JPH10332693A - Method for pre-treating specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for pre-treating specimen for measuring the latter-period product (AGE) of Maillard reaction which is watched as a novel clinical marker of diabetes mellitus or a complication of diabetes mellitus in the field of clinical inspection. SOLUTION: In a method for pre-treating specimen, a specimen for measuring the latter-period product (AGE) of Maillard reaction is heated to 50-100 deg.C under the presence of an anionic surface active agent. In addition, the AGF is detected by making an antibody which specifically reacts to the AGE to act on the heated specimen. The anionic surface active agent is sodium dodecyl sulfate and the anti-AGE antibody is a carboxymethylation antibody or an anticarboxymethylized peptide antibody. The AGE is a carboxymethylized protein or a carboxymethylation peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for pretreating an object for AGE measurement. More specifically, AGE in a biological component that can be a marker for diabetes or diabetic complications
The present invention relates to a pretreatment method of a subject for detecting or measuring the concentration.

[0002]

2. Description of the Related Art It is known that proteins in blood are non-enzymatically reacted with glucose and saccharified to form saccharified proteins. The saccharification reaction is called a Maillard reaction,
It is divided into early stage and late stage reactions. The reaction in the first stage is said to be until the amino group at the side chain or the N-terminal amino group of the protein reacts with the carbonyl group of the sugar to generate an Amadori rearrangement compound via a Schiff base. Examples of the first-stage reaction product include hemoglobin A1C
It is a well-known fact that glycated albumin and glycated albumin are known and used as clinical markers of diabetes.

Further, after the above-mentioned first-stage reaction, the Amadori rearrangement compound formed on the amino group at the side chain or the N-terminal amino group of the protein undergoes a complex reaction such as oxidation, dehydration, condensation, etc.
It is converted to GE to form AGE which is a late-stage reaction product.
The AGE has been reported to be involved in the development of vascular complications (Monnier, VM, et. Al., New England Jou
rnal of Medicine, vol. 314, p403, 1986), and has been noted as being involved in the development and progression of complications in diabetic patients. It has also been reported that AGE has a biological activity involved in the onset and progression of diabetic complications (Morizaki et al., The Newest Medicine, Vol. 49, p. 248, 1
994). Furthermore, it has been reported that AGE is present in lens proteins, skin collagen, etc. of the elderly and diabetic patients (Dunn, JA, et. Al., Biochemistry, vol.
30, p1205, 1991), AGE holds promise as a novel marker for diabetes or diabetic complications.

AGE is known to be an aggregate of a plurality of compounds. At present, the structure of AGE is a carboxymethylated protein (hereinafter sometimes abbreviated as "CM-modified protein"). , Pyralin, pentosidine, crossrin A & B, pyropyridine, X1 and the like have been proposed, and their qualitative and quantitative methods are detection using a specific antibody and hydrolysis of AGE to detect an AGE-converted amino acid by liquid chromatography. And AGE are hydrolyzed and analyzed by gas chromatography / mass spectrometry.
A method for detecting an amino acid converted into M may be used. However, the method of indirectly measuring AGE has a problem that the operation is complicated and the sensitivity is low. Therefore, a measurement method using a specific antibody that is easy to measure and has high sensitivity is often used.

However, when the specific antibody is used, AGEs prepared in vitro or AGEs in tissues
Can be detected, but it is difficult to detect AGE in body fluids by a method other than the dot blotting method. Therefore, in order to solve this problem, a pretreatment method of serum using a protease was proposed (39th Annual Meeting of the Japanese Diabetes Association, p248, 1996).
The pretreatment method has a problem that the operation is complicated.

On the other hand, the detection of AGE in a body fluid using a specific antibody by the dot blotting method can be performed without performing a pretreatment (Journal of the Japan Society of Nephrology, vol. 39, p.
243, 1997), but there is a problem that the operation is more complicated than the ELISA method.

[0007]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for detecting AGE in a subject, particularly a subject derived from a body fluid, using a specific antibody by a method other than the dot blotting method. An object of the present invention is to provide a simple preprocessing method that enables AGE to be detected.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, AGEs in a subject, particularly in a body fluid-derived subject, were analyzed for specific antibodies by a method other than the dot blotting method. In the case of detection using AGE, it has been found that AGE can be detected only by performing a simple treatment of heating the specimen in the presence of an anionic surfactant, and the present invention has been completed.

That is, the present invention provides a pretreatment of a subject, which comprises subjecting a subject for measuring the Maillard reaction late-stage product (AGE) to heat treatment at 50 to 100 ° C. in the presence of an anionic surfactant. Is the way. Another aspect of the present invention is a method for measuring AGE, wherein an anti-Maillard reaction late product antibody (anti-AGE antibody) is allowed to act on a sample pretreated by the above method, and AGE is detected by an immunological reaction. Is the way.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail, but the substance, production method, and operation method used in the present invention are not limited to the following.

The analyte used in the present invention is particularly a solution in which any component of protein, peptide and free amino acid (hereinafter, these are collectively referred to as "protein etc.") is dissolved or suspended. Not limited. Here, the peptide may be an oligopeptide or a polypeptide, for example, a degradation product of a protein. The above proteins and the like are generally derived from biological components. Examples of the biological components include blood, urine, lymph, amniotic fluid, peritoneal fluid, body fluids such as saliva, skin collagen, extracellular matrices such as fibronectin, and the like. Examples include lens proteins, tissues such as arteries and kidneys. The concentration of the protein or the like contained in the subject largely depends on the kind of the protein or the like contained therein (the order is several ng / ml when the protein or the like is free amino acid, and is several ng / ml when the protein or the like is a protein). On the order of several to several tens of weight percent), generally from about 0.001 ppm to about 30 weight percent.

