JPWO2014007367A1 - Method for detecting or measuring two substances in the same sample - Google Patents

Abstract

An object of the present invention is to provide a method for detecting or measuring each substance easily and with high sensitivity in a sample containing two substances having a common site and a non-common site that can be epitopes. The present invention relates to a method for detecting or measuring each substance in a sample containing two substances having a common site that can be an epitope and a non-common site, the first antibody recognizing the sample and the common site And incubating the substance, the substance bound to the first antibody and the second antibody recognizing a non-common part of one substance, and the common part And detecting or measuring a non-common site.

Description

  The present invention relates to a method for detecting or measuring two kinds of substances having a common site in the same sample.

Many proteins are translated and then subject to limited degradation by proteases and converted to active mature forms. This process is called processing. In the case of membrane proteins, limited degradation by proteases includes shedding that occurs directly above the membrane and processing that occurs in the extracellular region. The former converts a membrane protein into a secreted form, while the latter contributes to the activity control of the membrane protein.
In recent years, the importance of membrane protein processing has been gradually recognized, and it has become clear that it is involved in the development of various intractable diseases such as cancer, chronic inflammatory diseases, and Alzheimer's disease. For example, the processing fragment of HB-EGF by membrane-type MMP in ovarian cancer ascites correlates with the malignancy of ovarian cancer. The growth factor activity of HB-EGF is repressively controlled by the heparin-binding sequence at the N-terminus, but MT1-MMP cleaves the N-terminus of HB-EGF, thereby converting HB-EGF to heparin. It is converted into an independent growth factor, and the activation of the EGF receptor is strongly enhanced. Expression of both HB-EGF and MT1-MMP was observed in progressive ovarian cancer tissues, and HB-EGF and MT1-MMP expression was also immunized in ascites cancer cells infiltrated in ascites collected from ovarian cancer patients. Confirmed histologically. In addition, when molecular species of HB-EGF in ascites was analyzed by West blot with EGF-like domain antibody, HB-EGF processed by MT1-MMP was detected in 3 out of 10 ascites (non-patented) References 1 and 2). Therefore, it is thought that the processing fragment by the membrane type MMP of HB-EGF in the patient sample is effective as a method for judging the success of ovarian cancer treatment.

  Thus, by examining the presence / absence or abnormality of processing, it may be possible to diagnose a disease or judge the success of treatment. As a method for detecting a processing fragment of a membrane protein, a method using a neoepitope antibody that specifically recognizes a cleavage site is known. However, the production of neoepitope antibodies is very difficult. In addition, the cleavage site may be digested by endopeptidase, in which case the antigenicity is lost and the neoepitope antibody is not recognized. Furthermore, the detection sensitivity of neoepitope antibodies is generally not very high. Therefore, there is a need for a method for detecting processing fragments with higher sensitivity and ease.

Koshikawa, N. et al. Cancer Res, 2010, 70: 6093-6103 Koshikawa, N. et al. Cancer Sci, 2011, 102: 111-116

  An object of the present invention is to provide a method for detecting or measuring each substance easily and with high sensitivity in a sample containing two kinds of substances having a common site and a non-common site that can be epitopes. Such a method can be used, for example, for diagnosis of a disease involving protein processing and determination of the success of treatment.

