JP2011107064A - Immunoassay method of autoantibody to clathrin heavy chain, kit used for the same, and cancer determination method using the same - Google Patents

Abstract

An object of the present invention is to provide a method for measuring autoantibodies against clathrin heavy chain, which can be applied to cancer determination.
An anti-human immunoglobulin labeled with an auto-antibody produced by reacting an autoantibody against a clathrin heavy chain antigen in a specimen with a clathrin heavy chain antigen as a reagent. By measuring with an antibody, the autoantibody can be measured, and thereby cancer such as primary hepatocellular carcinoma can be determined.
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Description

  The present invention relates to an immunoassay method and an immunoassay kit for measuring autoantibodies against clathrin heavy chain. Autoantibodies against clathrin heavy chains appear specifically in the blood of cancer-bearing patients, and therefore the present invention not only provides a method for measuring autoantibodies, but also for cancer determination. Can also be used.

Clathrin is a protein that forms the skeleton that forms the outside of endosomes, which is formed when molecules in the extracellular matrix are taken up by endocytosis. The clathrin molecule has a triskelion structure, and during endosome formation, a plurality of clathrins are polymerized to form a lattice. The clathrin molecule has three legs and has a tripodal structure, each leg consisting of a clathrin heavy chain and a clathrin light chain. The clathrin heavy chain consists of 1675 amino acid residues and has a molecular weight of about 192 kDa. The molecular weight of the clathrin light chain is about 25-29 kDa (Non-patent Document 1).
On the other hand, by the improved agarose two-dimensional electrophoresis method (Non-patent Document 2) applying 2D-DIGE method (two-dimensional fluorescence difference gel electrophrosis) to agarose two-dimensional electrophoresis, the cancerous part of the primary hepatocellular carcinoma and the surrounding area Proteome analysis is performed to compare protein expression levels in non-cancerous tissues, and it has been clarified that clathrin heavy chains are often expressed in cancerous parts (Non-patent Document 3).

Dodge GR et al., Genomics 1991; 11: 174-178 Takeshi Tomonaga et al., Clin. Cancer Res. 2004; 10: 2007-2014 Masanori Seimiya et al., Hepatology 2008; 48: 519-30

  The present inventors can measure the expression level of clathrin heavy chain present in a tissue specimen derived from a patient suspected of cancer or a cancer patient to discriminate between a cancerous part and a non-cancerous part of those tissues. We found out what we could do and filed a patent application for this (Japanese Patent Application No. 264794). The present inventors further continued research on the presence of clathrin heavy chains in blood samples. Surprisingly, autoantibodies against clathrin heavy chains may exist in blood samples, and cancer patients In that case, the amount of the autoantibody was found to be large. Therefore, an object of the present invention is to provide a method for measuring autoantibodies against clathrin heavy chain, which can be applied to cancer determination.

The present inventors react an autoantibody against a clathrin heavy chain antigen in a specimen with a clathrin heavy chain antigen as a reagent, and label the resulting immunocomplex of the autoantibody and the clathrin heavy chain antigen with a labeled anti-human. By measuring with an immunoglobulin antibody, it was found that the autoantibody could be measured, thereby enabling cancer determination, and the present invention was completed.
Therefore, the present invention reacts with an autoantibody against a clathrin heavy chain in a sample and a clathrin heavy chain antigen as a reagent to produce an immune complex of the autoantibody against the clathrin heavy chain to be produced and the clathrin heavy chain antigen. The present invention relates to a method for immunoassay of autoantibodies against clathrin heavy chain, characterized in that the autoantibodies are measured by measuring.
Furthermore, the present invention relates to a kit for measuring autoantibody immunity against clathrin heavy chain, comprising at least clathrin heavy chain antigen as a reagent component.
Furthermore, this invention relates to the cancer determination method which determines that it is cancer by measuring the autoantibody with respect to a clathrin heavy chain.