When the biological component is a body fluid, since the protein or the like is present in the form of a solution or suspension from the beginning, it can be used as a subject as it is. For example,
Plasma usually contains about 7% by weight of protein, and urine usually contains protein at a concentration of about 10 to 15 mg / ml. In addition, the body fluid may be subjected to processing such as component adjustment or diluted with a buffer solution or the like to obtain a subject. For example, when blood is used as a subject, since blood usually contains 22% by weight of protein, red blood cells are lysed with distilled water or a buffer having a low salt concentration, and then diluted with a buffer. It is preferably used as a subject. When the biological component is a tissue or a cell other than a body fluid, it is preferable to dilute it with a buffer solution or the like to adjust the concentration of the protein or the like to be within the above range, and to use it as a subject. In the present invention, it is preferable to use, as the subject, a solution containing a protein derived from a bodily fluid, which is frequently used as a subject in a clinical test.

In the method of measuring AGE of the present invention, since an immunological reaction is used, AGE can be measured even if various proteins and the like are mixed in a subject.
The AGE of the specific protein or the like may be measured by separating and purifying the specific protein or the like in advance. In particular, the ratio of AGEs and non-AGEs of specific proteins etc. may have clinical significance,
In such a case, it is preferable to separate and purify in advance and use a solution containing the specific protein or the like as an analyte. Examples of such specific proteins include hemoglobin that has a long life and a high blood concentration, β2 microglobulin (β2M), which is a major component of deposited amyloid, and low-density lipoprotein (LDL) closely related to the onset of arteriosclerosis. )
And high-density lipoprotein (HDL).

The anionic surfactant used in the present invention may be any surfactant that dissolves in water and dissociates into ions, and forms an anion with an atomic group exhibiting surface activity, such as acyl sarcosine and alkyl sulfate. Sodium, alkylbenzene sulfonate, etc.,
In particular, when detecting AGE in a subject derived from a body fluid using a specific antibody by a method other than the dot blotting method,
Sodium dodecyl sulfate (SDS) having a large hydrophilic-hydrophobic balance (HLB) is particularly preferably used.

The concentration of the anionic surfactant used in the pretreatment of the subject is not particularly limited, but may be 0.01 to 10% by weight, more preferably 0.03 to 5% by weight, based on the weight of the subject. It is preferable to add so that

The specimen to which the anionic surfactant has been added is subjected to a heat treatment at a temperature in the range of 50 to 100 ° C. If the temperature is lower than this, the pretreatment effect is weak (see FIG. 3), and if the temperature is higher than this, the protein in the specimen tends to aggregate.
The heat treatment time may be appropriately determined according to the heat treatment temperature, but is preferably 15 minutes or more, more preferably 60 minutes or more, in order to stably obtain the effect of the pretreatment.

AGE in the subject is detected by an immunological reaction by allowing an anti-AGE antibody to act on the subject pretreated by the above method. At this time, the subject that has been subjected to the pretreatment may be subjected to an immunological reaction (antigen-antibody reaction) by directly acting the anti-AGE antibody, but in order to perform the antigen-antibody reaction favorably, after the pretreatment, It is preferable that the concentration of the anionic surfactant in the subject is adjusted to 0.1 wt% or less before the anti-AGE antibody is allowed to act. 0.1 wt% anionic surfactant concentration during antigen-antibody reaction
%, For example, a method of diluting the pretreated sample with a buffer solution or water containing no anionic surfactant, or ultrafiltration, gel filtration, ion exchange chromatography from the pretreated sample. For example, a method of removing an anionic surfactant by lithography or the like can be used.

The anti-AGE antibody used in the present invention is not particularly limited as long as it is a specific antibody that recognizes AGE. As such an anti-AGE antibody, an antiserum obtained by immunizing a host animal such as rabbit, goat, mouse, guinea pig or the like with AGE prepared by a known method as an immunogen (antigen), or the serum is conventionally used. Examples thereof include polyclonal antibodies obtained by purification by known methods such as salting out, gel filtration, ion exchange chromatography, affinity chromatography, and electrophoresis. For example, AGE (a model reaction of the Maillard reaction) prepared by incubating glucose and protein in vitro at 37 ° C. for 60 days is purified by dialysis, and an antiserum against AGE is obtained about 6 weeks after immunization of a rabbit.

When detecting a specific AGE structure in a subject, in addition to the antiserum and polyclonal antibody prepared as described above, mammalian spleen cells sensitized with the specific AGE structure, Monoclonal antibodies prepared from hybridomas obtained by fusing antibody-producing cells such as lymph node cells with myeloma cells are used as such or salting-out methods, gel filtration methods, ion-exchange chromatography, affinity chromatography, electrophoresis, which are conventionally known methods. It can be purified by electrophoresis or the like and used as an anti-AGE antibody.