As a result of repeated studies to solve the above problems, the present inventors have found that two types of fragments generated by shedding and processing of membrane proteins include a first antibody against a site that these fragments have in common, A second antibody recognizing a non-common site in one fragment and / or a third antibody recognizing a common site different from the first antibody or a non-common site different from the second antibody, and By detecting the binding between the antibody and the fragment, it was found that each of the processing fragments can be detected easily and with high sensitivity, and various applications thereof were studied, thereby completing the present invention.
That is, the present invention
[1] A method for detecting or measuring at least one substance in a sample containing two kinds of substances having a common part and a non-common part,
Contacting the sample with a first antibody that recognizes a common site and incubating;
Incubating the substance bound to the first antibody and the second antibody recognizing a non-common site of one substance in contact with each other;
Detecting or measuring the binding between the first and / or second antibody and the substance;
[2] The method according to [1] above, wherein the first antibody and the second antibody are labeled so as to be distinguishable from each other;
[3] The first antibody is immobilized on a solid support,
And a step of contacting and incubating the substance bound to the first antibody and a third antibody that recognizes a common site different from the first antibody,
The second antibody and the third antibody are labeled so as to be distinguishable from each other, and the detection or measurement of the binding between the first, second and / or third antibody and the substance is detected by the label. The method according to [1] or [2], wherein the method is performed by detecting or measuring
[4] The first antibody is immobilized on a solid support,
And a step of contacting and incubating the substance bound to the first antibody and a third antibody that recognizes a non-common site different from the second antibody,
The second antibody and the third antibody are labelably distinguishable from each other;
The method according to [1] or [2] above, wherein the detection or measurement of the binding between the first, second and / or third antibody and the substance is performed by detecting or measuring the label;
[5] The method according to any one of [1] to [4] above, wherein the two substances are fragments generated by processing and / or shedding of proteins;
[6] The method according to [5] above, wherein the recognition site of each antibody is not the end of the fragment;
[7] A kit for detecting or measuring at least one substance in a sample containing two kinds of substances having a common part and a non-common part,
A kit comprising: a first antibody that recognizes a common site; and a second antibody that recognizes a non-common site of one substance;
[8] The kit according to [7] above, wherein the first and second antibodies are fluorescently labeled with different colors, respectively.
[9] The kit according to [7] or [8] above, further comprising a third antibody that recognizes a common site different from the first antibody;
[10] The kit according to [7] or [8] above, further comprising a third antibody that recognizes a non-common site different from the second antibody;
[11] The kit according to [9] or [10], wherein the second and third antibodies are fluorescently labeled with different colors, respectively.
[12] The kit according to any one of [7] to [11], wherein the two kinds of substances are fragments generated by processing and / or shedding of proteins;
[13] A test method for Alzheimer's disease,
Incubating a sample derived from the subject and a first antibody that recognizes positions 672 to 687 of SEQ ID NO: 1 in contact;
A substance bound to the first antibody; a second antibody recognizing positions 1 to 671 of SEQ ID NO: 1; and / or a third antibody recognizing positions 688 to 711 of SEQ ID NO: 1. And incubating with, and
Detecting or measuring the binding between the second antibody and / or the third antibody and the substance;
[14] The method according to [13] above, wherein the second antibody and the third antibody are labeled so as to be distinguishable from each other; and [15] a test kit for Alzheimer's disease,
A first antibody that recognizes positions 672 to 687 of SEQ ID NO: 1;
A second antibody that recognizes positions 1 to 671 of SEQ ID NO: 1 and / or a third antibody that recognizes positions 688 to 711 of SEQ ID NO: 1.
About.

According to the present invention, at least one substance can be detected or measured easily and with high sensitivity from a sample containing two kinds of substances having a common part and a non-common part.
In particular, when the method of the present invention is used for detection of a processing fragment, it is not necessary to use a neoepitope antibody that recognizes a peptide end, so that an easy-to-make antibody can be used and is not affected by digestion with endopeptidase. Further, by immobilizing the first antibody on a solid phase carrier, it is possible to detect a minute amount of fragments with high sensitivity.
According to the method of the present invention, it is possible to determine the success of diagnosis or treatment by detecting the presence or absence or abnormality of processing or shedding.

FIG. 1 is a conceptual diagram illustrating processing of an amyloid precursor protein (APP) by an Aβ production pathway and a non-Aβ production pathway. FIG. 2 is a conceptual diagram illustrating the measurement of sAPPα and sAPPβ by the method of the present invention. FIG. 3 is a conceptual diagram illustrating the measurement of sAPPα and Aβ by the method of the present invention. FIG. 4 is a conceptual diagram illustrating the mechanism of the APP / Aβ detection method according to the embodiment. FIG. 5 shows the results obtained by adding Aβ and sAPP to human normal serum at various concentrations and measuring using the method according to the present invention. FIG. 6 shows the results of quantitative detection of Aβ and sAPP by the method according to the present invention. FIG. 7 shows the results of confirming the reactivity of the antibodies used in the examples by direct ELISA using Aβ and sAPP. FIG. 8 shows the results of confirming the reactivity of the antibodies used in the examples by sandwich ELISA using Aβ and sAPP.

(Detection or measurement method)
One aspect of the detection or measurement method according to the present invention is to detect or measure two kinds of substances contained in the same sample.
In the present specification, the two types of substances to be detected or measured are not particularly limited as long as they have a common site and a non-common site that can be epitopes, and examples thereof include peptides and nucleic acids. Is a peptide. In particular, two or more fragments generated from the same protein by processing or shedding can be mentioned.

