JP6074624B2 - Method, kit, and detection apparatus for detecting various types and / or mixed types of disease-related autoantibodies in biological samples - Google Patents

Description

  The present invention relates to a detection method, kit, and detection apparatus for individual and / or mixed various disease-related autoantibodies in a biological sample.

  Various disease-related antigens in biological samples are currently measured as indicators of the presence and progression of disease. For example, various tumor-associated antigens have been measured as tumor markers. However, in cancer patients, it has been revealed that autoantibodies against various tumor-associated antigens increase before tumor-associated antigens increase (see, for example, Non-Patent Documents 1 to 4). Therefore, detecting autoantibodies against tumor-associated antigens may lead to early detection of cancer rather than detecting tumor-associated antigens. In other diseases as well as cancer, disease-related autoantibodies (autoantibodies against disease-related antigens) are new disease markers that lead to early detection of diseases.

  In recent years, a test method for detecting autoantibodies against p53 protein in blood as a tumor marker has been approved as a subject of insurance application. Currently, detection of autoantibodies against p53 protein is performed only by enzyme immunoassay (ELISA method).

J. M. A. WHITEHOUSE and E. J. HOLBOROW, Br Med J, 4, 511-513, 1971 Imai H., et al., Clin Cancer Res, 1, 417-424, 1995 Murray A., et al., Ann Oncol, 21, 1687-1693, 2010 Werner S., et al., Int J Cancer, doi: 10. 1002 / ijc.28807, 2014 [Epub ahead of print]

However, the inventors have found that detection of disease-related autoantibodies by the ELISA method may have insufficient detection sensitivity.
Furthermore, detection methods that improve the detection rate of cancer and refractory diseases are desired, as well as methods for detecting autoantibodies against a single disease-related protein.

  Therefore, an object of the present invention is to provide a detection method capable of easily and simultaneously measuring autoantibodies against various disease-related proteins in a biological sample. Another object of the present invention is to provide a kit and a detection device for detecting disease-related autoantibodies for easily and simultaneously measuring autoantibodies against various disease-related proteins in a biological sample.

The present invention is as follows.
(1) A living body for a disease-related protein on a substrate having a plurality of spots in which individual disease-related proteins are spotted in one spot and / or a spot obtained by mixing a plurality of types of disease-related proteins in one spot A method of simultaneously detecting autoantibodies in a sample in the same reaction phase, wherein the disease is breast cancer, and the disease-related proteins are p53, ER, PgR, HER2, CEA, CYFRA, and CA15-3. A detection method comprising seven types of breast cancer-related proteins and using an evanescent wave excitation fluorescence method.
(2) The detection method according to (1), wherein the disease-related autoantibody is an antibody against at least a part of the disease-related protein.
(3) The disease-related autoantibody is an antibody against at least one selected from the group consisting of a wild-type disease-related protein, a mutant disease-related protein, and a modified disease-related protein, (1) or (2 ) Detection method.
(4) A large number of disease-related autoantibodies in the biological sample obtained by spotting individual disease-related antigens in one spot and / or spotting a mixture of multiple types of disease-related antigens in one spot Reacting with the disease-related antigen on the substrate having spots, and reacting a secondary antibody labeled with a fluorescent substance that binds to the disease-related autoantibody captured via the disease-related antigen on the substrate. The method according to any one of (1) to (3), including a step, a step of exciting the fluorescent material on the substrate with an evanescent wave, and a step of detecting fluorescence emitted from the fluorescent material on the substrate. Detection method.
(5) The detection method according to (4), wherein the secondary antibody labeled with the fluorescent substance reacts in a globulin type or subclass-specific manner.
(6) Using the evanescent wave excitation fluorescence method, there are multiple spots that spot individual disease-related proteins in one spot and / or those spotted by mixing multiple types of disease-related proteins in one spot A kit for simultaneously detecting autoantibodies in a biological sample against the disease-related protein on a substrate in the same reaction phase, wherein each disease-related protein is spotted in one spot and / or in one spot A spot obtained by mixing a plurality of types of disease-related proteins is a substrate in which multiple spots are immobilized in the same reaction phase , wherein the disease is breast cancer, and the disease-related proteins are p53, ER, PgR, HER2, CEA, a substrate comprising CYFRA, and seven types of breast cancer-associated protein CA15-3, their disease-related data Characterized by comprising a secondary antibody labeled with recognizing a fluorescent substance autoantibodies, the against Park protein, kits.
(7) The kit according to (6), wherein the disease-related autoantibody is an antibody against at least a part of the disease-related protein.
(8) The disease-related autoantibody is an antibody against at least one selected from the group consisting of a wild-type disease-related protein, a mutant disease-related protein, and a modified disease-related protein, (6) or (7 ) Kit.
(9) The kit according to any one of (6) to (8), wherein the secondary antibody labeled with the fluorescent substance reacts specifically with a globulin type or a subclass.
(10) A detection device for detection of disease-related autoantibodies, wherein one spot is spotted with individual disease-related proteins and / or one spot is a mixture of multiple types of disease-related proteins, A multispot-fixed substrate , wherein the disease is breast cancer, and the disease-related protein comprises seven types of breast cancer-related proteins of p53, ER, PgR, HER2, CEA, CYFRA, and CA15-3; A detection apparatus comprising: irradiation means for generating evanescent waves on the surface of the substrate; and detection means for detecting fluorescence emitted from the fluorescent material excited by the evanescent waves.