Specific AGEs include, for example, the above-mentioned CM-protein, CM-peptide, pyralin, pentosidine, crossrin A & B, pyropyridine, X1 and the like. Among them, CM-protein and CM-peptide are the main components of AGE. (Ike
da, K., et.al., Biochemistry, vol. 35, p8075, 199
6) When detecting a specific AGE in a subject, it is preferable to measure a CM-protein and a CM-peptide.
As described above, an anti-CM protein antibody or an anti-CM peptide antibody (hereinafter sometimes collectively referred to as an anti-CM antibody) for measuring a CM protein or a CM peptide is used in the presence of oxygen as described above. In addition to using an AGE prepared by incubating a reducing sugar and a protein at 37 ° C. for about 60 days as an immunogen, it is also possible to use a CM protein or a peptide synthesized using an organic synthetic technique as an immunogen. can get.

The method of carboxymethylating (CM-forming) the amino group of the amino acid constituting the protein or peptide, that is, the method of substituting the hydrogen of each amino group with a —CH ₂ —COOH group, is a known method. Used without restrictions.
For example, as in the reductive alkylation method described in "New Chemistry Experiment Course 1, Protein 4" (edited by The Biochemical Society of Japan, pp. 13-16, Tokyo Kagaku Dojin, published on March 20, 1991), CHO- C
An aldehyde compound represented by OOH and a protein or peptide are dissolved in an aqueous solution such as a borate buffer or a phosphate buffer, and the solution is adjusted to pH 8 to 10 in the presence of a hydride reducing agent such as sodium borohydride or sodium cyanoborohydride. The reaction may be carried out. If the pH is higher than this, proteins and the like may be denatured, and if the pH is lower than this, the hydride reducing agent becomes unstable. In order to allow the reaction to proceed specifically and quantitatively, the reaction is preferably performed at a temperature of 0 to 10 ° C.

Antibodies to specific AGEs including anti-CM antibodies and anti-AGE antibodies may be antibody molecules themselves, or active fragments of antibodies such as F (ab ') 2 obtained by enzymatic treatment of these antibodies (antibody of antibody). (A part including an antigen recognition site) may be used.

In the present invention, as a method for measuring AGE in a subject subjected to the above-mentioned pretreatment, an antigen-antibody reaction between an anti-AGE antibody such as an anti-CM antibody and an AGE such as a CM protein or a CM peptide is used. However, the embodiment is not particularly limited as long as the method can measure AGE in the subject. That is, anti-AGE antibodies, antibodies to biological components such as β2 microglobulin (β2M) and hemoglobin, and AGE were appropriately supported on an insoluble carrier so as to correspond to the non-competitive method, the competitive method, and the sandwich method described below. It can take a form or the like. The AGE and / or anti-A in the test sample may be determined according to the measurement method using the reagent having such an embodiment.
AGE can be measured by detecting an antigen-antibody reaction caused by contact with a GE antibody. Such an antigen-antibody reaction can be detected by using agglutination of an insoluble carrier or the like when the immunological measurement method is a so-called immunoagglutination method. In the case of a measurement method, it can be detected as a change in a physical quantity such as colorimetry, luminescence, or fluorescence.

To illustrate a specific embodiment of the method for measuring AGE in a specimen treated by the pretreatment method of the present invention, examples of the qualitative measurement method include a latex agglutination method and a microtiter method. Examples of the method include a radioimmunoassay, an enzyme immunoassay, a fluorescence immunoassay, a chemiluminescence immunoassay, and a latex quantification method.

The shape of the anti-AGE antibody or the antibody against the biological component, or the shape of the insoluble carrier carrying AGE in the subject may be appropriately selected depending on the purpose of use, such as beads, test plates, and the like. Examples include a sphere, a disk, a tube, and a filter. In addition, as the material thereof, those used as ordinary carriers for immunoassays, for example, synthesis of glass, polysaccharides or derivatives thereof, silica gel, porous ceramics, metal oxides, red blood cells, polypropylene, polystyrene, polyacrylamide, etc. Resins and those obtained by introducing a reactive functional group such as a sulfone group and an amino group into the resin by a known method are exemplified.

The method for immobilizing an anti-AGE antibody on an insoluble carrier or an antibody against a biological component, or an antigen in a subject is as follows.
Known methods such as a physical adsorption method, a covalent bonding method, an ionic bonding method, and a crosslinking method can be used without any limitation.

AGE in test sample in labeled immunoassay
The basic operation of the measurement can be performed by a conventional assay method, for example, an enzyme immunoassay (EIA) method such as a radioimmunoassay (RIA) method, an ELISA method, a Western blotting method, a dot blotting method, or the like. The operations, procedures, and the like in each of these assay methods are not particularly different from those generally employed, and can be in accordance with known non-competitive methods, competitive methods, sandwich methods, and the like. In non-competitive law,
After the AGE or anti-AGE antibody in the subject is carried on the insoluble carrier, the anti-AGE antibody or AGE may be contacted. In the competitive method, for example, after an artificially prepared AGE is supported on an insoluble carrier, the
What is necessary is just to contact the anti-AGE antibody made to react with. In the sandwich method, after an anti-AGE antibody or an antibody against a biological component such as albumin, β2M or hemoglobin is carried on an insoluble carrier, the AGE in the subject is contacted, and then an antibody against the biological component or an anti-AGE antibody is contacted. It should be done. By these measurement methods, the AGE conversion rate of a protein or peptide or the amount of AGE can be measured.

The labeling agent used in the labeling immunoassay includes:
Examples thereof include radioactive substances such as radioactive iodine and radioactive carbon, fluorescent substances such as fluorescein isothiocyanate and tetramethylrhodamine, and enzymes such as alkaline phosphatase and peroxidase. The antigen-antibody reaction product obtained by such a method is detected using radioactivity, colorimetry, fluorescence, luminescence and the like.

