CN111273012B - Method for combined detection of serum autoantibodies - Google Patents

Method for combined detection of serum autoantibodies Download PDF

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CN111273012B
CN111273012B CN202010142815.9A CN202010142815A CN111273012B CN 111273012 B CN111273012 B CN 111273012B CN 202010142815 A CN202010142815 A CN 202010142815A CN 111273012 B CN111273012 B CN 111273012B
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ctag1a
elavl4
uchl1
autoantibody
gage7
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CN111273012A (en
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王红叶
李永哲
韩晓红
姚林
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Yiling Beijing Biotechnology Co ltd
Beijing Sanpin Medical Technology Co ltd
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Yiling Beijing Biotechnology Co ltd
Beijing Sanpin Medical Technology Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung

Abstract

The invention belongs to the technical field of diagnosis or detection of tumor-related markers, and particularly relates to an application of an autoantibody or a detection reagent of the autoantibody in preparation of a product for diagnosing tumors and a method for combined detection of serum autoantibody. The antigen protein used in the method is selected from the combination of more than two of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ETHE 1. The invention coats antigen protein on solid phase carrier, and can detect the level of autoantibody with affinity to protein by detecting signal molecule strength. The method can well distinguish cancer from healthy people, and provides an optimization method for cancer screening by taking the autoantibody as an index.

Description

Method for combined detection of serum autoantibodies
Technical Field
The invention belongs to the technical field of diagnosis or detection of tumor-related markers, and particularly relates to an application of an autoantibody or a detection reagent of the autoantibody in preparation of a product for diagnosing tumors and a method for combined detection of serum autoantibody.
Background
According to the data of the world health organization, in 2018, the number of new cancer patients reaches 1810 ten thousand, and 960 ten thousand cancer patients die. 209 ten thousand new cases of lung cancer occurred globally in 2018, ranked first among all cancer types. According to the statistics of the national cancer center in 2017, the incidence and mortality of lung cancer are ranked first in malignant tumors. The incidence was first ranked in men (20.27%) and second only to breast cancer in women (17.07%) at 14.94%. Mortality rates were first in both men and women, 23.89% and 17.70%, respectively. It is predicted that lung cancer mortality in china may increase by about 40% in 2015 to 2030 years.
Lung cancer can be divided histologically into two main categories: small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 79% of the diagnosed lung cancers, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Despite the great advances in recent years in targeted therapies and immunotherapy for lung cancer, surgical resection, adjuvant radiotherapy and/or chemotherapy remain the first-choice methods for early treatment of NSCLC patients. Despite the improved survival rates of all cancers in recent years, the survival rates of lung cancer are still at a low level. In 2012 and 2015, the survival rate of lung cancer in men was 16.8%, which was 62.5% lower than that of thyroid cancer with the highest survival rate, and the survival rate of women was only 25.1%. The low survival rate of lung cancer is associated with limitations in diagnostic techniques, and most lung cancer patients are found at an advanced stage at the time of diagnosis, losing the best treatment opportunity. Currently, high-resolution (or low-dose) computed tomography (LDCT) of the breast is the only screening test that is effective in reducing early mortality from lung cancer. Many regions have established a project for free screening of high risk populations by LDCT. One study in the united states has shown that LDCT screening has a better early screening potential for lung cancer, or permits a 20% reduction in mortality in high risk groups with lung cancer. Clinical transformation remains a problem. LDCT also has the problem that a high false positive rate can lead to over-diagnosis to some extent. Repeated exposure to radiation may also lead to potential health hazards. Therefore, the discovery of non-invasive serological biomarkers for early lung cancer diagnosis would greatly facilitate lung cancer intervention and prevention. The method of LDCT after initial screening based on biomarkers or combining biomarker testing with LDCT becomes a better means for diagnosing lung cancer.