  In the present invention, autoantibodies against clathrin heavy chains present in specimens, particularly blood-derived specimens, can be easily measured, and are effective in determining cancer patients such as primary hepatocellular carcinoma.

It is the result of having measured the autoantibody with respect to the clathrin heavy chain of a healthy subject sample, a primary primary hepatocellular carcinoma patient sample, and a recurrent primary hepatocellular carcinoma patient sample using the ELISA plate which sensitized the clathrin heavy chain antigen.

  The method for immunoassay of an autoantibody against a clathrin heavy chain according to the present invention comprises reacting an autoantibody against a clathrin heavy chain in a specimen with a clathrin heavy chain antigen as a reagent, It is characterized by measuring autoantibodies against clathrin heavy chains by measuring immune complexes with clathrin heavy chain antigens.

  In the present invention, the sample is preferably a biological sample, particularly a blood-derived sample, and examples of the blood-derived sample include whole blood, plasma, and serum.

  The measurement target of the immunoassay method of the present invention is an autoantibody against clathrin heavy chain, which is a receptor for intracellular transport and various macromolecular substances on the surface of cell membrane and organelle. It is one of the components of clathrin, a protein involved in phagocytosis. A clathrin heavy chain is a protein composed of 1675 amino acids having a molecular weight of 192 kDa, which is also designated as CHC and is encoded by Swiss-Prottory No, Q00160, and includes Hc; CHC; CHC17; CLH-17; CLTCL2; KIAA0034; Has an abbreviation.

  In order to carry out the immunoassay method of the present invention, a clathrin heavy chain antigen is used as a reagent. The clathrin heavy chain antigen as a reagent is not particularly limited as long as it is an antigen capable of antigen-antibody reaction with an autoantibody against the clathrin heavy chain, but it is a clathrin heavy chain full-length protein or a clathrin heavy chain full-length protein variant. 1 in the amino acid sequence of a protein or a clathrin heavy chain full-length protein having the same function as that of the protein and capable of antigen-antibody reaction with an autoantibody against a clathrin heavy chain and having 90% or more homology with the amino acid sequence. Mutant, a protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or added, a fragment peptide of clathrin heavy chain, and a peptide capable of antigen-antibody reaction with a clathrin heavy chain autoantibody Can be illustrated. The clathrin heavy chain full-length protein is available from Sigma, but since the entire amino acid sequence is known as described above, the clathrin heavy chain full-length protein and its mutants can also be synthesized by gene recombination techniques. When a fragment peptide of clathrin heavy chain is used in the present invention, the clathrin heavy chain full-length protein may be prepared by cleaving into various peptide fragments by enzymatic degradation or the like, or using a commercially available automatic peptide synthesizer. Can also be created easily. In addition, a fragment peptide of the target clathrin heavy chain can be prepared by a gene recombination technique.

  In the present invention, a variant or fragment peptide of the thus obtained clathrin heavy chain full-length protein is reacted with an autoantibody against the clathrin heavy chain to select an antigen-antibody reaction, and a class as a reagent. It can be used as a phosphorus heavy chain antigen. In the present invention, in addition to the whole peptide fragments described above, a part of them can be used, or a mixture thereof can be used, and these are also included in the clathrin heavy chain antigen as a reagent.

  In the present invention, for immunoassay of an autoantibody against clathrin heavy chain, for example, clathrin heavy chain antigen as a reagent is solid-phased on a microplate or other carrier, and the autoantibody is then applied to the resulting water-insolubilized carrier. A sample expected to be contained is applied and reacted with a clathrin heavy chain antigen as a reagent for antigen-antibody reaction, and then an anti-human immunoglobulin antibody labeled with an enzyme or the like is applied for reaction-binding to the autoantibody.