For example, an anti-AGE antibody or A in a subject
GE is supported on an insoluble carrier at a rate of 0.01 to 1000 μg / cm ² , and 0.001 to 1000 μg of AGE or anti-AGE antibody in the subject is brought into contact with the GE for measurement.
When an antibody against a biological component is carried on an insoluble carrier, as described above, after contacting AGE in the subject, an anti-AGE antibody is further contacted. It is preferable to use an antibody that is not carried on an insoluble carrier and that is labeled with a labeling agent.

The basic operation for measuring AGE in a subject using an immunoagglutination reagent can be performed according to a conventional assay method, for example, a hemagglutination method, a passive agglutination method, an immunoassay method, an immunoturbidimetry method, or the like. The operations, procedures, and the like in each of these assay methods can be based on those generally employed. For example, 0.001 per gram of the particulate insoluble carrier.
0.001 to 15% by weight of particles (hereinafter, abbreviated as sensitized particles) carrying １００100 mg of anti-CM antibody by the above method.
What is necessary is just to disperse in an aqueous medium so that it may be used as an active ingredient of an immunoreagent. The particle size of the insoluble carrier that carries the antibody has an average particle size of 0.05 to 10 μm from the viewpoint of ease of aggregation after the antigen-antibody reaction and easiness of discrimination of aggregation.
It is preferred to use an insoluble carrier of The sensitized particles prepared by such a method may be brought into contact with an antigen in a sample, and the degree of aggregation of the sensitized particles may be measured. Conventional methods such as visual observation and optical measurement can be used without limitation for the degree of aggregation of particles.

As shown in the examples described below, when a subject consisting of blood from a patient with diabetes or diabetic complications was pretreated by the pretreatment method of the present invention, when an untreated subject was used, Is not detected, the competition E
It became possible to detect by the LISA method. In addition, since the pretreatment method was simpler than the conventional method, the present invention seems to be particularly useful in the field of clinical examination.

[0032]

As described above, by subjecting a subject to heat treatment in the presence of an anionic surfactant, biological components, especially AGEs derived from body fluids, which could not be detected by other than dot blotting, can be detected. . This means that AGE, which is likely to be a novel marker for diabetes, diabetic complications or dialysis amyloidosis, can be collected easily and in a short time with blood, serum, plasma, urine, etc., which can be collected without causing much pain to the patient. It means that it can be measured directly, and its industrial significance is great.

[0033]

EXAMPLES The present invention is described below in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 and Comparative Example 1 (1) Preparation of AGE-converted human serum albumin (HSA) In order to convert HSA into AGE, the pH was adjusted to 7.4.
In 0 mM phosphate buffer, H was adjusted to 60 mg / ml.
SA (fraction V, manufactured by Sigma) and 1.67M
Glucose (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved so that Next, after sterile ultrafiltration with a 0.2 μm filter, the mixture was incubated at 37 ° C. for 90 days.

After 90 days, the above solution was washed with 0.15 M NaC.
20 mM phosphate buffer (pH 7.4) containing
(Sometimes referred to as 20 mM PBS) at 4 ° C. for 60 hours. During this time, the 20 mM PBS was changed four times every 12 hours.

(2) Preparation of antibody against AGE-conjugated HSA The above-mentioned AGE-conjugated HSA was added to rabbits weighing 2 kg or more.
Immunization was performed as follows using SA as an antigen.

In 0.5 ml of the antigen solution prepared to be 2 mg / ml, complete Freund's adjuvant was added to 0.5 ml.
The addition of 5 ml was injected into the rabbit ear vein. Thereafter, every two weeks, 0.25 ml of the 2 mg / ml antigen and 0.25 ml of Freund's incomplete adjuvant were boosted. During this period, in order to confirm whether antibodies against AGE-conjugated HSA were produced,
The rabbits were partially bled from the outer ear vein. Six weeks later, A
The production of an antibody against GE HSA was confirmed by enzyme-linked immunosorbent assay (ELISA), and whole blood was collected.

(3) Preparation of affinity purification column 5 ml of Affigel 15 (manufactured by BIO-RAD) was added to 15 ml of 1
After washing with 0 mM acetate buffer (pH 4.5),
5 mg / ml HSA solution in M PBS
1.6 ml was added, followed by gentle stirring at room temperature for 1 hour. Next, unreacted HSA was removed by filtration, 30 ml of 1M ethanolamine was added, and the mixture was gently stirred at room temperature to block unreacted N-hydroxysuccinimide ester. The support on which the HSA is immobilized is packed in a column,
The column was washed with ion-exchanged water until the absorbance at 280 nm became zero. Further, the column was equilibrated with 20 mM PBS.

Similarly, a column on which the above-mentioned AGE-modified HSA was immobilized was prepared.

(4) Antibody to AGE-conjugated HSA (anti-A
Affinity purification of GE-HSA antibody) The prepared anti-AGE-HSA antibody diluted with 20 mM PBS so as to have a concentration of 1 mg / ml was applied to a column on which the HSA was immobilized so as to have a concentration of about 100 mg. Then, 2 until the absorbance at 280 nm becomes zero.
0 mM PBS was flowed at a flow rate of 0.5 ml / min. Antibodies that did not bind to the column were recovered as anti-AGE-HSA antibodies. When the absorbance at 280 nm becomes 0, a 20 mM PBS to 0.1 M glycine buffer (pH
3.0), elute unnecessary proteins bound to the column, equilibrate the column with 20 mM PBS,
The recovered antibody was applied to the column again, and the antibody not bound to the column was recovered. This operation was repeated once more.

