabetes marker.

  First, the ionic strength of S-cysteinylated transthyretin (measured value: M / Z = 13,863, theoretical value: M / Z = 13880.53) in diabetic patients using the protocol described in the above example. And the HbA1c value were confirmed.

Measurement conditions and statistics
IMAC30: chelate metal (Cu) immobilization chip,
Fr4: FIG. See serum expression analysis protocol.

CHCA: MS measurement matrix
Measurement value: M / Z: 13,863
P-Value: 0.0004, ROC area: 0.86.

  The results are shown in FIG. FIG. 35 shows the relationship between ionic strength and HbA1c value of S-cysteinylated transthyretin (measured value: M / Z = 13,863, theoretical value: M / Z = 13880.53) in diabetic patients. Vertical axis: I-intensity of S-systemylated TTR measured by SELDI-TOF-MS; Horizontal axis: C: control (healthy person), P: diabetic patient.

  Next, the relationship between the ionic strength of the S-cysteinylated transthyretin (measured value: M / Z = 13,874, theoretical value: M / Z = 13880.53) and the HbA1c value in diabetic patients was measured.

Measurement conditions and statistics
IMAC30: chelate metal (Cu) immobilization chip,
Fr4: FIG. See serum expression analysis protocol.

MS measurement matrix: SPA-H
Measurement value: M / Z: 13,874,
P-Value: 0.007, ROC area: 0.82.

  FIG. 36 shows the result. FIG. 36 shows the relationship between the ionic strength and HbA1c value of S-cysteinylated transthyretin (measured value: M / Z = 13,874, theoretical value: M / Z = 13880.53) in diabetic patients. The vertical axis represents the ionic strength of S-cysteine TTR measured by SELDI-TOF-MS; the horizontal axis represents C: control (healthy person), P: diabetic patient.

  Next, the relationship between the ionic strength of S-cysteinylated transthyretin (measured value: M / Z = 13,884, theoretical value: M / Z = 13880.53) and HbA1c value in diabetic patients was examined.

Measurement conditions and statistical values CM10 (pH 7): weak cation exchange tip (cleaning conditions)
Fr4: FIG. See serum expression analysis protocol.

SPA-H :: MS measurement matrix Measurement value: M / Z: 13884
P-Value: 0.0005, ROC area: 0.90
The result is shown in FIG. FIG. 37 shows the relationship between ionic strength and HbA1c value of S-cysteinylated transthyretin (measured value: M / Z = 13,884, theoretical value: M / Z = 13880.53) in diabetic patients. Vertical axis: I-intensity of S-systemylated TTR measured by SELDI-TOF-MS; Horizontal axis: C: control (healthy person), P: diabetic patient.

  Next, actual measurement data of M / Z: 13,863 in diabetic patients is shown (FIG. 38).

Thus, the behavior of S-cysteinylated transthyretin was found to be an indicator of diabetes and was also found to correlate with its severity.
[Example 10]

(Apolipoprotein is a good pre-diagnostic marker.)
In this example, 8.3K predicted as a prophylactic (pre-) marker in the above example was identified as Apo CII.

(Purification and identification scheme of rat apolipoprotein C2)
(A. Purification method by ion exchange)
A sample obtained by diluting 750 μL of rat serum with 50 mM Tris-HCl (pH 6.0) and filtering using Millipore Millex-HV (0.45 μm) filter unit was used as a sample. The obtained sample was fractionated using 1 mL of Amersham HiTrapQ HP which is a strong anion exchange column. First, 5 CV washing was performed with 50 mM Tris-HCl (pH 6.0), then 12 CV washing was performed with 50 mM Tris-HCl (pH 6.0) and 200 mM NaCl, and then 5 CV was washed with 50 mM Tris-HCl (pH 6.0). Washing was performed. Elution was performed at 2 CV using 50 mM Na-Acetate (pH 3.0). The elution sample was subjected to acetone precipitation with 10 times the amount of acetone. Dissolved and used as a sample for SDS-PAGE.

(B. Purification method by SDS-PAGE)
A. The sample prepared in the above procedure was used at 20 · L, and fractionation was performed at a constant voltage (100 V, 2.5 hr) using DRC Perfect NT Gel polypeptide analysis. As the running buffer, 100 mM Tris, 100 mM Tricine, 0.1% SDS was used. After completion of the electrophoresis, fixation with 10% AcOH in 40% MeOH, washing with ultrapure water, and staining with CBB (C. extraction from gel and SELDI-TOF-MS analysis)
Extraction from the gel was performed using a mixed solution of equal amounts of methanol and 100 mM ammonium bicarbonate, and mixed at a ratio of ultrapure water: 100 mM ammonium bicarbonate: MeCN = 2: 5: 3 (15 μL) It was performed using.

  15 μL of the obtained extract was placed on a CIPHERGEN H50 ProteinChipArray, washed twice with 5 μL of 0.1% TFA, and the matrix was measured twice with 0.5 μL of 25% SPA.

(D. Identification method by in-gel digestion)
The sample whose molecular weight was measured using SELDI-TOF-MS was subjected to in-gel digestion and identification. (See protocol below)
(1) Preparation of reaction solution Adjustment of reduction solution <Essential adjustment>
Dissolve DTT (1.5 mg) in 100 mM ammonium bicarbonate (1 mL).
Adjustment of alkylation solution
Dissolve iodoacetamide (10 mg) in 100 mM ammonium bicarbonate (1 mL).
Adjustment of trypsin solution
-Trypsin (20 μg) is dissolved in 50 mM acetic acid (0.1 mL) to make a trypsin stock solution. To a trypsin stock solution (25 μL), add ultrapure water (200 μL), 100 mM ammonium bicarbonate (500 μL), and MeCN (300 μL) to make a trypsin solution.
(2) Cut out the band and put it in a 500 μL tube.
(3) Add 100 μL of an equal volume mixed solution of methanol and 100 mM ammonium bicarbonate and decolorize.
(4) Add 100 μL of the reducing solution and incubate for 30 minutes.
(5) Add 100 μL of alkylation solution and incubate for 30 minutes.
(6) Add 15 μL of trypsin solution and incubate at 37 ° C. overnight.
(7) 1/10 times the amount of 1% TFA is added to stop the reaction.