For example, Alzheimer's disease is mentioned as a disease in which abnormalities in processing and fragments generated by processing are involved in the onset. Alzheimer's disease occurs when amyloid β protein (Aβ) produced by processing of amyloid precursor protein (APP) accumulates in the brain to form senile plaques, and senile plaques develop by killing nerve cells. It is considered.
Processing of APP, which is a type I transmembrane protein, has an Aβ production pathway and a non-Aβ production pathway. As shown in FIG. 1, in the Aβ production pathway, APP is cleaved by β secretase and γ secretase to produce sAPPβ and Aβ. On the other hand, in the non-Aβ production pathway, APP is cleaved by α-secretase and γ-secretase, resulting in fragments called sAPPα and P3.
Control of secretase has become an important issue for the prevention and treatment of Alzheimer's disease, and studies have been conducted to inhibit Aβ production by inhibiting β-secretase and γ-secretase. Therefore, if fragments such as sAPPα, sAPPβ, Aβ, and P3 can be detected, they can be used for evaluation of therapeutic drug candidates.
Aβ also includes Aβ40, which consists of 40 amino acids, and Aβ42, which consists of 42 amino acids. Among them, Aβ42 serves as the nucleus of amyloid aggregation in the brain and triggers the onset of Alzheimer's disease. Molecule. Therefore, if Aβ42 can be detected in particular, it is considered more useful for evaluation of therapeutic drug candidates.
Furthermore, if Aβ and sAPP in the serum and cerebrospinal fluid of a subject can be detected, it can be used for the evaluation of the presence or absence of Alzheimer's disease.

In the present specification, the phrase “two substances have a common site” means a state in which the two substances have an epitope recognized by the same antibody. When the substance is a peptide, the common site that can be an epitope may be the same site only in the amino acid sequence, or it may be the same site including the modification with sugar chains and lipids, the presence or absence of phosphorylation, etc. Also good.
In the present specification, “the two substances have a non-common site” means a state in which the two substances have epitopes recognized by different antibodies. Non-common sites that can be epitopes include, when the substance is a peptide, sites with different amino acid sequences, sites with the same amino acid sequence but different modifications with carbohydrates and lipids, and the same amino acid sequence, but different phosphorylation status There may be a site.
As described above, when the two substances are peptides, the method according to the present invention has two substances having a common sequence and a non-common sequence; two common sequences; Two different substances; two substances that have two common sequences, one of which is different in lipid modification; one that has two common sequences, and one of which differs in the presence or absence of phosphorylation, etc. Can be detected or measured.

  The method according to the present invention is a method for detecting or measuring each of two kinds of substances, and may be used for examining only the presence or absence of each of the two kinds of substances, or for measuring the respective amounts. Or may be used to determine the ratio of the amounts of the two substances. The method according to the present invention can also be applied to detect or measure each of three or more substances in a sample.

In the method according to the present invention, first, a sample containing two kinds of substances and a first antibody that recognizes a common site are brought into contact with each other and incubated.
In the present specification, the “sample” is not particularly limited as long as it may contain a substance to be detected or measured. For example, blood, urine, spinal fluid, puncture fluid, tissue, serum derived from humans or animals And cerebrospinal fluid. These samples may be appropriately diluted with a buffer solution or the like, or may be subjected to treatment with a drug or the like prior to the step of contacting with the first antibody.

The step of bringing the sample into contact with the first antibody and incubating can be performed according to a known method. Conditions such as incubation time and temperature can be appropriately determined by those skilled in the art.
By this step, the first antibody binds to both of the two substances.

Subsequently, the substance bound to the first antibody and the second antibody that recognizes a non-common site existing only in one substance are brought into contact with each other and incubated. This step can also be performed according to a known method, and conditions can be appropriately determined by those skilled in the art.
By this step, the second antibody binds only to a substance having a non-common site recognized by the second antibody among substances bound to the first antibody.

  Next, the binding between the first antibody and / or the second antibody and the substance is detected or measured. The binding between the antibody and the substance can be detected or measured according to various known methods. By the steps so far, the first antibody and the second antibody are bound to one of the two substances, and only the first antibody is bound to one. Therefore, for example, by labeling the first antibody and the second antibody so that they can be distinguished from each other, only the first antibody is bound to the substance to which the first and second antibodies are bound. A substance can be detected or measured.