Next, the recovered antibody was applied to the column on which the above-mentioned AGE-modified HSA was immobilized, and 20 mM PBS was added.
At a flow rate of 0.5 ml / min. The antibody bound to the column by this operation was defined as an anti-AGE-HSA antibody by 20 m
M PBS from 0.1 M glycine buffer (pH 3.
In place of 0), the antibody bound to the column was eluted and collected. Immediately after the collection, the pH was returned from 3.0 to 7.4, followed by dialysis with 20 mM PBS. The anti-AGE-HSA
The antibody was used as an antibody for biotin labeling.

(5) Biotin Labeling of Anti-AGE-HSA Antibody Biotin labeling of the purified antibody was performed using a protein biotinylation system (GIBCO).

1.5 μm of the purified anti-AGE-HSA antibody
A sodium carbonate buffer (pH 9.0) was added to a concentration of 0.05 M to a solution diluted or concentrated with 20 mM PBS to give a g / ml. Next, 50 mg / ml prepared according to the instructions was added to 6.7 ml of the antibody solution.
Was added, and the mixture was gently stirred at room temperature for 1 hour, and ammonium chloride was added to a concentration of 0.11 M to terminate the reaction. Thereafter, the antibody solution was desalted using a column attached to this kit. Further, when the number of moles of biotin introduced by avidin / HABA included in the kit was calculated, 14 moles of biotin was bound to 1 mole of the anti-AGE-HSA antibody.

(6) Antigen specificity of anti-AGE-HSA antibody Antigen specificity of anti-AGE-HSA antibody was determined by competitive ELISA.
Confirmed.

20 mM PB so that the concentration becomes 1 μg / ml.
The prepared AGE-conjugated HSA was added to the biotin-labeled anti-AGE-HSA antibody diluted with
It was added so as to be 00 μg / ml. 37
The solution was left at ℃ for 1 hour and used as an antibody solution inhibited by AGE-conjugated HSA.

In the same manner as in the preparation of AGE-modified HSA, AGE-modified Hb prepared from hemoglobin (manufactured by Sigma: fraction V, hereinafter sometimes abbreviated as Hb) was prepared. 1μ
To the g / ml anti-AGE-HSA antibody solution, the above-mentioned AGE-conjugated Hb was added at a concentration of 0.1, 1, 10, and 100 μg / ml, respectively. This solution was left at 37 ° C. for 1 hour and used as an antibody solution inhibited by AGE-conjugated Hb.

In performing the competitive ELISA, the prepared AGE-formed HSA was added to 20 mM so as to have a concentration of 1 μg / ml.
Diluted with carbonate buffer (pH 9.6). Subsequently, the diluted AGE-ized HSA solution was added to a 96-well immunoplate (NUN
C) (100 μl per well) and 37 ° C.
For 1 hour, and the AGE-ized HSA was fixed on an immunoplate. One hour later, the AGE-bound HSA not bound to the immunoplate was removed, and the wells were washed three times with 20 mM PBS, and then 1 wt% of bovine serum albumin (hereinafter referred to as B).
20 mM carbonate buffer (p.
H9.6) was applied in an amount of 100 μl per well, and 3
The mixture was allowed to stand at 7 ° C. for 1 hour to block a portion where AGE-bound HSA was not bound. One hour later, the BSA solution was removed, and the plate was washed three times with 20 mM PBS containing 0.05 wt% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.).
Antibody solution inhibited by GE HSA, or A at the above concentration
The antibody solution inhibited by GEylated Hb was added at 10 / well.
0 μl was applied and left at 37 ° C. for 1 hour. afterwards,
3% in 20 mM PBS containing 0.05 wt% Tween 20
After washing twice, a mixed solution of biotin and avidin labeled with alkaline phosphatase (manufactured by Funakoshi: vectastain ABC kit) prepared according to the instruction manual was applied in an amount of 100 μl per well and left at 37 ° C. for 1 hour. Furthermore, 20 mM PBS containing 0.05 wt% Tween 20
3 times with alkaline phosphatase substrate kit (BIO
-RAD) (100 μl per well). After standing at room temperature for 5 minutes, a 0.4 M sodium hydroxide solution was added at 100 μl per well to stop the reaction of alkaline phosphatase, and the absorbance at 405 nm was measured. Figure 1 shows the results
Shown in From these results, the prepared anti-AGE-HSA antibody can be used not only for AGE-modified HSA but also for AGE-modified Hb.
The inhibition of the reaction between E-conjugated HSA and the anti-AGE-HSA antibody indicated that the compound also showed reactivity with AGE-conjugated Hb.

(7) Optimal Surfactant Concentration During Pretreatment Blood from a diabetic patient was treated with sodium dodecyl sulfate (SD
S) Pretreatment was performed in the presence. Further, the optimum surfactant concentration at the time of the pretreatment was confirmed by a competitive ELISA.

The blood (protein concentration: 200 mg /
ml) was lysed with 30 μl of distilled water containing 0.67 wt% SDS. The hemolyzed sample is placed at 80 ° C.
The sample incubated for 1 hour was used as a subject.
The SDS concentration at this time was 0.0
It was 12 wt%.