  1 μL of the digested sample was placed on a metal plate, and 0.4 μL of saturated CHCA was used as a matrix, and measurement was performed with an Applied Biosystems 4700 Proteomics Analyzer.

A database search of Mascot (http://www.matrixscience.com/search_form_select.html) was performed on the obtained data, and it was identified as Rat Simulator to Apolipoprotein C2.

(E. Identification method by Western blotting)
B. After performing SDS-PAGE as described above, staining was not performed with CBB, but blotting was performed on a PVDF membrane as described below, and BIO-RAD Amplified Alkaline Phosphatate Goat Anti-Rabbit Immun-Blot Assay Kit, 170-64 Detection was performed according to 300002087.
(1) Reagent preparation: Anode buffer 60 mM Tris, 40 mM CAPS, 0.1% SDS, pH 9.6
Cathode buffer 60 mM Tris, 40 mM CAPS, 15% MeOH, pH 9.6
・ Tris-buffer sale (TBS)
20 mM Tris-HCl, 500 mM NaCl, pH 7.5
・ Cleaning solution (TTBS)
20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5
・ 5% non-fat dry milk in blocking buffer TBS ・ Primary antibody solution Santa Cruz Biotechnology, anti Apo C2 (Q-20) Goat polyclonal IgG, sc-19014, Lot C1904 diluted 1000 times with TTBS (final concentration 0.2 μg) / ML)
・ Secondary antibody solution Zymed Laboratories, Rabbit Anti-Goat IgG (H + L) Biotin-Conjugate, 81-1640, Lot 50494328 was diluted 3000 times with TTBS. Streptavidin-biotinylated alkaline phosphatase complex Streptavidin 30 mL Add 10 μL of biotinylated alkaline phosphatase and incubate for 2 hours at room temperature.
-AP color developing solution 25x stock is diluted 25 times with ultrapure water to make AP color developing buffer. 200 μL of AP coloring reagent A and 200 μL of AP coloring reagent B are added to 20 mL of AP coloring buffer solution to prepare an AP coloring solution.
(2) A filter paper wetted with an anode buffer solution on an anode plate, a PVDF membrane (7 × 6 cm) wetted with methanol for 5 minutes, and a gel placed thereon to wet the cathode buffer solution. And a cathode plate was placed thereon, and a current was applied at a constant current of 105 mA for 30 minutes.
(3) After energization, the PVDF membrane was immersed in ultrapure water and shaken for 5 minutes.
(4) The PVDF membrane was air-dried and then blocked with a blocking buffer. Blocking was performed at room temperature under shaking for 1 hour.
(5) After blocking, the substrate was immersed in TTBS and shaken at room temperature for 5 minutes × 3 times to wash the blocking buffer.
(6) It was immersed in a primary antibody solution and shaken at room temperature for 2 hours.
(7) After the primary antibody reaction, it was immersed in TTBS and washed with shaking for 5 minutes × 3 times at room temperature.
(8) It was immersed in a secondary antibody solution and shaken at room temperature for 2 hours.
(9) During the operation of (8), streptavidin-biotinylated alkaline phosphatase complex was prepared.
(10) After the secondary antibody reaction, it was immersed in TTBS and washed by shaking for 5 minutes × 3 times at room temperature.
(11) It was immersed in a streptavidin-biotinylated alkaline phosphatase complex solution and shaken at room temperature for 2 hours.
(12) Streptavidin-biotinylated alkaline phosphatase After the complex reaction, it was immersed in TTBS and washed by shaking for 5 minutes × 3 times at room temperature.
(13) It was confirmed by dipping in an AP coloring solution.

(F. Identification method using antibody chip (CIPHERGENPS10 / PS20 ProteinChipArray))
The sample extracted by the procedure of C was identified using an antibody chip. (See protocol below)
(1) Preparation of reagent Blocking buffer: 1 M Tris-HCl pH 8.0 + 140 mM NaCl
Wash buffer 1: 0.1% Triton X-100 / PBS
Wash buffer 2: 1% Triton X-100 / PBS + 150 mM NaCl
(2) Apply 200 μL of 50% MeCN to each spot and leave it for 1 min, RT, and leave (only PS10).
(3) Remove MeCN, apply 200 μL of PBS to each spot, and let stand for 1 min, RT, and leave (only PS10).
(4) Remove PBS and apply 100 μL of 125 μg / mL protein A solution to each spot and let stand at 4 ° C. overnight.
(5) Remove protein A solution <SIGMA Aldrich, Protein A from Staphylococcus aureus, P7837-5MG, Lot 074K10321>, apply 200 μL of blocking buffer, and allow to stand for 30 min, RT (room temperature).
(6) Remove blocking buffer and apply 200 μL of wash buffer 1 and allow to stand for 5 min, RT, (2 times).
(7) The washing buffer 1 is removed, and the antibody solution <Santa Cruz Biotechnology, anti Apo C2 (Q-20) Goat polyclonal IgG, sc-19014, Lot C1904> is adjusted to a final concentration of 30 μg / mL with PBS. Dilute and apply 100 μL of antibody solution for 2 hr, RT, and shake.
(8) The antibody solution is removed, 200 μL of washing buffer 1 is applied, and shaken for 5 minutes, RT, and 2 minutes.
(9) The washing buffer 1 is removed, 200 μL of PBS is applied, and the mixture is shaken for 5 minutes, RT.
(10) Remove PBS, apply 200 μL of 10 mM HEPES (pH 7.5), perform 30 sec, RT, and shake.
(11) Remove HEPES, apply 20 μL of the antigen solution, and perform 2 hr, RT, and shake.
(12) Remove the antigen solution, apply 200 μL of Wash Buffer 2 and shake for 3 min, RT, and shake (2 times).
(13) The washing buffer 2 is removed, 200 μL of PBS is applied, and the mixture is shaken for 3 minutes, RT.
(14) PBS is removed, and 200 μL of 10 mM HEPES (pH 7.4) is applied, shaken for 30 seconds, RT (room temperature), and shaken.
(15) Remove HEPES, air dry, apply EAM (50% SPA 0.5 μL × 2 times), and perform measurement.