People have perceived that the immune system of the body has possible prevention and treatment effects on tumors more than 100 years ago. Tumor growth is accompanied by the production of some substance with antigenic properties. These substances are no longer treated by the body as "self" but as antigens. The immune system will produce antibodies to destroy these antigens. These antibodies are called autoantibodies. Since the last 70 th century when autoantibodies were discovered, hundreds of autoantibodies have been identified. Tumor-associated autoantibody detection provides an alternative to cancer detection. This approach is expected to be achieved by detecting high expression (relative to healthy humans and benign tumors) of autoantibodies in the serum of cancer patients. Currently used in the clinic are tumor associated antigens such as Carbohydrate Antigen (CA)125, CA19-9, carcinoembryonic antigen (CEA) and Alpha Fetoprotein (AFP). The content of these markers in serum is increased with the increase in tumor size, so that the sensitivity and specificity of tumor-associated antigens in early stages of cancer are very poor and false positive results may occur in infections, benign tumors or pregnancy. Autoantibodies are produced early in tumorigenesis and can be detected five years before clinical symptoms appear. In early stages of cancer, the signal of the detected tumor-associated antigen is very weak, but since the concentration of autoantibodies is much higher than that of the antigen, detection of autoantibodies is equivalent to obtaining an amplified signal. Autoantibodies have a half-life of up to 30 days in blood circulation and are more stable in vitro than other markers. The autoantibody detection has the advantages of economy, low cost, simple operation and the like. Autoantibody detection can be achieved directly on existing clinical detection platforms using archived serum.
For a single antigenic protein, its autoantibodies appear only in 10% -30% of patients and are less sensitive as a diagnostic tool. This may be associated with tumor heterogeneity and diversity of immune responses. Since immune system responses vary among patients even for the same cancer, it is necessary to bind a plurality of autoantibodies to improve detection sensitivity while maintaining high specificity. In recent years, many studies have been directed to the detection of a range of autoantibodies for cancer diagnosis. For example, Zhang et al established a combination of seven autoantibody markers (14-3-3 ζ, c-Myc, MDM2, NPM1, p16, p53 and cyclin B1) to detect the corresponding autoantibody levels in the sera of lung cancer patients and healthy controls. The results show that this combination can increase the sensitivity to 68.9% and the specificity to 79.5%. They also tested a series of sera from lung cancer patients before they were diagnosed and found that elevated autoantibody levels could be detected more than four years before lung cancer diagnosis. The sensitivity and specificity were 76.0% and 73.2%, respectively (Dai, Liping, et al, "Autoantibodies against molecular-associated antigens in the early detection of Long Cancer. Lung Cancer99(2016):172- > 179.).
Autoantibodies to tumor-associated antigens can increase significantly months or even years prior to clinical symptoms. Autoantibodies are more easily detected than antigens through multiple amplification of B cell proliferation. The problem of low sensitivity of a single autoantibody is solved by simultaneously detecting a plurality of autoantibodies, and a method with high specificity and high sensitivity is provided for early diagnosis and postoperative monitoring of cancer.
Disclosure of Invention
The invention aims to provide a marker for diagnosing tumor, wherein the marker is an autoantibody, and early diagnosis of a tumor patient is realized by detecting the autoantibody. Solves the problem of poor specificity and sensitivity of taking antigen protein as a diagnostic marker, can be diagnosed five years before clinical symptoms appear, and provides a method with high specificity and high sensitivity for early diagnosis and postoperative detection of cancer.
In order to achieve the above object, the present invention provides an autoantibody or an application of a detection reagent for the autoantibody in preparing a product for diagnosing a tumor, wherein the autoantibody is selected from a combination of two or more of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ete 1.
Wherein, the two or more can be two, three, four, five, six, seven or eight.
Preferably, the autoantibody is IgG and/or IgA. Further preferably, the autoantibody comprises IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgE or IgD.
Preferably, the autoantibody is selected from the group consisting of five, six, seven or eight of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ete 1.
Preferably, the detection reagent comprises an antigenic protein selected from the group consisting of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ete 1.
Wherein, the two or more can be two, three, four, five, six, seven or eight.
Further preferably, the antigenic protein is selected from the group consisting of five, six, seven or eight of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ete 1.