20 mM PB so that the concentration becomes 5 μg / ml.
The samples lysed with the biotin-labeled anti-AGE-HSA antibody diluted with
It was added so as to be 100 or 1000 μg / ml. This solution was left at 37 ° C. for 1 hour, and used as an antibody solution inhibited by blood AGE.

The competitive ELISA was performed according to the method described above. That is, AGE-bound HSA diluted with a 20 mM carbonate buffer (pH 9.6) was immobilized on a 96-well immunoplate at 50 ng / well. Thereafter, AGE-bound HSA not bound to the immunoplate was removed, and 20 m
After washing the wells three times with MPBS, the wells containing 1 wt% of bovine serum albumin (hereinafter sometimes abbreviated as BSA) were added.
0 mM carbonate buffer (pH 9.6)
0 μl was applied and left at 37 ° C. for 1 hour.
The portion where SA was not bound was blocked. One hour later, the BSA solution was removed, and 0.05% by weight of Tween 20 was added.
After washing three times with 20 mM PBS containing the above, the antibody solution inhibited by the analyte at the above concentration was added to 100 μl per well.
1 was applied and left at 37 ° C. for 1 hour. Then, 0.
After washing three times with 20 mM PBS containing 05 wt% Tween 20, a mixed solution of biotin and avidin labeled with alkaline phosphatase (Funakosi: vectastain ABC kit) prepared according to the instruction manual was added for 10 times per well.
0 μl was applied and left at 37 ° C. for 1 hour. Furthermore,
3% in 20 mM PBS containing 0.05 wt% Tween 20
After washing twice, a substrate solution prepared according to the specifications using a substrate for alkaline phosphatase (manufactured by DAKO: AMPAK) was applied in an amount of 100 μl per well. After standing at room temperature for 20 minutes, add the reaction stop solution provided in the kit to 2 wells per well.
The reaction of alkaline phosphatase was stopped by adding 5 μl, and the absorbance at 492 nm was measured. The results are shown in FIG.

Similarly, 10 μl of the above blood was added to 0.0
4, 0.4, 0.67, and 6.7 wt% (0.03, 0.3, 0.
Hemolyzed with distilled water containing SDS (corresponding to 5 and 5 wt%) (Example 1), or 30 μl of distilled water without SDS (Comparative Example 1: SDS concentration 0 wt%), and lysed at 80 ° C.
After the incubation for an hour, a competitive ELISA was performed. The results are shown in FIG.

From the above results, it was found that the subject heat-treated in the presence of SDS inhibited the reaction between the AGE-HSA and the anti-AGE-HSA antibody (Example 1), while the sample treated in the absence of SDS showed no reaction with AGE-HSA. Since the reaction of the anti-AGE-HSA antibody was not inhibited (Comparative Example 1), it became clear that AGE in human blood could be detected by the treatment.

(8) Optimal Pretreatment Temperature As described above, blood from a diabetic patient was pretreated in the presence of sodium dodecyl sulfate (SDS). Further, the optimum surfactant concentration at the time of the pretreatment was confirmed by a competitive ELISA.

The above blood (protein concentration: 200 mg /
ml) was lysed with 30 μl of distilled water containing 0.67 wt% SDS. The lysed samples were taken at room temperature, 37, 50, 60, 70, 80, 90 and 1 respectively.
What was incubated at 00 ° C. for 1 hour was used as a subject.

20 mM PB so that the concentration becomes 5 μg / ml.
Each sample lysed with the biotin-labeled anti-AGE-HSA antibody diluted with
g / ml. This solution is heated at 37 ° C for 1 hour.
It was left for a time and used as an antibody solution inhibited by blood AGE.

The competitive ELISA was performed in the same manner as described above. The results are shown in FIG.

From the above results, it was found that the sample heat-treated at 50 ° C. or higher in the presence of SDS had a higher AG value than the untreated sample.
Since the reaction between ESA and the anti-AGE-HSA antibody was significantly inhibited, it was clarified that the treatment should be performed at 50 ° C. or higher. On the other hand, when the temperature exceeds 100 ° C., the aggregation of the protein in the specimen becomes intense, and the competitive ELISA
Has become difficult to use as a subject.

Example 2 and Comparative Example 2 (1) Preparation of CM-Produced Protein In order to convert the amino group of the protein into CM, 1 ml of 1 mg / ml HSA adjusted to pH 9 and 0.25 M glyoxyl adjusted to pH 9 were used. 1 ml of acid (Sigma)
And left at 0 ° C. for 12 hours. Thereafter, 1 mg of sodium cyanoborohydride was added, and the mixture was further left for 12 hours.

As a control, HSA was treated in the same manner except that glyoxylic acid was not added.

The CM ratio of HSA after each of the above treatments was determined by measuring the unreacted amino group with trinitrobenzenesulfonic acid (hereinafter referred to as T
NBS) and measured by the following method.

That is, 0.5 ml of each sample was added to 0.5 ml of a 0.1 M aqueous sodium hydroxide solution containing 0.1 M sodium tetraborate. Next, after recrystallization and washing with diluted hydrochloric acid, TNB adjusted to 1.1 M with distilled water was used.
20 μl of S was added and stirred. After 30 minutes, the reaction was stopped by adding 2 ml of 98.5 mM sodium dihydrogen phosphate containing 1.5 mM sodium sulfite, and the reaction was stopped with distilled water for 10 minutes.
When the absorbance at 420 nm was measured after diluting by a factor of 2, the absorbance of CM HSA was 0.03, and the absorbance of HSA not treated with glyoxylic acid (control) was 1.
25. When the same measurement was carried out in a system not containing any of the above HSA, the absorbance was 0.03, and it was found that the CM conversion rate of the CM-modified HSA was 100%.