  The results are shown in FIGS. 39 and 40.

  FIG. 39 shows a diabetic rat serum 8.3K (Apo CII) box diagram.

  FIG. 40 shows the above time-series data. Apo CII was found to be a good pre-diagnostic marker.

  FIGS. 41 to 43 show the results showing that the obtained serum is rat apolipoprotein CII. FIG. 41 shows the SELDI-MS results of the bands on the gel. FIG. 42 shows the confirmation of the obtained band by Western blot. FIG. 43 shows a state of being enhanced by the anti-ApoC2 antibody in the chromatographic fractionation.

Figures 44-47 demonstrate that apolipoprotein CII in humans is also a pre-diagnostic marker.
[Example 11: Screening]

  First, a substance that can be changed (preferably normalized) to a marker substance found in the present invention is screened.

In this example, a test substance is ingested by an animal that develops diabetes or an animal that has a high risk of developing in the future, and at least one concentration of the marker substance in the body fluid of the animal is compared with a reference value, Evaluate the effect of the substance on improving diabetes or reducing the risk of future onset.
[Example 12: Human screening]

  In Example 11, it is evaluated whether a substance that has an effect of improving diabetes or reducing the risk of future onset in an animal model is actually effective in a human subject. This is done in accordance with the same procedure as in a normal clinical trial.

It will be appreciated that such screening can actually diagnose diabetes in animals.
[Example 13: Production of an antibody capable of differentially distinguishing a marker substance found in healthy subjects from a marker substance found in affected individuals]
In the above-described examples, transthyretin that has been revealed to be a marker substance for diabetes includes an unmodified one and a modified one at a cysteine residue that is present solely in the amino acid sequence. In addition to cysteinylation, sulfonylation, glutathioneation, homocysteinylation, and the like are known as modifications, but cysteinylated transthyretin was found as a marker for diabetes in the above examples.

  The purpose of this example was to create a system that specifically detects this cysteinylated transthyretin and does not detect unmodified transthyretin.

The peptide sequence (especially in the vicinity of the Cys residue) as an antigen is well conserved between humans and typical immunized animals, and since a large amount of transthyretin is contained in the serum, antibodies can be used even when immunized normally. Therefore, it was decided that the technology of ADLib (registered trademark) of Chiome was used. The alignment of amino acid sequences near cysteine residues is shown below.
[Homo sapiens] GPTGTGESKCPLMVK (SEQ ID NO: 25)
[Musmusculus] GPAGAGESKCPLMVK (SEQ ID NO: 26)
[Rattusnorvegicus] GPGGAGESKCPLMVK (SEQ ID NO: 27)
[GallusGallus] APLVSHGSVDSKCPLMVK (SEQ ID NO: 28)

(Materials and methods)
The antigens used are as follows. Antibody production was carried out upon request from Chiome (Tokyo, Bunkyo).

(antigen)
Name and origin of the protein from which the peptide is derived: transthyretin (human)
Length: 15 residues Peptide sequence: GPTGTGESKCPLMVK (15 residues from the first amino acid residue at the N-terminus of transthyretin; SEQ ID NO: 25)
Modification: Cys residue in peptide is linked to free cysteine by disulfide bond (cysteinylation modified).

(Antigen preparation method)
The antigen was prepared based on a conventional method.

  As a result of the above procedure, about 20 mg of each peptide (both Cys peptide and unmodified peptide) was produced. This was dissolved in MilliQ (distilled water), this solution was stored at −80 ° C., and dispensed to about 4 mg each.

(Antibody preparation method)
An outline of the antibody selection process is shown below.

(Magnetic beads production)
First, antigen fixation to magnetic beads was performed. Immobilize cysteinyl-transthyretin partial peptide on magnetic beads, perform selection, and then perform ELISA for cysteinyl-transthyretin partial peptide and non-cysteinyl-transthyretin partial peptide using culture supernatant after 1 week It was.

(Antibody selection from library)
(Antibody selection method)
The antibody selection method was in principle according to the method described in JP-A-2006-149383. Briefly, antibodies that specifically bind to streptavidin from a population of DT40 cells (see WO2004 / 011644) that induces somatic recombination at the immunoglobulin locus and produces various immunoglobulin molecules. This was done by sorting the molecules. This will be described in detail below.