Antibody labeling methods include enzymes such as peroxidase and alkaline phosphatase, radioactive materials such as ¹²⁵ I, ¹³¹ I, ³⁵ S, and ³ H, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, near red Examples include, but are not limited to, methods using fluorescent materials such as outer fluorescent materials, luminescent materials such as luciferase, luciferin, and aequorin, gold colloids, nanoparticles such as quantum dots, and the like. The labeling of the antibody can be performed according to a known method. Alternatively, the antibody may be labeled with biotin and detected by binding avidin or streptavidin labeled with an enzyme or the like.
For example, if the first antibody is labeled with a red fluorescent substance and the second antibody is labeled with a green fluorescent substance, the substance to which both the first antibody and the second antibody are bound is yellow, A substance bound only to the first antibody is red, and both can be identified and detected or measured.

In one embodiment of the method according to the present invention, the first antibody is used by being immobilized on a solid phase carrier. As a result, in the step of binding the sample and the first antibody, the two substances that bind to the first antibody can be concentrated on the surface of the solid phase carrier, so that even if the amount of the substance is very small, it is detected with high sensitivity. be able to.
In the present specification, the “solid phase carrier” is not particularly limited as long as it is a carrier capable of immobilizing an antibody, glass, metallic, resin-made microtiter plates, substrates, beads, nitrocellulose membranes, nylon membranes, A PVDF membrane etc. are mentioned, An antibody can be fix | immobilized to these solid-phase carriers according to a well-known method.

When the first antibody is immobilized on the solid phase carrier, the step of bringing the sample and the first antibody into contact is performed, for example, by immobilizing the first antibody on a microtiter plate and dropping the sample in each well. Alternatively, the first antibody-immobilized beads may be introduced into the sample.
By this step, the common site of the two kinds of substances in the sample binds to the first antibody and is captured on the solid surface. Thereafter, a solid phase surface washing step (for example, when a microtiter plate is used as the solid phase carrier) and a solid phase carrier recovery step (for example, when beads are used as the solid phase carrier) are performed to fix the solid phase surface. Only the substance trapped on the phase carrier may be recovered.

  When the first antibody is immobilized on a solid phase carrier, the step of bringing the substance bound to the first antibody into contact with the second antibody and incubating the second antibody and the solid phase carrier surface It can be performed by contacting and incubating.

In this embodiment, a third antibody is used to detect or measure two substances. The third antibody recognizes a common site different from the first antibody in the two substances and is labeled so as to be distinguishable from the second antibody.
The first antibody captures two kinds of substances on the surface of the solid phase carrier, the non-common site is labeled with the second antibody, and the common site is labeled with the third antibody. Two substances having only a common site can be detected and measured. That is, the number of molecules labeled with the second antibody represents the number of molecules having non-common sites, and the number of molecules labeled with the second antibody is subtracted from the number of molecules labeled with the third antibody. It is possible to determine the number of molecules having only (see FIG. 2 for example).

As the third antibody, one that recognizes a non-common site different from the second antibody in the two substances and is labeled so as to be distinguishable from the second antibody may be used.
Two substances can be detected by capturing two substances on the surface of the solid phase carrier with the first antibody, labeling a non-common part with the second antibody, and labeling another non-common part with the third antibody. Can be measured (see FIG. 3).

The “antibody” used in the method of the present invention may be either a monoclonal antibody or a polyclonal antibody, and both can be prepared according to known methods.
For example, when the first antibody is a monoclonal antibody, first, a non-human mammal is immunized using one of two substances to be detected and measured, and antibody-producing cells (lymph node cells) are immunized from the immunized animal. Or spleen cells). This is fused with myeloma cells using polyethylene glycol or the like to produce a hybridoma. Subsequently, screening is performed using the culture supernatant to determine whether the hybridoma that has become a colony secretes a desired antibody, that is, an antibody that binds to the common site of the two substances. The cells producing the desired antibody can be cloned by limiting dilution, and further screening can be performed to establish a hybridoma clone that secretes the first antibody that recognizes the common site of the two substances. The first antibody can be obtained by purification.
When the substance to be detected and measured is a peptide, it is desirable to select an antibody that recognizes a site that is not the terminal of the peptide during screening. Thereby, even if the terminal of the peptide to be detected / measured is degraded by endopeptidase, an antibody capable of binding to the peptide of interest can be obtained.
In the above method, when immunizing a non-human mammal, only a fragment that is a common site of two substances may be used as an antigen.