The present invention can provide a detection method capable of easily and simultaneously measuring autoantibodies against various disease-related proteins in a biological sample. In addition, according to the present invention, it is possible to provide a kit and a detection device for detecting disease-related autoantibodies for easily and simultaneously measuring autoantibodies against various disease-related proteins in a biological sample.
Furthermore, according to the present invention, it is possible to provide a technique that enables early detection of cancer, intractable diseases, and the like.

FIG. 1 is a graph summarizing the detection results of autoantibodies against seven types of proteins by the evanescent wave excitation fluorescence method.

[Method for detecting individual and / or mixed types of disease-related autoantibodies in biological samples]
In one embodiment, the present invention is a method for simultaneously detecting autoantibodies in biological samples against individual and / or mixed types of disease-related proteins in the same reaction phase, using evanescent wave excitation fluorescence. A detection method is provided.
In the present embodiment, autoantibodies against various tumor-related proteins and disease-related proteins are simultaneously measured by a batch operation, and the detection rate of cancer and refractory diseases is improved by combining the results.

Currently, the only insurance-applicable tumor-related autoantibody test in Japan is anti-p53 autoantibody. Research has reported autoantibodies against many tumor-related proteins other than the p53 protein, and the effectiveness of simultaneous measurement of various autoantibodies has been described. In the present embodiment, the simultaneous measurement can be easily performed using an evanescent wave for the first time.
Until now, most of the measurement of tumor-related autoantibodies including anti-p53 autoantibodies has been carried out by ELISA method. In order to carry out multiple simultaneous measurements by ELISA method, the amount of patient specimens and the solid phase in the kit The required amount of antigen and the like has become quite large. Moreover, the problem of false negativeization of low affinity autoantibodies remains.
This embodiment is superior to low-affinity autoantibody detection in comparison with measurement by ELISA, and requires only a small amount of patient sample and disease-related protein (solid-phase antigen), which is the same as all types of disease-related proteins. It is excellent in operability because of simultaneous reaction in phases. As a result, since autoantibodies against various tumor-related proteins can be confirmed simultaneously, the detection (positive) rate becomes higher, and early detection of cancer can be expected in many patients.
On the other hand, the relationship between refractory diseases and autoantibodies has also been reported, and this embodiment is very useful not only for cancer but also for diseases in which detection of autoantibodies is effective, such as refractory diseases and autoimmune diseases. .
The detection method of this embodiment will be described in detail below.

(Evanescent wave excitation fluorescence method)
For example, when the incident angle of the excitation light incident on the glass substrate is increased to cause total reflection, light called evanescent light oozes out about several hundred nm from the opposite interface of the total reflection surface. The evanescent wave excitation fluorescence method is a fluorescence detection method applying this principle. By placing the fluorescent dye in the evanescent field and exciting the fluorescence with the evanescent light, only the fluorescent dye existing near the surface of the glass substrate is obtained. This is a detection method that emits fluorescence and can dramatically reduce background light in fluorescence observation.