(Selection of antibodies that specifically bind to streptavidin)
(Cultured cells)
DT40 cells were cultured at 59.5% CO 2 at 39.5 ° C. in a 5% CO 2 thermostat. The medium was IMDM medium (Invitrogen), and 10% FBS, 1% chicken serum, penicillin 100 units / ml, streptomycin 100 μg / ml, 2-mercaptoethanol 55 μM were used. Trichostatin A (Wako Pure Chemical Industries) was dissolved in methanol at 5 mg / ml as a stock, and diluted with a medium as appropriate so that the final concentration was 2.5 ng / ml. The culture was continued while the cell concentration was maintained at 105 to 10 6 cells / ml.

(Production of streptavidin magnetic beads)
Dynabeads M-280 Tosylactivated (Dynal) was used as the magnetic beads, and DynaMPC (Dynal) was used as the magnetic stand. 200 μl of beads were washed 3 times with 500 μl of buffer A (0.1 M sodium phosphate, pH 7.4), then in buffer A, 400 μl with 240 μg streptavidin (Nacalai Tesque) for 24 hours at 37 ° C. The reaction was allowed to stir with rotation. The beads were then washed twice with 500 μl of buffer C (10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 0.1% BSA). Thereafter, 500 μl of Buffer D (0.2 M Tris-HCl, pH 8.5, 0.1% BSA) was added, and the mixture was allowed to react at 37 ° C. for 4 hours with stirring by rotation to perform blocking. Thereafter, the cells were washed twice with 500 μl of buffer C and then suspended in 400 μl of buffer C containing 0.02% sodium azide.

(Preparation of rabbit IgG magnetic beads)
Dynabeads M-280 Tosylactivated (Dynal) was used for the magnetic beads, and DynaMPC (Dynal) was used for the magnetic stand. 200 μl of beads were washed 3 times with 500 μl of buffer A (0.1 M sodium phosphate, pH 7.4) and then rotated overnight at 37 ° C. with 120 μg rabbit IgG (SIGMA) in 200 μl of buffer A The reaction was carried out with stirring. The beads were then washed twice with 200 μl of buffer C (10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 0.1% BSA). Thereafter, 200 μl of buffer solution D (0.2 M Tris-HCl, pH 8.5, 0.1% BSA) was added, and the mixture was allowed to react at 37 ° C. for 4 hours while stirring by rotation to perform blocking. Thereafter, the cells were washed twice with 500 μl of buffer C and then suspended in 200 μl of buffer C containing 0.02% sodium azide.

(Selection using streptavidin magnetic beads and rabbit IgG magnetic beads)
About 5 × 10 7 wild-type DT40 cells treated with trichostatin A, 2.5 ng / ml for 7 weeks were washed once with 10 ml of a washing buffer (PBS containing 1% BSA) and once with 1 ml, and then 1 ml. Were mixed with 5 × 10 6 streptavidin magnetic beads (rabbit IgG magnetic beads in the case of selection with rabbit IgG magnetic beads) in a washing buffer of 4 and incubated at 4 ° C. for 30 minutes with gentle rotation. Thereafter, it was washed 5 times with 1 ml of washing buffer. Finally, the cells bound to the magnetic beads were suspended in 500 μl, added to 30 ml of medium, dispensed 300 μl in 96-well plates, and cultured at 39.5 ° C. One week later, ELISA was performed on the culture supernatant.

(ELISA with two plates)
Prepare two 96-well immunoplate maxisorp (NalgeNunc), and add 200 μl of 5 μg / ml streptavidin (Nacalai Tesque) and ovalbumin (Sigma) (both dissolved in PBS) to room temperature. It was left to stand overnight and fixed to the plate (in the case of selection with rabbit IgG magnetic beads, rabbit IgG (SIGMA) and egg white lysozyme (SIGMA) were used). Thereafter, the mixture was blocked with 200 μl of 0.5% skim luc for 1 hour at room temperature, and washed 3 times with 200 μl of ELISA washing buffer (PBS supplemented with 0.05% Tween 20). To this was added 100 μl of cell culture supernatant and allowed to react at room temperature for 1 hour. Here, 100 μl of the same culture supernatant was dispensed to both the streptavidin-fixed plate and the ovalbumin-fixed plate (in the case of rabbit IgG magnetic bead selection, rabbit IgG-fixed plate and egg white lysozyme-fixed plate). Thereafter, the plate was washed 5 times with 200 μl of ELISA washing buffer, and horseradish peroxidase-conjugated goat anti-chicken IgM antibody (Bethyl) was diluted 2000 times with PBS, and 100 μl was added. After reacting at room temperature for 45 minutes, the plate was washed 5 times with an ELISA washing buffer, and then 100 μl of TMB + (Dako) was added and reacted at room temperature for 4 minutes, and then the reaction was stopped with 100 μl of 1N sulfuric acid. The quantification was performed by measuring the absorbance at 450 nm with an mQuantBiomolecular Spectrometer (Bio-Tek Instruments).

  The results of the ELISA show that the O.D. D. The value is evaluated by simultaneously showing the result of ELISA concerning the reactivity with ovalbumin of the same clone.

  Since all clones showing high affinity for streptavidin react strongly with ovalbumin, selection using only the affinity for streptavidin as an index is nonspecific for any ligand. It has been experimentally found that the probability of selecting antibodies that bind automatically is very high. On the other hand, using this method, it was confirmed that such non-specific antibodies can be efficiently excluded from the beginning. In fact, when assaying with streptavidin alone, 28 clones are selected as candidates, but most of them also bind strongly to ovalbumin. By excluding such clones, it is possible to immediately limit to one clone.