In one embodiment of the invention, the autoantibody is selected from any one of the following combinations:
(a)CTAG1A，ELAVL4，UCHL1，p53，ETHE1；
(b)CTAG1A，ELAVL4，UCHL1，p53，ETHE1，ANXA1；
(c)CTAG1A，ELAVL4，UCHL1，p53，ETHE1，MAGEA4；
(d)CTAG1A，ELAVL4，UCHL1，p53，ETHE1，GAGE7；
(e)ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1；
(f)CTAG1A，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1；
(g)CTAG1A，ANXA1，ELAVL4，UCHL1，GAGE7，p53，ETHE1；
(h)CTAG1A，ANXA1，MAGEA4，UCHL1，GAGE7，p53，ETHE1；
(i)CTAG1A，ANXA1，MAGEA4，ELAVL4，GAGE7，p53，ETHE1；
(j)CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，p53，ETHE1；
(k)CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，ETHE1；
(l) CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p 53; or
(m)CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1。
Preferably, the tumor is lung cancer. The lung cancer includes Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). Further preferred include, but are not limited to, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer or small cell lung cancer.
The invention also provides a product for diagnosing tumors, which comprises a detection reagent of the autoantibody, wherein the autoantibody is selected from the combination of more than two of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ETHE 1.
Wherein, the two or more can be two, three, four, five, six, seven or eight.
Preferably, the autoantibody is IgG and/or IgA. Further preferably, the autoantibody comprises IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgE or IgD.
Preferably, the autoantibody is selected from the group consisting of five, six, seven or eight of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ete 1.
Preferably, the detection reagent comprises an antigenic protein selected from the group consisting of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ete 1.
Wherein, the two or more can be two, three, four, five, six, seven or eight.
Further preferably, the antigenic protein is selected from the group consisting of five, six, seven or eight of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ete 1.
In one embodiment of the invention, the autoantibody is selected from any one of the following combinations:
(a)CTAG1A，ELAVL4，UCHL1，p53，ETHE1；
(b)CTAG1A，ELAVL4，UCHL1，p53，ETHE1，ANXA1；
(c)CTAG1A，ELAVL4，UCHL1，p53，ETHE1，MAGEA4；
(d)CTAG1A，ELAVL4，UCHL1，p53，ETHE1，GAGE7；
(e)ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1；
(f)CTAG1A，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1；
(g)CTAG1A，ANXA1，ELAVL4，UCHL1，GAGE7，p53，ETHE1；
(h)CTAG1A，ANXA1，MAGEA4，UCHL1，GAGE7，p53，ETHE1；
(i)CTAG1A，ANXA1，MAGEA4，ELAVL4，GAGE7，p53，ETHE1；
(j)CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，p53，ETHE1；
(k)CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，ETHE1；
(l) CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p 53; or
(m)CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1。
Preferably, the tumor is lung cancer. The lung cancer includes Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). Further preferred include, but are not limited to, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer or small cell lung cancer.
The invention further provides a method for detecting the autoantibody, wherein the autoantibody is selected from the combination of more than two of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ETHE 1.
Wherein, the two or more can be two, three, four, five, six, seven or eight.
Preferably, the autoantibody is IgG and/or IgA. Further preferably, the autoantibody comprises IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgE or IgD.
Further preferably, the autoantibody is selected from the group consisting of five, six, seven or eight of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ete 1.
Preferably, the autoantibody is selected from any one of the following combinations:
(a)CTAG1A，ELAVL4，UCHL1，p53，ETHE1；
(b)CTAG1A，ELAVL4，UCHL1，p53，ETHE1，ANXA1；
(c)CTAG1A，ELAVL4，UCHL1，p53，ETHE1，MAGEA4；
(d)CTAG1A，ELAVL4，UCHL1，p53，ETHE1，GAGE7；
(e)ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1；
(f)CTAG1A，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1；
(g)CTAG1A，ANXA1，ELAVL4，UCHL1，GAGE7，p53，ETHE1；
(h)CTAG1A，ANXA1，MAGEA4，UCHL1，GAGE7，p53，ETHE1；
(i)CTAG1A，ANXA1，MAGEA4，ELAVL4，GAGE7，p53，ETHE1；
(j)CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，p53，ETHE1；
(k)CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，ETHE1；
(l) CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p 53; or
(m)CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1。
Preferably, the detection method comprises immobilizing the antigenic protein on a solid support. Wherein the antigen protein is selected from combination of more than two of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ETHE 1. Wherein, the two or more can be two, three, four, five, six, seven or eight. Further preferably, the antigenic protein is selected from the group consisting of five, six, seven or eight of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ete 1.