In one embodiment, the above-described detection method comprises a step of reacting a disease-related autoantibody in a biological sample with individual and / or mixed types of disease-related antigens immobilized on a substrate, and the disease on the substrate. Reacting a secondary antibody labeled with a fluorescent substance that binds to the disease-related autoantibody captured via the relevant antigen, exciting the fluorescent substance on the substrate with an evanescent wave, and on the substrate Detecting fluorescence emitted from the fluorescent substance.
The disease-related antigen may be not only a self-component but also a foreign molecule such as a bacterial cell component or a viral antigen.
A substrate on which a variety of individual disease-related antigens are immobilized means a substrate on which a plurality of spots prepared by spotting individual disease-related antigens in one spot are prepared.
The substrate on which various mixed disease-related antigens are immobilized means a substrate in which multiple spots prepared by mixing a plurality of types of disease-related antigens in one spot are prepared. As such a substrate, for example, a spot in which a plurality of types of antigens related to lung cancer are mixed, and a plurality of types of proteins specifically expressed for each cancer, such as a breast cancer-related antigen or a colon cancer-related antigen, are used as antigens. And a substrate spotted by mixing.
For example, the substrate on which a variety of individual and mixed types of disease-related antigens are immobilized includes, for example, one in which individual disease-related antigens are spotted in one spot and a mixture of spots in which a plurality of types of disease-related antigens are spotted in one spot. This means a substrate prepared with spots.

Examples of the living body include humans and mammals other than humans. Examples of biological samples include blood, serum, plasma, urine, cerebrospinal fluid, nasal fluid, saliva and the like.
For example, the amount of patient specimen is 50 to 100 μL that is optimally diluted for one measurement item (autoantibody measurement against one disease-related protein) in ELISA, but in this embodiment, it is in 135 or more spots (items). On the other hand, it can be measured with 100 μL. The amount of labeled secondary antibody used is the same as the amount of patient specimen, and this embodiment is a patient-friendly measurement method that is much less expensive than the ELISA method.

  A disease-related autoantibody means an antibody against a disease-related antigen contained in a biological sample. Examples of the disease-related autoantibodies include antibodies whose globulin type (class) is IgG type, IgM type, IgA type, IgD type, IgE type, and IgG and IgA types also include subclass-specific antibodies.

  Fluorescently labeled secondary antibodies for detecting disease-related autoantibodies include fluorescently labeled anti-human IgG antibodies, anti-human IgM antibodies, anti-human IgA antibodies, anti-human IgD antibodies, and anti-human IgE antibodies. Human IgG and IgA antibodies also include antibodies that react specifically with each subclass.

  Fluorescent substances labeled with secondary antibodies include Cy3, Cy5, FITC, AMCA, PerCP, R-phycoerythrin (RPE), allophycoerythrin (APC), Texas Red, Princeton Red, quantum dots, and green fluorescent protein (GFP) Blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and the like.

(Step of capturing a disease-related autoantibody using a secondary antibody labeled with the fluorescent substance on a substrate by contacting a biological sample with a substrate on which various and / or mixed disease-related antigens are immobilized. )
The substrate is obtained by immobilizing a disease-related antigen on an evanescent wave excitation fluorescence method. Examples of the substrate include chemical products such as glass and plastic. Disease-related antigens include malignant tumors, gastrointestinal diseases (such as ulcerative colitis), and respiratory diseases (such as idiopathic interstitial pneumonia) that are designated by the Ministry of Health, Labor and Welfare as intractable diseases (intractable diseases), for example. , Cardiovascular diseases (such as idiopathic dilated cardiomyopathy), renal / urological diseases (such as IgA nephropathy), blood system diseases (such as idiopathic thrombocytopenic purpura), immune system diseases (such as systemic lupus erythematosus) , Endocrine diseases (such as Cushing's syndrome), metabolic diseases (such as amyloidosis), neurological / muscular diseases (such as Guillain-Barre syndrome), skin / connective tissue diseases (such as pustular psoriasis), bone / joint diseases (idiopathic femur) And proteins that produce autoantibodies due to osteonecrosis of the head. In addition to these proteins (self-components), disease-related antigens include foreign molecules such as bacterial cell components and virus antigens.

  Examples of tumor-associated antigens from which autoantibodies are produced include many proteins such as p53, HER2, CEA, and Cyclin B1. The disease-related antigen may be a protein purified from a biological sample, and is cell-free such as in E. coli, yeast, insect cells and other host cells, or in E. coli extract, rabbit reticulocyte extract, wheat germ extract, etc. It may be a recombinant protein expressed in an expression system.