(2. Confirmation of specificity)
(Preparation of culture supernatant for ELISA for verification of streptavidin specificity)
The culture supernatant used for analyzing the further specificity by ELISA was a medium prepared as follows in order to remove serum-derived IgM and the like. Immunoglobulin was removed from chicken serum (Invitrogen) with 50% saturated ammonium sulfate as a precipitate, and the supernatant was dialyzed against PBS. Volume increase caused by dialysis was corrected by concentration with CentriPrep (Amicon) to obtain antibody-removed chicken serum. This was added to AIM-V serum-free medium (Invitrogen) at a concentration of 6%. Cells were added thereto at a concentration of about 106 / ml, cultured for 2 days, and the culture supernatant was removed and subjected to ELISA.

(ELISA for verification of streptavidin specificity)
Add 200 μl of 5 μg / ml streptavidin, ovalbumin, human IgG (Sigma) and 0.5% skim milk (Difco) (both dissolved in PBS) to 96-well immunoplate maxi soap, and overnight at room temperature It was left to fix to the plate. Thereafter, the mixture was blocked with 200 μl of 0.5% skim luc for 1 hour at room temperature, and washed 3 times with 200 μl of ELISA washing buffer. 100 μl of the cell culture supernatant diluted 1-fold to 3125-fold in 5-fold increments was added to this and allowed to react at room temperature for 1 hour. The clone used here was clone SD10-1, which was obtained by limiting dilution of the previous SD10 once. Thereafter, the plate was washed 5 times with 200 μl of ELISA wash buffer, and horseradish peroxidase-conjugated goat anti-chicken IgM antibody was diluted 2,000 times with PBS and added in 100 μl aliquots. After reacting at room temperature for 45 minutes, the plate was washed 5 times with an ELISA washing buffer, and then 100 μl of TMB + was added and reacted at room temperature for 4 minutes, and then the reaction was stopped with 100 μl of 1N sulfuric acid. Quantification was performed by measuring the absorbance at 450 nm using an mQuant Biomolecular Spectrometer.

(result)
(Examination of specificity by ELISA)
The reactivity of cysteinylated transthyretin partial peptide and non-cysteinylated transthyretin partial peptide was compared.

  As a result, most of the clones that reacted with the cysteinylated transthyretin partial peptide reacted in the same manner with the non-cysteinylated transthyretin partial peptide. A different thing was obtained.

If the selection is further advanced, a Cys peptide-specific antibody can be produced. Therefore, an antibody can be produced that specifically detects cysteinylated transthyretin but not unmodified transthyretin. For that purpose, it was found that it can be obtained by selecting a peptide that reacts with the antigen peptide (Cys-modified peptide) used in this example and does not react with an unmodified peptide of the same sequence.
[Example 14: Antibody production using Asp-modified TTR peptide]

  In Example 13, similar antibody production was carried out using Asp-modified TTR peptide as an antigen.

  The antigens used are as follows.

  This was conjugated to BSA using “ImjectImmunogen EDC Kit with BSA” (PIERCE), and an antibody was produced according to the procedure described in Example 13.

By examining the results as described in Example 13, Cysylated peptide-specific antibodies can be made. Therefore, an antibody can be produced that specifically detects cysteinylated transthyretin but not unmodified transthyretin.
[Example 15: Antibody production using HuCAL]

  In the above-described examples, transthyretin that has been revealed to be a marker substance for diabetes includes an unmodified one and a modified one at a cysteine residue that is present solely in the amino acid sequence. In addition to cysteinylation, sulfonylation, glutathioneation, homocysteinylation, and the like are known as modifications, but cysteinylated transthyretin was found as a marker for diabetes in the above examples.

  The purpose of this example was to create another system that specifically detects this cysteinylated transthyretin and does not detect unmodified transthyretin.

The peptide sequence (especially in the vicinity of the Cys residue) as an antigen is well conserved between humans and typical immunized animals, and since a large amount of transthyretin is contained in the serum, antibodies can be used even when immunized normally. Use Morphosys (Germany) MorphoSys (registered trademark), HuCAL (registered trademark), HuCAL GOLD (registered trademark), AutoPan (registered trademark), and AutoScreen (registered trademark) technology. It was.
The alignment of amino acid sequences near cysteine residues is shown below.
[Homosapiens] GPTGTGESKCPLMVK (SEQ ID NO: 25)
[Musmusculus] GPAGAGESKCPLMVK (SEQ ID NO: 26)
[Rattusnorvegicus] GPGGAGESKCPLMVK (SEQ ID NO: 27)
[GallusGallus] APLVSHGSVDSKCPLMVK (SEQ ID NO: 28)

(Materials and methods)
The antigens used are as follows. Antibody production was performed by requesting GeneFrontier (Tokyo, Chuo).

(antigen)
Name and origin of the protein from which the peptide is derived: transthyretin (human)
Length: 15 residues Peptide sequence: TGESKCPLMVK (15 residues from the first amino acid residue at the N-terminus of transthyretin; positions 5 to 15 in SEQ ID NO: 25)
Modification: Cys residue in peptide is linked to free cysteine by disulfide bond (modified with cysteinylation) or biotinylated.

  Therefore, the following four types of sequences were prepared.

1) Biotin-TGESK [C (Cys)] PLMVK-COOH
2) NH2-TGESK [C (Cys)] PLMVK (biotin) -COOH
3) Biotin-TGESKCPLMVK-COOH
4) TGESKCPLMVK-COOH
1) and 2) are antigenic peptides, and 3) and 4) are control peptides. In 1) and 2), the purity was 95% or more, and 5 mg each was used. The control used 1 mg with a purity of 90%.

(Antigen preparation method)
The antigen was prepared based on a conventional method.

  As a result of the above procedure, about 20 mg of each peptide (both Cys peptide and unmodified peptide) was produced. This was dissolved in MilliQ (distilled water), this solution was stored at −80 ° C., and dispensed to about 4 mg each.