The commercialized HSA obtained by such a method has 2
After dialysis against 0 mM PBS at 4 ° C. for 2 days to remove unreacted glyoxylic acid and sodium cyanoborohydride, it was used as an immunogen for preparing an antibody against the CM-protein.

(2) Antibody to CM-conjugated HSA (anti-CM
Preparation of CM-HSA) A rabbit weighing 2 kg or more was treated with the CM-HS prepared above.
A was used as an antigen to immunize in the following manner.

To 0.5 ml of the antigen solution prepared to be 2 mg / ml, complete Freund's adjuvant was added to 0.5 ml.
The addition of 5 ml was injected into the rabbit ear vein. Thereafter, every two weeks, 0.25 ml of the 2 mg / ml antigen and 0.25 ml of Freund's incomplete adjuvant were boosted. During this period, rabbits were partially bled once every two weeks from the peripheral ear vein to confirm whether antibodies against CM-HSA were produced. Six weeks later, anti-C
The production of M-HSA antibody was determined by enzyme immunoassay (EL
ISA) method, and whole blood was collected.

(3) Preparation of Affinity Purification Column After washing 25 ml of Affigel 15 with 75 ml of 10 mM acetate buffer (pH 4.5), 10 mg / ml of H
62.5 ml of the SA solution was added, followed by gentle stirring at room temperature for 1 hour. Next, unreacted HSA was removed by filtration, and 1
M ethanolamine (30 ml) was added, and the mixture was gently stirred at room temperature to block unreacted N-hydroxysuccinimide ester. The support having the HSA immobilized thereon was packed in a column, and washed with ion-exchanged water until the absorbance at 280 nm became zero. Further, the column was equilibrated with 20 mM PBS.

(4) Affinity Purification of Anti-CM-HSA Antibody The prepared anti-CM-HSA antibody diluted with 20 mM PBS to 1 mg / ml was applied to the affinity purification column to about 100 mg. . Then 20m until the absorbance at 280nm becomes 0
MPBS was flowed at a flow rate of 0.5 ml / min. Antibodies that did not bind to the column were recovered as anti-CM-HSA antibodies. 20m when the absorbance at 280nm becomes 0
M PBS from 0.1 M glycine buffer (pH 3.
In place of 0), unnecessary proteins bound to the column were eluted, the column was equilibrated with 20 mM PBS, the recovered antibody was applied to the column again, and the antibody not bound to the column was recovered. This operation was repeated once more, and the resultant was used as an antibody for biotin labeling.

(5) Biotin Labeling of Anti-CM-HSA Antibody Biotin labeling of the purified antibody was performed in the same manner as in Example 1 using a protein biotinylation system (GIBCO). 14 mol of biotin was bound to 1 mol of the anti-CM-HSA antibody.

(6) Antigen specificity of anti-CM-HSA antibody 20 mM P was added so that the amount of CM HSA became 1 μg / ml.
0.1, 1, 10, and 100 μg / ml of the prepared CM-conjugated HSA was added to the anti-CM-HSA antibody diluted with BS, respectively.
Was added so that This solution was left at 37 ° C. for 1 hour and used as an antibody solution inhibited by CM-conjugated HSA.

In the same manner as in the preparation of CM HSA, CM Hb prepared from hemoglobin (Hb: Sigma) was prepared. The CM-modified Hb was added to 1 μg / ml of anti-CM-HSA
0.1, 1, 10, and 100 μg /
ml. This solution was allowed to stand at 37 ° C. for 1 hour and used as an antibody solution inhibited by CMb Hb.

In performing the competitive ELISA, the prepared CM-modified HSA was added to 20 mM so as to have a concentration of 1 μg / ml.
Diluted in PBS. Then, the diluted CM HSA
The solution was applied to a 96-well immunoplate (manufactured by NUNC) at 100 μl per well, left at 37 ° C. for 1 hour, and the CM-modified HSA was fixed to the immunoplate. After one hour, the CMSA which was not bound to the immunoplate was removed, and 20 mM PB containing 0.5 wt% gelatin was removed.
S was applied at 100 μl per well,
After allowing to stand for a period of time, the portion where CMSA was not bound was blocked. After 1 hour, the gelatin solution was removed and 2
After washing three times with 0 mM PBS, the CM-H
Antibody solution inhibited by SA, or CMHb at the above concentration
100 μl per well of the antibody solution inhibited by was applied and left at 37 ° C. for 1 hour. After that, 20 mM
After washing three times with PBS, the biotin-labeled anti-CM prepared above
-Apply 0.1 μg of HSA antibody per well,
It was left at 37 ° C. for 1 hour. In addition, 3 mM with 20 mM PBS
After washing twice, a mixed solution of biotin and avidin labeled with alkaline phosphatase (manufactured by Funakoshi: vectastain ABC kit) prepared according to the instruction manual was applied in an amount of 100 μl per well and left at 37 ° C. for 1 hour. Furthermore, 20 mM PBS containing 0.05 wt% Tween 20
3 times with alkaline phosphatase substrate kit (BIO
-RAD) (100 μl / well). After standing at room temperature for 5 minutes, a reaction of alkaline phosphatase was stopped by adding 100 μl of 0.4 M sodium hydroxide solution per well, and the absorbance at 405 nm was measured. FIG. 4 shows the results. From these results, the prepared anti-CM-HSA antibody was
Not only CM HSA but also CM Hb
And the reaction of anti-CM-HSA antibody with
It was found that Mb Hb also showed reactivity.