  Examples of the method for immobilizing a tumor-associated antigen on a substrate include a method using physical adsorption, a method using a chemical linker having a reactive group such as an epoxy group, a succinimide group, and a maleimide group.

For example, a total of 135 proteins of 9 × 15 can be immobilized on the substrate, and the layout can be designed freely.
In this embodiment, the amount of the disease-related protein required for the solid phase is, for example, 4 nL per spot at a concentration of 1 to 10 μg / mL, and the concentration of 1 to 10 μg / mL of the standard ELISA method. Compared with the need for 50 to 100 μL, it is much less. That is, the amount of antigenic protein that increases the cost can be suppressed to an overwhelmingly small amount in this embodiment.

  When the substrate on which the disease-related antigen is immobilized is brought into contact with the biological sample, the disease-related autoantibodies contained in the biological sample are bound to the disease-related antigen immobilized on the substrate. Thereby, disease-related autoantibodies can be captured on the substrate. Next, a fluorescently labeled secondary antibody that binds to the antibody is reacted.

(Step of exciting the fluorescent material with evanescent waves)
Subsequently, the fluorescent substance of the fluorescently labeled secondary antibody fixed on the substrate is excited by an evanescent wave through the disease-related autoantibody. In this step, for example, a fluorescence detection apparatus (trade name: GlycoStation (trademark) Reader 1200, manufactured by Glyco Technica) using an evanescent wave excitation fluorescence method can be used.

(Step of detecting the fluorescence emitted from the fluorescent substance)
Subsequently, the fluorescence emitted from the fluorescent material is detected. Since the evanescent wave excitation fluorescence method is a homogeneous assay in which the measurement is performed in a solution state throughout, a B / F separation (Bond / Free separation) operation such as washing is not required during the detection process. For this reason, for example, it does not affect the detection of a low-affinity antibody having a weak binding force. Measurement incorporating B / F separation is also possible, and in the case of medium to high affinity antibodies that are not affected by the binding to disease-related antigens by B / F separation, further enhancement of sensitivity is possible. Become. Since the amount of the disease-related autoantibody in the serum sample is proportional to the amount of the fluorescently labeled secondary antibody that binds to it, the fluorescence intensity can be quantified and the disease-related autoantibody can be quantified. A detection method in which a reaction or washing is performed using an immobilized antibody or the like, such as an ELISA method, is called a heterogeneous assay.

In one embodiment, the disease-related autoantibody may be a refractory disease-related autoantibody, a tumor-related autoantibody, or a combination thereof.
The disease-related autoantibody is preferably an antibody against at least a part of the disease-related protein, and is selected from the group consisting of a wild-type disease-related protein, a mutant disease-related protein, and a modified disease-related protein. More preferred is an antibody against at least one.
Examples of the disease-related protein having no abnormality in the gene and the like and subjected to post-translational modification include modified proteins such as phosphorylation, acetylation, methylation, myristoylation, glycosylation, and the like.

(Disease-related antigen)
Disease-related antigens include refractory disease-related proteins and tumor-related proteins.
The tumor-associated protein is preferably a protein encoded by an oncogene or a tumor suppressor gene.
Oncogenes include a gene group encoding a growth factor such as sis; a gene group encoding a receptor tyrosine kinase such as erbB, fms, ret; a gene group encoding a non-receptor tyrosine kinase such as ves; A gene group encoding a GTP / GDP-binding protein of Gr; a gene group encoding a serine / threonine kinase such as src, mos, raf; a gene group encoding a nuclear protein such as myc, myb, fos, jun, erbA; crk A gene group encoding a signal transduction adapter molecule such as; a fusion gene such as Bcr-Abl.
Furthermore, as oncogenes, Ras-MAP kinase pathway-related genes such as Shc, Grb2, Sos, MEK, Rho, and Rac genes; phospholipase C gamma-protein kinase C pathway-related genes such as PLCγ and PKC; PI3K, Akt, Bad PI3K-Akt pathway-related genes such as JAK-STAT pathway-related genes such as JAK and STAT; GAP pathway-related genes such as GAP, p180, and p62.

  Examples of the tumor suppressor gene include RB, p53, WT1, NF1, APC, VHL, NF2, p16, p19, BRCA1, BRCA2, PTEN, and E cadherin gene.