(Antibody preparation method)
(Primary screening)
In principle, temporary screening was performed using AutoPan (registered trademark).

  The phage library used was HuCAL GOLD (registered trademark), which is a phage library in which a human Fab antibody was expressed on the surface. Phages with various Fabs exceeding 15 billion are included. Obtained from a human combinatorial antibody library (HuCAL; WO 97/08320; Knappik et al., J. Mol. Biol. 296: 55, 2000). This approach is a method of assembling one or more nucleic acid sequences encoding one or more (poly) peptide sequences suitable for the production of a (poly) peptide library, wherein the (poly) peptide sequences are amino acid consensus. A method comprising a sequence and comprising the following steps: (a) deriving from a collection of at least three homologous proteins one or more (poly) peptide sequences comprising at least one amino acid consensus sequence; Optionally modified to remove unfavorable interactions between amino acids within the (poly) peptide sequence or between the (poly) peptide or other (poly) peptide sequences, Identifying an amino acid within the peptide sequence (c) at least one component in each said (poly) peptide sequence; Identifying a specific sub-element; (d) back-translating each said (poly) peptide sequence into a corresponding nucleic acid coding sequence; (e) a region adjacent to the end of the sub-sequence encoding said sub-element or In the step of assembling cleavage sites in the region between the ends, each of the cleavage sites is (ea) unique within each of the nucleic acid coding sequences; (eb) the corresponding subsequence of all the nucleic acid coding sequences It has the characteristic that it is common to.

  About the library obtained in this way, an antigen is solid-phased and made to react with a HuCAL (trademark) library. Collection (elution) of the bound phage is performed using a unique CysDisplay. The number of phage expressing candidate antibodies is narrowed down to hundreds to thousands by the primary screening. Here, CycDisplay ™, which is a method based on the method described in JP-T-2003-505025, was used. In this technique, an antibody (Fab) expressed on a phage is expressed by breaking a disulfide bond, and the phage portion is eluted with a reducing agent. Therefore, many specific antibodies can be selected as compared with the elution method depending only on affinity.

  Alternatively, phagemid rescue, phage amplification and purification are performed as follows. HuCAL® Fab was amplified in 2 × TY medium (2 × TY-CG) containing 34 μg / ml chloramphenicol and 1% glucose. After infecting helper phage (VCSM13) at 37 ° C. and OD600 of about 0.5, the cells were centrifuged and resuspended in 2 × TY / 34 μg / ml chloramphenicol / 50 μg / ml kanamycin, and then the cells were treated at 30 ° C. Grow overnight. Phages were PEG precipitated from the supernatant (Ausubel et al., 1998), then resuspended in PBS / 20% glycerol and stored at −80 ° C. Phage growth between the two rounds of selection was performed as follows: the eluted phage was infected with log-phase TG1 cells and seeded on LB-agar supplemented with 1% glucose and 34 μg / ml chloramphenicol. did. After overnight incubation at 30 ° C., the colonies were scraped and adjusted to OD6000.5. Helper phage was added as described above.

  Wells of MaxiSorp ™ microtiter plates (Nunc) were coated with rat or human TIMP protein dissolved in PBS and diluted to 50 μg / ml (2 μg / well). After blocking with 5% non-fat dry milk in PBS, 1-5 × 1012 HuCAL® Fab phages purified as described above were added at 20 ° C. for 1 hour. After several washing steps, bound phage was released by pH elution with 100 mM triethylamine and then neutralized with 1M Tris.HCl, pH 7.0. Krebs et al. Immunol. Meth. 254: 67, 2001. A few rounds of selection were performed and phage was propagated between each round as described above.

(Secondary screening)
Next, secondary screening by ELISA is performed. This is done using AutoScreen (registered trademark). The phages selected in the primary screening are cloned, each Fab antibody is expressed, and an antigen-specific ELISA positive clone is selected.

    Fab-encoded inserts of selected HuCAL® Fab1 fragments were used to promote rapid expression of soluble Fabs using the expression vector pMORPH® × 7FS (Knappik et al., J. Mol. Biol. 296: 55, 2000). The selected HuCAL® Fab1 clone DNA preparation was digested with the appropriate restriction enzymes and then the Fab code insert was excised. Subcloning the purified insert into the restriction enzyme cleavage vector pMORPH® × 7 already having an scFv-insert creates a Fab expression vector. The Fab expressed in this vector has two C-terminal tags (FLAG ™ and Strep-tag II) for detection and purification.

  This is selected by ELISA.

(Production and purification of candidate antibodies)
Next, after confirming that the ELISA positive clone is a single clone by sequencing, a large amount of antibody is produced. The obtained Fab antibody is purified by affinity chromatography.

  The antibody produced was expressed in TG-1 cells of Fab fragment encoded by pMORPH® × 9FS in shake flask culture with 1 L of 2 × TY medium supplemented with 34 μg / ml chloramphenicol. . After induction with 0.5 mM IPTG, the cells were grown at 22 ° C. for 16 hours. Periplasmic extracts of cell sediments were prepared and Fab fragments were isolated by Strep-tactin® chromatography (IBA, Gottingen, Germany). Apparent molecular weight was determined by size exclusion chromatography (SEC) using calibration standards. The concentration was determined by UV spectrophotometry.

(2. Confirmation of specificity)
(Preparation of culture supernatant for ELISA for verification of streptavidin specificity)
The culture supernatant used for analyzing the further specificity by ELISA was a medium prepared as follows in order to remove serum-derived IgM and the like. Immunoglobulin was removed from chicken serum (Invitrogen) with 50% saturated ammonium sulfate as a precipitate, and the supernatant was dialyzed against PBS. Volume increase caused by dialysis was corrected by concentration with CentriPrep (Amicon) to obtain antibody-removed chicken serum. This was added to AIM-V serum-free medium (Invitrogen) at a concentration of 6%. Cells were added thereto at a concentration of about 106 / ml, cultured for 2 days, and the culture supernatant was removed and subjected to ELISA.