The water-insolubilized carrier can be easily prepared using a known method for binding a protein to a solid phase surface. For example, as the solid-phase support, beads, microplates, tubes and the like are usually used. As a method for binding a clathrin heavy chain antigen as a reagent to these solid phase surfaces, known immobilization techniques such as physical adsorption and chemical binding can be appropriately used.
When the clathrin heavy chain antigen thus immobilized is brought into contact with the autoantibody-containing specimen, only the autoantibody against the clathrin heavy chain specifically binds to the clathrin heavy chain antigen as a reagent. Therefore, when a labeled anti-human immunoglobulin antibody is added, the anti-human immunoglobulin antibody binds to an autoantibody against the clathrin heavy chain, and thus measurement can be performed using this label.

As the label, commonly used labels such as fluorescent substances such as enzymes, radioisotopes, FITC, rhodamine and luminol, and chemiluminescent substances are appropriately used. By using these various labels, autoantibodies against clathrin heavy chain can be measured by methods such as enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay and the like.
As the labeling enzyme used in the enzyme immunoassay, enzymes commonly used in enzyme immunoassay (EIA) such as horseradish peroxidase, bovine small intestine alkaline phosphatase, β-galactosidase, urease, glucose oxidase and the like are appropriately used. A chromogenic substrate that is suitable for EIA and commonly used in EIA is appropriately used. Examples of chromogenic substrates include 3,3 ', 5,5'-tetramethylbenzidine (TMBZ), TMBZ.HCl, TMBZ.PS, ABTS, o-phenylenediamine, p-hydroxyphenylacetic acid and the like in the case of HRP. In the case of alkaline phosphatase, p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate and the like are used, and in the case of β-galactosidase, o-nitrophenyl-β-D-galactopyranoside, 4-Methylumbelliferyl β-D-galactopyranoside and the like are used.
In the radioimmunoassay method, the fluorescence immunoassay method, the chemiluminescence immunoassay method, and the like, a commonly used known label can be employed.
In the immunoassay method of the present invention, in addition to the above-described method, the clathrin heavy chain may also be obtained by Western blot method, immunohistochemical staining method, immunoassay method such as latex immunoturbidimetric method and immunoprecipitation method, and liquid chromatography method. Autoantibodies against can be measured.

  The immunoassay method of the present invention can be carried out by a kit for measuring autoantibody immunity against clathrin heavy chain, which contains at least clathrin heavy chain antigen as a reagent component. The clathrin heavy chain antigen can be used as a reagent component of the kit in a form bound to a water-insoluble carrier such as a microplate. Other reagent components of the kit include an anti-human immunoglobulin antibody, such as an enzyme immunoassay method, a radioimmunoassay method, a fluorescence immunoassay method, a chemiluminescence immunoassay method, etc. to be adopted. Depending on the measurement method, those labeled with a label such as an enzyme, a radioisotope, a fluorescent substance, or a chemiluminescent substance are used. As other reagent components, a surfactant and a buffer may be added as appropriate.

  In the present invention, cancer can be determined by measuring autoantibodies against clathrin heavy chain. When the amount of autoantibodies to clathrin heavy chain is large, primary liver cancer (primary hepatocellular carcinoma, primary cholangiocellular carcinoma, etc.), liver cancer such as metastatic liver cancer, bladder cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer Cancers such as thyroid cancer and skin cancer are suspected, and measurement of autoantibodies against clathrin heavy chain according to the present invention is effective for discrimination of cancer diseases in patients. In the present invention, it is effective for discriminating primary hepatocellular carcinoma. For example, it is effective for discriminating between healthy individuals and patients with primary primary hepatocellular carcinoma or patients with recurrent primary hepatocellular carcinoma.

EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not restrict | limited to these Examples at all.
Example 1
Measurement of autoantibodies against clathrin heavy chains Autoantibodies against clathrin heavy chains are explained in detail below for serum samples collected from healthy individuals, patients with primary primary hepatocellular carcinoma, and patients with recurrent primary hepatocellular carcinoma. Measured by enzyme immunoassay (ELISA method).