  The disease-related antigen may be a plurality of tumor-related proteins extracted from one type of carcinoma. That is, in one embodiment, the above-described detection method may be for cancer diagnosis.

  Examples of the carcinoma include skin cancer, lung cancer, colon cancer, stomach cancer, breast cancer, prostate cancer, thyroid cancer and the like. It has been reported that there are expression / mutation patterns of genes specific to carcinomas, including the oncogenes and tumor suppressor genes described above. Therefore, the disease detection rate can be improved by selecting a disease-related antigen based on a gene expression profile for each carcinoma.

  As an example, breast cancer-related antigens include p53, ER (Estrogen Receptor), PgR (Progesterone Receptor), HER2, CEA (Carcinoembryonic antigen), CYFRA (Cytokeratin 19-fragment) -3, CA15 15 IGFBP2 (IGF binding protein 2), Top 2 (Topoisomerase II), MUC1 (Mucin-1), Cyclin B1, Cyclin D1, HSP-27 (Heat shock protein 27), HSP-60 (o s 60) , GIPC- 1 (GAIP-interacting protein-1), c-myc, c-myb, NY-ESO-1, BRCA1 (breast cancer susceptibility gene, 1) It is done.

  As a tumor-related protein, it is preferable to select a protein that has already been measured by a routine test as a tumor marker. In addition to anti-p53 autoantibodies, the significance of detecting autoantibodies against known tumor markers is high, and considering the fact that anti-p53 autoantibodies become positive earlier than general tumor markers, many other known tumor markers Autoantibodies are also likely to have been produced from the early stage of the cancer stage before those tumor markers became positive. By combining optimal antigens, the early detection rate of cancer is significantly improved.

[Kit for detecting disease-related autoantibodies in biological samples]
In one embodiment, the present invention is a kit for simultaneously detecting autoantibodies in a biological sample against individual and / or mixed types of disease-related proteins using the evanescent wave excitation fluorescence method in the same reaction phase. A kit comprising a substrate on which a variety of individual and / or mixed types of disease-related proteins are immobilized in the same reaction phase, and a secondary antibody labeled with a fluorescent substance that recognizes autoantibodies against these disease-related proteins. provide.

(Antibodies to anti-disease related autoantibodies)
As described above, the disease-related autoantibody in the kit may be an antibody against at least a part of the disease-related protein. In addition, as described above, the disease-related autoantibody in this kit is an antibody against at least one selected from the group consisting of a wild-type disease-related protein, a mutant disease-related protein, and a modified disease-related protein. Also good. Further, as described above, the secondary antibody labeled with a fluorescent substance in this kit may react with a globulin type or subclass specific.

  The kit according to the present embodiment may further include a buffer solution for diluting the antibody against the biological sample or the anti-disease related autoantibody.

[Detection device for anti-disease-related autoantibody detection]
In one embodiment, the present invention is a detection apparatus for detecting a disease-related autoantibody, wherein a substrate on which various and / or mixed disease-related antigens are immobilized, and irradiation for generating evanescent waves on the surface of the substrate. There is provided a detection apparatus comprising: means; and detection means for detecting fluorescence emitted from the fluorescent material excited by the evanescent wave.

  The detection device is for detecting a disease-related autoantibody, and the disease-related antigen may be an intractable disease-related antigen. The detection device may be for detecting tumor-related autoantibodies, and the disease-related antigen may be a tumor-related antigen.

(Substrate on which disease-related antigen is immobilized)
In the detection apparatus of the present embodiment, the material of the substrate on which the disease-related antigen is fixed, the disease-related antigen to be fixed, the fixing method, and the like may be the same as those of the above-described substrate.

(Irradiation means for generating evanescent waves on the surface of the substrate)
Any irradiation means (generating means) for generating evanescent waves on the surface of the substrate may be used as long as it generates evanescent waves by total reflection. Specifically, for example, an evanescent wave irradiation unit of a fluorescence detection device (trade name: GlycoStation (trademark) Reader 1200, manufactured by Glyco Technica) using an evanescent wave excitation fluorescence method can be used as such irradiation means. . Examples of the light source of the evanescent wave include a laser, an LED, a mercury lamp, and a halogen lamp. The wavelength of the evanescent wave to be irradiated is preferably changeable by changing a light source, a dichroic mirror, a filter, or the like.