(7) Pretreatment of the Subject The blood from the diabetic patient treated in Example 1 was confirmed in the same manner as in Example 1 by a competitive ELISA. FIG. 5 shows the results.

Similarly, add 10 μl of the above blood to 0.0
Distilled water containing 4, 0.4, 0.67, 6.7 wt% SDS (Example 2) or distilled water 30 without SDS
The sample was subjected to hemolysis with μl (Comparative Example 2), incubated at 80 ° C. for 1 hour, and subjected to competitive ELISA. FIG. 5 shows the results.

From the results, it was found that the sample heat-treated in the presence of SDS inhibited the reaction between CM-HSA and the anti-CM-HSA antibody (Example 2), while the sample treated in the absence of SDS showed no reaction with CM-HSA. Since the reaction of the CM-HSA antibody was not inhibited (Comparative Example 2), it became clear that the treatment enabled detection of the CM-protein in human blood.

(8) Measurement of CM-protein in Pretreated Blood EDTA-2K from two diabetic patients and two healthy subjects
Was pretreated by the same operation as in Example 1, and the CM-protein in the blood was measured by a competitive ELISA. That is, 10 μl of blood is added to 0.6
Hemolysis with 30 μl of distilled water containing 7 wt% SDS
Incubate for 1 hour at C
A competition ELISA was performed.

The competitive ELISA was performed in the same manner as in Example 1. After measuring the hemoglobin concentration by the cyanmethemoglobin method, the hemoglobin was 20 μg / we.
After applying to a blocked 96-well immunoplate (manufactured by NUNC) in which CM-modified HSA was immobilized so as to be 1 l, then applying 0.5 μg of biotin-labeled anti-CM-HSA antibody per well, and then at 37 ° C. for 1 hour I left it.
Furthermore, the mixture was washed three times with 20 mM PBS, and a mixed solution of biotin and avidin labeled with alkaline phosphatase prepared according to the instruction manual (Vectastain AB manufactured by Funakoshi)
C kit) was applied at 100 μl per well, and 37
It was left at 0 ° C. for 1 hour. Furthermore, Tween 20 of 0.05 wt%
Was washed three times with 20 mM PBS containing, and 100 μl per well of a substrate solution prepared according to a protocol using an alkaline phosphatase substrate kit (manufactured by BIO-RAD) was applied. After standing at room temperature for 5 minutes, a reaction of alkaline phosphatase was stopped by adding 100 μl of 0.4 M sodium hydroxide solution per well, and the absorbance at 405 nm was measured. Table 1 shows the measurement results.

[0077]

[Table 1]

[Brief description of the drawings]

FIG. 1 shows the results of examining the antigen specificity of the prepared anti-AGE-HSA antibody by competitive ELISA, with the vertical axis representing 4 points.
The absorbance at 05 nm, the horizontal axis is the amount of each inhibitor added.

FIG. 2 shows the results of examining the optimal surfactant concentration at the time of pretreatment of a test sample using an anti-AGE-HSA antibody by a competitive ELISA. The vertical axis represents the absorbance at 405 nm, and the horizontal axis represents the absorbance. The amount of the pretreated sample added {the amount of added protein (inhibitor) per well}. In addition, the percentage display of each line in the figure represents the concentration (wt%) of SDS with respect to the weight of the subject at the time of the pretreatment.

FIG. 3 shows that the optimum temperature during the pretreatment of the subject is determined by anti-CM-H
As a result of examination by competitive ELISA using SA antibody,
The vertical axis indicates the absorbance at 405 nm, and the horizontal axis indicates the temperature during pretreatment.

FIG. 4 shows the results of examining the antigen specificity of the prepared anti-CM-HSA antibody by competitive ELISA, with the vertical axis representing 40%.
Absorbance at 5 nm, the horizontal axis is the amount of each inhibitor added / each well
The amount of added protein (inhibitor) per｝.

FIG. 5 is a graph showing the effect of pretreatment of a subject on anti-CM-HSA.
As a result of examination by a competitive ELISA using antibodies, the vertical axis indicates the absorbance at 405 nm, and the horizontal axis indicates the amount of the pretreated analyte {the amount of added protein (inhibitor) per well}. .

Continued on the front page (72) Inventor Yoshinori Yoshimura 1-1, Mikage-cho, Tokuyama-shi, Yamaguchi Pref.

Claims (4)

[Claims] 1. An analyte for measuring a Maillard reaction late product (AGE) is reacted with an analyte at 50 to 50% in the presence of an anionic surfactant.
A pretreatment method for a subject, which comprises performing a heat treatment at 100 ° C. 2. The method according to claim 1, wherein the anionic surfactant is sodium dodecyl sulfate. 3. An anti-Maillard reaction late-product antibody (anti-AGE antibody) is added to the sample pretreated by the method according to claim 1.
AGE is detected by an immunological reaction. 4. The AG according to claim 3, wherein the anti-AGE antibody is an anti-carboxymethylated protein antibody or an anti-carboxymethylated peptide antibody, and the AGE is a carboxymethylated protein or a carboxymethylated peptide.
Method for measuring E.