1. Method (1) Preparation of clathrin heavy chain ELISA plate An ELISA plate (manufactured by Nunc, Maxisorp) was used as a water-insoluble carrier, and a clathrin heavy chain full-length body (Sigma, 12.5 μg / mL, 100 μL / well). The mixture was allowed to stand at 4 ° C. overnight for sensitization, and then washed three times with PBS containing 0.05% Tween 20 (200 μL / well). Subsequently, the clathrin heavy chain ELISA plate was prepared by coating overnight with PBS (200 μL / well) containing 1.5% BSA and 10% saccharose.

(2) Measurement of autoantibodies against clathrin heavy chain Serum of each sample was diluted 100 times with PBS, and 100 μL / well was added to the clathrin heavy chain ELISA plate and allowed to stand at 37 ° C. for 1 hour. Thereafter, the plate was washed three times with PBS containing 0.05% Tween 20 (200 μL / well). HRP-labeled immunoglobulin (HRP-labeled anti-Human IgG (manufactured by Zymed) diluted 4000-fold with PBS containing 0.05% Tween 20) was added to each plate at a rate of 100 μL / well for 30 minutes at 37 ° C. Left to stand. Next, the plate was washed 3 times with PBS containing 0.05% Tween 20 (200 μL / well), 100 μL / well of TMB was added, and the plate was allowed to stand at room temperature for 10 minutes, and then 100 μL / well as a reaction terminator. 1N sulfuric acid was added. Absorbance was measured at a wavelength of 450 nm using a microplate reader (BioRad).
The specimens used were 48 healthy subjects, 20 primary primary hepatocellular carcinoma patient specimens, and 20 recurrent primary hepatocellular carcinoma patient specimens.

2. Results The results of measuring autoantibodies against clathrin heavy chain using clathrin heavy chain ELISA plate are shown in FIG. For the significant difference test, KaleidaGraph 4.0 was used, and statistical processing was performed by Wilcoxon two-sample test.
As shown in FIG. 1, compared with the healthy subject group, a clear significant difference was observed between the primary primary hepatocellular carcinoma patient sample group and the recurrent primary hepatocellular carcinoma patient sample group. There was also a significant difference between the primary primary hepatocellular carcinoma patient sample group and the recurrent primary hepatocellular carcinoma patient sample group.

  As explained in detail above, autoantibodies against clathrin heavy chain in a specimen such as a blood-derived specimen react with clathrin heavy chain antigen as a reagent, By measuring the immune complex, it is possible to measure the autoantibodies, thereby making it possible to determine cancers such as primary hepatocellular carcinoma.

Claims (12)

  By reacting an autoantibody against a clathrin heavy chain in a specimen with a clathrin heavy chain antigen as a reagent, and measuring an immune complex of the autoantibody against the clathrin heavy chain to be produced and the clathrin heavy chain antigen, A method for immunoassay of an autoantibody against a clathrin heavy chain, which comprises measuring the autoantibody.   The immunoassay method according to claim 1, wherein the specimen is a blood-derived specimen.   The immunoassay method according to claim 1 or 2, wherein the immunoassay method is an enzyme immunoassay method, a fluorescence immunoassay method, a chemiluminescence immunoassay method, or a radioimmunoassay method.   The immunoassay method according to any one of claims 1 to 3, which is for cancer determination.   The immunoassay method according to claim 4, wherein the cancer is primary hepatocellular carcinoma.   A kit for measuring autoantibody immunity against clathrin heavy chain, comprising at least clathrin heavy chain antigen as a reagent component.   The kit according to claim 6, wherein the clathrin heavy chain antigen is bound to a water-insoluble carrier.   The kit according to claim 7, further comprising an anti-human immunoglobulin antibody.   The kit according to claim 8, wherein the anti-human immunoglobulin antibody is labeled with a label.   A method for determining cancer, wherein it is determined that the cancer is by measuring an autoantibody against a clathrin heavy chain.   The cancer determination method according to claim 10, wherein autoantibodies in a blood-derived specimen are measured.   The cancer determination method according to claim 11, wherein the cancer is primary hepatocellular carcinoma.