(Detection means for detecting the fluorescence emitted from the fluorescent material excited by the evanescent wave)
As a detection means for detecting the fluorescence emitted from the fluorescent material excited by the generation (irradiation) of the evanescent wave, for example, a CCD camera can be cited. The detection apparatus may include a program for acquiring a fluorescence image of the substrate and digitizing the fluorescence intensity of a specific spot on the substrate. Specifically, for example, a fluorescence detection unit of a fluorescence detection apparatus (trade name: GlycoStation (trademark) Reader 1200, manufactured by Glyco Technica) using an evanescent wave excitation fluorescence method can be used as such a detection means.

  According to the present embodiment, a technology that enables earlier detection of cancer, refractory disease, etc. than before by detecting autoantibodies in biological samples against various disease-related proteins simultaneously with high sensitivity. Can be provided.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

  In 64 breast cancer patients, autoantibodies against 7 kinds of tumor-related proteins including p53 protein were detected and compared by evanescent wave excitation fluorescence method.

(Detection of autoantibodies against p53 protein by ELISA)
Serum samples were collected from 64 breast cancer patients. Autoantibodies against the p53 protein present in these serum samples were measured by ELISA. For the measurement, a measurement kit (MESACUP anti-p53 test, manufactured by Medical Biology Laboratory Co., Ltd.) currently used in daily clinical tests was used. Serum to be tested was added to the well of a plastic plate on which wild-type full-length p53 protein was immobilized and reacted, and autoantibodies against p53 protein were detected with a peroxidase-conjugated anti-human IgG antibody.

  As a result, autoantibodies against p53 protein were detected in 10 out of 64 samples. In addition, autoantibodies against p53 protein were not detected from the remaining 54 samples.

(Detection of autoantibodies against 7 kinds of proteins including p53 protein by evanescent wave excitation fluorescence method)
Autoantibodies against p53, ER, PgR, HER2, CEA, CYFRA, and CA15-3 were detected from 64 serum samples identical to those examined by the ELISA method described above by evanescent wave excitation fluorescence.

First, a wild-type full-length p53 protein (Industry Research Institute, Entry Clone ID) is placed in a well of a glass substrate for a fluorescence detection apparatus (trade name: GlycoStation (trademark) Reader 1200, manufactured by Glyco Technica) using an evanescent wave excitation fluorescence method. FLJ92943AAAF), ER protein (Industry Research Institute, Entry clone ID; FLJ94220AAAN), PgR protein (Industry Research Institute, Entry clone ID; 100016179_F), HER2 protein (Industry Research Institute, Entry clone ID; FLJFLAN802A8, A12, A12) , CEA protein (National Institute of Advanced Industrial Science and Technology, Entry clone ID; 93953AAAF), CYFRA (CYTOKER) TIN19FRAGMENT) protein (Industry Research Institute, Entry Clone ID; FLJ93770AAAF), CA15-3 (MUC1) protein (Industry Research Institute, Entry Clone ID; FLJ60927AAAN) was dropped in a 4 nL spot at a concentration of 10 μg / mL. Then, the epoxy group on the substrate and the amino group of each protein were reacted and immobilized. Subsequently, after washing the glass substrate with a surfactant-added phosphate buffer, 100 μL of the above-mentioned serum sample diluted 10-fold with a surfactant-added phosphate buffer is added per 135 spots and reacted at room temperature for 1 hour. I let you. Subsequently, Cy3-labeled goat anti-human IgG (Fc) antibody (Jackson, model number 109-165-098) diluted 100-fold per 135 spots was added and reacted at room temperature for 0.5 hour. Subsequently, the fluorescence of Cy3 was detected with a fluorescence detection apparatus (trade name: GlycoStation (trademark) Reader 1200, manufactured by Glyco Technica) using an evanescent wave excitation fluorescence method, and the fluorescence intensity was digitized.
A cut-off value was set to 2000 RLU (relative fluorescence unit), and a sample in which fluorescence of 2000 RLU or more was detected was judged as positive, and a sample in which fluorescence of less than 2000 RLU was detected was judged as negative. The results of simultaneous detection of autoantibodies against 7 disease-related proteins are shown in FIG.

  In the Examples, the positive rate of anti-p53 autoantibodies that are effective in breast cancer patients was 15.6% (10/64) in the ELISA method, whereas, as shown in FIG. Among them, the positive rate of any of the cases in which any of the tumor-related autoantibodies was positive was 45.3% (29/64), which is a measurement of various tumor-related autoantibodies. It also increased by 2.90 times (45.3 / 15.6).