The second antibody can be similarly produced. When immunizing a non-human mammal, a second antibody can be obtained by selecting a substance that binds to only one of the two substances during screening. it can.
Moreover, when immunizing a non-human mammal, a fragment consisting of a non-common site that exists only in one of the two substances may be used as an antigen.

  The third antibody can be prepared in the same manner as the first antibody. At the time of screening, a substance that recognizes an epitope different from that of the first antibody and binds to both of the two substances may be selected.

  The antibody may be a polyclonal antibody. In the case of a polyclonal antibody, a non-human mammal is immunized with an appropriately selected antigen, and can be obtained from the serum of the immunized animal.

(Detection or measurement kit)
The kit according to the present invention is for detecting or measuring each substance in a sample containing two substances having a common site that can be an epitope and a non-common site.
The terms used in the detection or measurement method according to the present invention are also used synonymously for the kit.

  One embodiment of the kit according to the present invention includes a first antibody and a second antibody. The first antibody and the second antibody may be labeled so as to be distinguishable from each other, or a reagent necessary for labeling separately from the antibody may be provided in the kit. In addition, a buffer solution, a reagent, a microtiter plate, a solid phase carrier such as a microplate reader and beads, an instruction manual, and the like necessary for the detection or measurement method according to the present invention may be provided.

  Another embodiment of the kit according to the present invention includes first to third antibodies. Each antibody may be labeled so as to be distinguishable from each other, or a reagent necessary for labeling separately from the antibody may be provided in the kit. In addition, a buffer solution, a reagent, a microtiter plate, a solid phase carrier such as a microplate reader and beads, an instruction manual, and the like necessary for the detection or measurement method according to the present invention may be provided.

(Testing method for Alzheimer's disease)
The method for examining Alzheimer's disease according to the present invention includes:
Incubating a sample derived from the subject and a first antibody that recognizes positions 672 to 687 of SEQ ID NO: 1 in contact;
A substance bound to the first antibody, a second antibody recognizing positions 1 to 671 of SEQ ID NO: 1, and / or a third antibody recognizing positions 688 to 711 of SEQ ID NO: 1 Incubating with contact, and
Detecting or measuring the binding between the second antibody and / or the third antibody and the substance.

In this specification, the sample derived from the subject can be blood, urine, spinal fluid, puncture fluid, tissue, serum, cerebrospinal fluid collected from the subject, particularly serum or cerebrospinal fluid. Liquid is preferred. The sample may be diluted with an appropriate buffer or may be treated with a drug or the like.
SEQ ID NO: 1 shows the amino acid sequence of human amyloid β precursor protein. As shown in FIG. 1, normal shedding with α-secretase results in secreted APPα (sAPPα), which is a fragment from position 1 to position 687 of SEQ ID NO: 1. On the other hand, by β and γ secretase, Aβ40 which is a fragment from positions 672 to 711, Aβ42 which is a fragment from positions 672 to 713, Aβ43 which is a fragment between positions 672 and 714, and the like are generated.
When detecting sAPPα and Aβ40, 42 or 43, positions 672 to 687 of SEQ ID NO: 1 are common sites, positions 1 to 671, and positions 688 to 711 to 713 are non-common sites.

  Therefore, when the antibody that recognizes the common site is used as the first antibody and is immobilized on, for example, a solid phase and brought into contact with a sample, sAPPα, Aβ 40, 42, or 43 is captured on the surface of the solid phase. After washing appropriately, when a second antibody that recognizes positions 1 to 671 of SEQ ID NO: 1 is incubated in contact with the solid surface, it binds to sAPPα if present. In addition, when a third antibody that recognizes positions 688 to 711 of SEQ ID NO: 1 is brought into contact with the solid phase surface and incubated, if Aβ40, 42, or 43 is present, it binds thereto.

Incubation with the second and third antibodies may be performed simultaneously or separately. If the second and third antibodies are labeled in a distinguishable manner, the ratio of sAPPα and Aβ40 can be easily determined from the same sample. The second and third antibodies may be labeled, or may be detected using a secondary antibody.
By measuring changes in sAPPα and Aβ40 over time, it is possible to evaluate the progression of Alzheimer's disease and the like, and it is also possible to evaluate the effects of therapeutic agents and treatment methods.