(ELISA for verification of streptavidin specificity)
Add 200 μl of 5 μg / ml streptavidin, ovalbumin, human IgG (Sigma) and 0.5% skim milk (Difco) (both dissolved in PBS) to 96-well immunoplate maxi soap, and overnight at room temperature It was left to fix to the plate. Thereafter, the mixture was blocked with 200 μl of 0.5% skim luc for 1 hour at room temperature, and washed 3 times with 200 μl of ELISA washing buffer. 100 μl of the cell culture supernatant diluted 1-fold to 3125-fold in 5-fold increments was added to this and allowed to react at room temperature for 1 hour. The clone used here was clone SD10-1, which was obtained by limiting dilution of the previous SD10 once. Thereafter, the plate was washed 5 times with 200 μl of ELISA wash buffer, and horseradish peroxidase-conjugated goat anti-chicken IgM antibody was diluted 2,000 times with PBS and added in 100 μl aliquots. After reacting at room temperature for 45 minutes, the plate was washed 5 times with an ELISA washing buffer, and then 100 μl of TMB + was added and reacted at room temperature for 4 minutes, and then the reaction was stopped with 100 μl of 1N sulfuric acid. Quantification was performed by measuring the absorbance at 450 nm using an mQuant Biomolecular Spectrometer.

  As evaluation criteria, five (5-50 μg each) antibodies that give an ELISA specific activity of 5 times or more with respect to the prescribed ELISA positive (control peptide) are selected, and appropriate antibodies selected from these are produced. . Fab antibody formatting or bivalent Fab miniantibody (with Myc-His tag) format may be selected.

(result)
(Examination of specificity by ELISA)
The reactivity of the cysteinylated transthyretin partial peptide and the non-cysteinylated transthyretin partial peptide is compared.

  As a result, most of the clones that react with the cysteinylated transthyretin partial peptide can be obtained that react differentially with those that react similarly with the non-cysteinylated transthyretin partial peptide.

  If the selection is further advanced, a Cys peptide-specific antibody can be produced. Therefore, an antibody can be produced that specifically detects cysteinylated transthyretin but not unmodified transthyretin. For that purpose, it can be obtained by selecting those that react with the antigenic peptide (Cys-modified peptide) used in this Example and do not react with an unmodified peptide of the same sequence.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

The present invention has utility in the pharmaceutical industry and the like. In particular, it has industrial applicability in the manufacture of diagnostic agents.

Claims (53)