According to the present invention, there is provided a detection method capable of easily and simultaneously measuring autoantibodies against various disease-related proteins in a biological sample, as can be seen from the examples of autoantibody detection against seven kinds of proteins including p53 protein. Can do. In addition, according to the present invention, it is possible to provide a kit and an anti-disease-related autoantibody detection detection apparatus for easily and simultaneously measuring autoantibodies against various disease-related proteins in a biological sample.
The significance of measuring autoantibodies is high in cancer and intractable diseases. By examining disease-related proteins that are likely to produce efficient autoantibodies for early detection of diseases and measuring autoantibodies in combination, high positive rates can be strongly expected. Many proteins that produce autoantibodies in cancer and intractable diseases have been reported. If autoantibody profiling according to the present invention can be performed easily and simultaneously by measuring autoantibodies against various disease-related proteins, it will provide useful information for early detection of cancer and intractable diseases, judgment of therapeutic effects, etc. it can.

Claims (10)

In a biological sample for a disease-related protein on a substrate having multiple spots, one spot-spotted individual disease-related protein and / or one spot mixed with a plurality of types of disease-related proteins A method for detecting autoantibodies simultaneously in the same reaction phase,
The disease is breast cancer, and the disease-related protein includes seven types of breast cancer-related proteins p53, ER, PgR, HER2, CEA, CYFRA, and CA15-3;
A detection method using an evanescent wave excitation fluorescence method.   The detection method according to claim 1, wherein the disease-related autoantibody is an antibody against at least a part of the disease-related protein.   The detection according to claim 1 or 2, wherein the disease-related autoantibody is an antibody against at least one selected from the group consisting of a wild-type disease-related protein, a mutant disease-related protein, and a modified disease-related protein. Method.   A substrate having multiple spots of the disease-related autoantibodies in the biological sample, wherein one spot is spotted with individual disease-related antigens and / or one spot is a spot obtained by mixing a plurality of types of disease-related antigens. Reacting with the disease-related antigen above, and reacting a secondary antibody labeled with a fluorescent substance that binds to the disease-related autoantibody captured via the disease-related antigen on the substrate; The detection method according to any one of claims 1 to 3, comprising a step of exciting the fluorescent material on the substrate with an evanescent wave and a step of detecting fluorescence emitted from the fluorescent material on the substrate. .   The detection method according to claim 4, wherein the secondary antibody labeled with the fluorescent substance reacts specifically with a globulin type or a subclass. Using an evanescent wave excitation fluorescence method, on a substrate having multiple spots, one spot spotted with individual disease-related proteins and / or one spot spotted with multiple types of disease-related proteins mixed A kit for simultaneously detecting autoantibodies in a biological sample against the disease-related protein in the same reaction phase,
1 obtained by spot individual disease-associated proteins in spots, and / or 1 by mixing a plurality of types of disease-related protein spots those spots, a multi-spot fixed substrate in the same reaction phase, the The disease is breast cancer, and the disease-related protein comprises 7 types of breast cancer-related proteins p53, ER, PgR, HER2, CEA, CYFRA, and CA15-3;
Its characterized by comprising a secondary antibody labeled with recognizing a fluorescent substance autoantibodies, the against these disease-associated proteins, kits.   The kit according to claim 6, wherein the disease-related autoantibody is an antibody against at least a part of a disease-related protein.   The kit according to claim 6 or 7, wherein the disease-related autoantibody is an antibody against at least one selected from the group consisting of a wild-type disease-related protein, a mutant disease-related protein, and a modified disease-related protein. .   The kit according to any one of claims 6 to 8, wherein the secondary antibody labeled with the fluorescent substance reacts specifically with a globulin type or a subclass. A detection device for detecting disease-related autoantibodies, in which an individual disease-related protein is spotted in one spot and / or a mixture of multiple types of disease-related proteins is spotted in one spot. A substrate containing 7 types of breast cancer-related proteins of p53, ER, PgR, HER2, CEA, CYFRA, and CA15-3, wherein the disease is breast cancer,
Irradiating means for generating an evanescent wave on the surface before Symbol substrate,
Comprising detecting means for detecting the fluorescence emitted from the fluorescent material excited by pre Symbol evanescent wave, the detection device.

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