  As the first to third antibodies, commercially available antibodies may be used, or they may be prepared by the method described above. These antibodies preferably recognize sites that are not at the end of each fragment.

In the present specification, “examination” means examining a sample collected from a subject in order to obtain information necessary for diagnosis, and the detection method of the present invention can be carried out, for example, by an examination company or the like. .
In addition, in the present invention, the “test” is a test for confirming whether or not Alzheimer's disease is caused, in addition to a test for confirming whether or not Alzheimer's disease is likely to occur. This includes screening the possibility and examining the degree of progression, prognosis, therapeutic effect, etc. after it has been revealed that the patient has Alzheimer's disease.
The test method according to the present invention may be combined with other test methods for Alzheimer's disease.

  The present invention also includes a method for diagnosing Alzheimer's disease, which comprises carrying out the above-described method for testing Alzheimer's disease.

(Test kit for Alzheimer's disease)
The test kit for Alzheimer's disease according to the present invention includes a first antibody, a second antibody, and / or a third antibody used in the Alzheimer's disease test method. It is preferable to have both the second antibody and the third antibody.
In the test kit for Alzheimer's disease according to the present invention, each antibody may be labeled so as to be distinguishable from each other, or a reagent necessary for labeling separately from the antibody may be provided in the kit. The first antibody for capture may not be labeled. The second or third antibody is not labeled, and a labeled secondary antibody for detecting them may be provided.
In addition, a buffer solution, a reagent, a microtiter plate, a solid phase carrier such as a microplate reader and beads, instructions for use, and the like necessary for the detection or measurement method according to the present invention may be provided.

  The disclosures of all patent and non-patent documents cited herein are hereby incorporated by reference in their entirety.

(Embodiment 1)
The embodiment of the present invention will be described by taking a method of detecting an APP processing product as an example.
As described above, APP is processed by the Aβ production pathway and the non-Aβ production pathway to produce sAPPα, sAPPβ, Aβ, and P3. Therefore, for example, if sAPPα and sAPPβ are the “two substances” referred to in the present invention, the processing by each production pathway can be quantitatively evaluated. This embodiment is shown in FIG.
In the method of FIG. 2, a first antibody that recognizes a common site, a second antibody that recognizes a non-common site, and a third antibody that recognizes a common site different from the first antibody are used. For example, the second and third antibodies are labeled with red and green fluorescent substances, respectively.

  When the first antibody is immobilized on a microtiter plate and a sample from an appropriately treated patient is dropped into the well, sAPPβ and sAPPα are captured by the first antibody. Once the microtiter plate is washed, the second and third antibodies are added. The order in which the antibodies are added is not particularly limited. Thereafter, when detection is performed by washing treatment, color development treatment, and the like, since the second and third antibodies are bound to sAPPα, they are observed in yellow. On the other hand, since only the third antibody is bound to sAPPβ, it is observed in green, and both can be quantified.

  Further, for example, even when sAPPα and Aβ are “two substances” referred to in the present invention, the processing by each production pathway can be quantitatively evaluated. This embodiment is shown in FIG. In the method of FIG. 3, a first antibody that recognizes a common site, a second antibody that recognizes a non-common site, and a third antibody that recognizes a non-common site different from the second antibody are used. For example, the second and third antibodies are labeled with red and green fluorescent substances, respectively.

  When the first antibody is immobilized on a microtiter plate and a sample from an appropriately treated patient is dropped into the well, sAPPα and Aβ are captured by the first antibody. Once the microtiter plate is washed, the second and third antibodies are added. The order in which the antibodies are added is not particularly limited. Thereafter, when sAPPα is detected by performing washing treatment, color development treatment, etc., the second antibody is bound, and therefore, it is observed in red. On the other hand, since only the third antibody is bound to Aβ, it is observed in green. This makes it possible to quantify both.

  By repeating the method of FIG. 2 and FIG. 3 over time, a change in which of the Aβ production pathway and the non-Aβ production pathway is dominant can be seen, so that the disease worsening and the effect of treatment can be evaluated. Is also possible.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to this at all. Those skilled in the art can change the present invention into various modes without departing from the meaning of the present invention, and such changes are also included in the scope of the present invention.