Comprising selectively recognize means marker substance in a sample from a subject, a system for a subject to advance diagnostic or diagnostic whether diabetes, the marker substance, transthyretin and trans A system comprising at least one of thyretin derivatives, wherein the means for recognizing has the ability to distinguish between transthyretin and transthyretin derivatives. The system further comprises an agent that interacts specifically with the marker substance, said agent is a nucleic acid molecule, a protein, a polypeptide, a lipid, a sugar chain is selected from the group consisting of organic molecules and a composite molecule thereof The system according to claim 1. The system of claim 2 , wherein the factor is an antibody. The system of claim 2 , wherein the agent is labeled or labelable. The means include mass spectrometer, nuclear magnetic resonance measuring apparatus, X-ray analyzer, SPR, chromatography, immunological means, biochemical means, electrophoresis equipment, chemical analysis equipment, fluorescence two-dimensional differential electrophoresis The system of claim 1, wherein the system is selected from the group consisting of: isotope labeling, tandem affinity purification, physical means, laser microdissection, and combinations thereof. The system of claim 1, further comprising a standard for the marker substance. The system of claim 1, further comprising means for purifying the sample. The system of claim 1, wherein the subject comprises a mammal. The system of claim 1, wherein the subject comprises a rodent. The system of claim 1, wherein the subject comprises a human. The hand stage is capable of a quantitative determination of the marker substance, according to claim 1 system. The system according to claim 1, further comprising a quantification unit for quantifying the marker substance. 13. The system according to claim 12, wherein the quantification unit includes a determination unit that compares a standard curve with a measurement result to determine whether the marker substance is within a normal value range. The system according to claim 13, wherein the determination unit is a computer. A composition for pre-diagnosing or diagnosing whether a subject comprising an agent that specifically interacts with a marker substance is diabetic, wherein the marker substance comprises at least one of transthyretin and a transthyretin derivative. A composition comprising a species, wherein the factor is an antibody having the ability to distinguish between transthyretin and a transthyretin derivative.   The transthyretin derivatives include S-cysteinyl transthyretin, glutathioneated transthyretin, SS bond-forming transthyretin, oxidized transthyretin, formylated transthyretin, acetylated transthyretin, phosphorylated 2. The system of claim 1, wherein the system is selected from the group consisting of transthyretin, glycosylated transthyretin, myristylated transthyretin and complex derivatives thereof.   The system of claim 1, wherein the transthyretin derivative is S-cysteinyl transthyretin. The transthyretin derivatives are S- cysteinyl transthyretin, the means for recognizing is an antibody, according to claim 1 system. The hand stage reacts differentially with respect to the transthyretin and transthyretin derivative, according to claim 1 system. The hand stage does not substantially react only with either one of transthyretin or transthyretin derivative, according to claim 1 system.   The at least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of developing diabetes or a high risk of developing it in the future. The system described in.   The at least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of the degree of progress of diabetes or the risk of future onset. The system described.   The transthyretin is encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or shown in SEQ ID NO: 2 or SEQ ID NO: 4. The system according to claim 1, which has an amino acid sequence in which the first 20 amino acids of the amino acid sequence to be removed are excised or has a modified sequence thereof.   The transthyretin derivative corresponds to the cysteine at position 30 or the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, respectively. The cysteine of the cysteine is cysteinylated, or the cysteine at position 30 or the corresponding cysteine is cysteinylated in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, respectively. The system according to claim 1, wherein the first 20 amino acids of the derivative are excised. The hand stage has the ability to distinguish between the monomer and tetramer of transthyretin, according to claim 1 system. The hand stage has the ability to distinguish between transthyretin and S- cysteinyl transthyretin system of claim 1. The hand stage includes an antibody having the ability to distinguish between transthyretin and S- cysteinyl transthyretin system of claim 1. The hand stage recognizes and transthyretin and S- cysteinyl transthyretin, and said system further comprises means for identifying and transthyretin and S- cysteinyl transthyretin claim The system according to 1. The hand stage recognizes and transthyretin and S- cysteinyl transthyretin, means for identifying a molecular weight of the system transformer molecular weight of transthyretin and S- cysteinyl transthyretin, and trans Sai The system of claim 1, further comprising means for measuring a relative ratio of retin and S-cysteinyl transthyretin. A diagnostic agent for pre-diagnosing or diagnosing whether a subject containing an agent that specifically interacts with a marker substance is diabetic, wherein the marker substance comprises at least one of transthyretin and a transthyretin derivative. A diagnostic agent comprising a species, wherein the factor is an antibody having the ability to distinguish between transthyretin and a transthyretin derivative. A method for measuring a marker substance in a sample derived from a subject in order to detect or detect in advance whether or not the subject is diabetic, or to support the pre-diagnosis or diagnosis,
The method
A) a step of measuring a marker substance in a sample derived from the subject and discriminating between transthyretin and a transthyretin derivative; and B) whether the subject is diabetic or possible from the measurement result. The process of determining whether
It encompasses,
The marker substance includes at least one of transthyretin and transthyretin derivatives.
Method. 32. At least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of developing diabetes or a high risk of developing it in the future. The method described in 1. The at least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of the degree of progress of diabetes or the risk of future onset. The method described. Of selectively recognizing means marker substance in a sample from a subject, in the manufacture of a medicament for a subject to advance diagnostic or diagnostic whether diabetes, to the use, the marker substance, trans Use comprising at least one of thyretin and transthyretin derivatives, wherein the means for recognizing has the ability to distinguish between transthyretin and transthyretin derivatives. 35. At least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of developing diabetes or an increased risk of developing in the future. Use as described in. 35. At least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of the degree of development of diabetes or the risk of future onset. The method described. Of selectively recognizing means marker substance in a sample from a subject to the use for which the subject pre-diagnostic or diagnostic whether diabetes, the marker substance, transthyretin and trans Sai Use comprising at least one of the retin derivatives, wherein the means for recognizing has the ability to distinguish between transthyretin and transthyretin derivatives. 38. At least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of developing diabetes or a high risk of developing it in the future. Use as described in. 38. At least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of the degree of development of diabetes or the risk of future onset. Use of description.   32. The method of claim 31, wherein the sample is blood.   The sample is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, the marker substance in the body fluid is captured on the carrier, and the sample substance in the body fluid is based on the amount of the captured marker substance. 32. The method according to claim 31, wherein the concentration of the marker substance is calculated.   The method according to claim 41, wherein the carrier has a flat portion, and the substance having affinity for the marker substance is immobilized on a part of the flat portion.   43. The method according to claim 41 or 42, wherein the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody. A method for evaluating a substance characterized by evaluating an improvement effect of diabetes or a reduction effect of future risk of developing a test substance,
Step to feed a test substance to a non-human animal or future risk has developed a) diabetes is highly non-human animal; and b) a reference value of at least one of the concentration of marker substances in the non-human animal in a body fluid Compared to the above, the step of evaluating the effect of improving diabetes or reducing the future risk of developing the test substance, wherein the marker substance comprises at least one of transthyretin and a transthyretin derivative, Process,
Including the method. 45. At least one phenomenon selected from the group consisting of a decrease in the transthyretin and an increase in the transthyretin derivative is an indicator of developing diabetes or a high risk of developing it in the future. The method described in 1. 45. The method according to claim 44, wherein at least one phenomenon selected from the group consisting of a decrease in transthyretin and an increase in the transthyretin derivative is an indicator of the degree of progress of diabetes or the risk of future onset. The method described.   The reference value is obtained when a non-human animal that has developed diabetes or a non-human animal that has a high future risk of ingesting a known substance that does not have an effect of improving diabetes or reducing the risk of developing future disease. 45. The method according to claim 44, wherein the concentration of the marker substance is in the body fluid of the non-human animal.   48. The method according to any one of claims 44 to 47, wherein the body fluid is blood.   49. The method according to any one of claims 44 to 48, wherein the test substance is a food material.   The bodily fluid or bodily fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, and the marker substance in the bodily fluid is captured on the carrier, and based on the amount of the captured marker substance The method according to any one of claims 44 to 49, wherein the concentration of the marker substance in a body fluid is calculated.   51. The method according to claim 50, wherein the carrier has a flat part, and the substance having affinity for the marker substance is immobilized on a part of the flat part.   52. The method according to claim 50 or 51, wherein the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody.   A substance screening method comprising: evaluating a test substance by the substance evaluation method according to any one of claims 44 to 52; and screening a substance having an effect of improving diabetes or a risk of reducing future risk of onset. .