FIG. 4 shows an outline of the APP / Aβ detection method in this example. An antibody that specifically binds to the region from position 672 to position 687 of the amyloid precursor protein (SEQ ID NO: 1) as a capture antibody, and an antibody that specifically binds to the region from position 1 to position 671, and position 688 The antibody specifically binding to the region at position 713 was used as a detection antibody.
As a sample, human normal serum added with sAPPα and Aβ40 was used.
The materials used are shown in the table below.

The experimental procedure is shown in the table below.

  FIG. 5 shows the results of the above experiment with various concentrations of sAPPα and Aβ40 added to normal human serum. SAPPα could be detected quantitatively by IBL (# 28053). On the other hand, Abcam (# 62658) was able to detect Aβ quantitatively.

  Based on the results shown in FIG. 5, the logarithm of concentration is plotted on the horizontal axis and the fluorescence intensity is plotted on the vertical axis. Aβ is 10 ng / ml or more, and sAPPα is 100 ng / ml or more. there were.

  FIG. 7 shows the result of confirming the reactivity of the antibody used in the above-mentioned experiment by direct ELISA using Aβ and sAPPα. It was confirmed that the capture antibody bound to both, and the detection antibody specifically bound to each antigen.

  FIG. 7 shows the result of confirming the reactivity of the antibody used in the above-mentioned experiment by sandwich ELISA using Aβ and sAPPα. It was confirmed that the capture antibody bound to both, and the detection antibody specifically bound to each antigen.

Claims (15)

A method for detecting or measuring at least one substance in a sample containing two kinds of substances having a common part and a non-common part,
Contacting the sample with a first antibody that recognizes a common site and incubating;
Incubating the substance bound to the first antibody and the second antibody recognizing a non-common site of one substance in contact with each other;
Detecting or measuring the binding between the first and / or second antibody and the substance.   The method according to claim 1, wherein the first antibody and the second antibody are labelably distinguishable from each other. The first antibody is immobilized on a solid support;
And a step of contacting and incubating the substance bound to the first antibody and a third antibody that recognizes a common site different from the first antibody,
The second antibody and the third antibody are labelably distinguishable from each other;
The method according to claim 1 or 2, wherein the detection or measurement of the binding between the first, second and / or third antibody and the substance is performed by detecting or measuring the label. The first antibody is immobilized on a solid support;
And a step of contacting and incubating the substance bound to the first antibody and a third antibody that recognizes a non-common site different from the second antibody,
The second antibody and the third antibody are labelably distinguishable from each other;
The method according to claim 1 or 2, wherein the detection or measurement of the binding between the first, second and / or third antibody and the substance is performed by detecting or measuring the label.   The method according to any one of claims 1 to 4, wherein the two kinds of substances are fragments generated by processing and / or shedding of proteins.   6. The method of claim 5, wherein the recognition site of each antibody is not the end of the fragment. A kit for detecting or measuring at least one substance in a sample containing two kinds of substances having a common part and a non-common part,
A kit comprising: a first antibody that recognizes a common site; and a second antibody that recognizes a non-common site of one substance.   The kit according to claim 7, wherein the first and second antibodies are fluorescently labeled with different colors.   The kit according to claim 7 or 8, further comprising a third antibody that recognizes a common site different from the first antibody.   The kit according to claim 7 or 8, further comprising a third antibody that recognizes a non-common site different from the second antibody.   The kit according to claim 9 or 10, wherein the second and third antibodies are fluorescently labeled with different colors.   The kit according to any one of claims 7 to 11, wherein the two substances are fragments generated by processing and / or shedding of proteins. A test method for Alzheimer's disease,
Incubating a sample derived from the subject and a first antibody that recognizes positions 672 to 687 of SEQ ID NO: 1 in contact;
A substance bound to the first antibody; a second antibody recognizing positions 1 to 671 of SEQ ID NO: 1; and / or a third antibody recognizing positions 688 to 711 of SEQ ID NO: 1. And incubating with, and
Detecting or measuring the binding between the second antibody and / or the third antibody and the substance.   The method of claim 13, wherein the second antibody and the third antibody are distinguishably labeled with each other. A test kit for Alzheimer's disease,
A first antibody that recognizes positions 672 to 687 of SEQ ID NO: 1;
And a second antibody that recognizes positions 1 to 671 of SEQ ID NO: 1 and / or a third antibody that recognizes positions 688 to 711 of SEQ ID NO: 1.