Preferably, the solid phase carrier includes but is not limited to one or a combination of more than two of an enzyme-labeled micro-porous plate, a micro-particle, a micro-sphere, an affinity membrane, a liquid phase chip, a glass slide, a test strip and a plastic sphere.
Preferably, the method for immobilizing the antigen protein includes a direct coating method and an indirect coating method. Wherein, the direct coating method is to directly fix the antigen protein on a solid phase carrier. The indirect coating method is to indirectly fix the antigen on the solid phase carrier through the specific reaction between biotin and streptavidin.
Preferably, the detection method includes signal detection by visible light color development, chemiluminescence, fluorescence, or the like.
Preferably, the detection reagent further comprises a secondary antibody containing a marker, a blocking buffer and/or a washing buffer, and the like.
Preferably, the use thereof is in the diagnosis of tumors; further preferably, the detection method is used for auxiliary diagnosis of lung cancer.
Preferably, the tumor is lung cancer. The lung cancer includes Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). Further preferred include, but are not limited to, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer or small cell lung cancer.
The antigen protein of the invention can be natural or artificial. In a specific embodiment of the present invention, the antigen protein is artificially synthesized, and specifically comprises the steps of using a cloning plasmid containing a target gene as a template, amplifying a desired gene fragment by PCR, introducing the amplified gene fragment into escherichia coli, yeast or mammalian cells, expressing and purifying the amplified gene fragment to obtain the antigen protein. The autoantibodies of the invention may be in the serum, plasma, interstitial fluid, cerebrospinal fluid or urine of a human or non-human animal. The non-human animal is a non-human mammal, including but not limited to mouse, rat, monkey, zebrafish, pig, chicken, rabbit, etc.
The "product" of the present invention includes, but is not limited to, a kit containing reagents required for the reaction of an antigenic protein with an autoantibody, reagents required for the binding of a solid support and an antigenic protein to a solid support, and reagents conventionally selected such as a reagent for eluting unbound substances.
"tumors" as referred to herein include, but are not limited to, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and biliary tract cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; said lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. Preferably, the tumor is lung cancer. In one embodiment of the invention, the tumor is lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer or small cell lung cancer.
"diagnosis" as used herein refers to the determination of whether a patient has suffered from a disease or condition in the past, at the time of diagnosis, or in the future, or the determination of the progression or likely progression of a disease in the future, or the assessment of a patient's response to a therapy.
The invention provides a novel cancer detection method, which improves the sensitivity and specificity of detection by detecting a plurality of autoantibodies, and provides possibility for clinical cancer screening and auxiliary diagnosis; the operation is convenient and simple, the cost is low, and the serum can be used as a detection sample, so that the harm to patients is low. The invention has good application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The foregoing aspects of the present invention are explained in further detail below with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: comparing the levels of a plurality of autoantibodies in lung cancer patients to healthy controls, wherein figure 1A is CTAG 1A; FIG. 1B is ANXA 1; FIG. 1C is MAGEA 4; FIG. 1D is ELAVL 4; fig. 1E is UCHL 1; FIG. 1F is GAGE 7; FIG. 1G is p 53; fig. 1H is ete 1.
FIG. 2: ROC analysis of autoantibodies in plasma of lung cancer patients and healthy control sera, wherein FIG. 2A is CTAG 1A; FIG. 2B is ANXA 1; FIG. 2C is MAGEA 4; FIG. 2D is ELAVL 4; fig. 2E is UCHL 1; FIG. 2F is GAGE 7; FIG. 2G is p 53; fig. 2H is ete 1.
FIG. 3: ROC profiles of different antigen combinations in lung cancer and healthy patients, where fig. 3A is CTAG1A, ELAVL4, UCHL1, p53, ete 1; FIG. 3B shows CTAG1A, ELAVL4, UCHL1, p53, ETHE1, ANXA 1; FIG. 3C shows CTAG1A, ELAVL4, UCHL1, p53, ETHE1, MAGEA 4; FIG. 3D shows CTAG1A, ELAVL4, UCHL1, p53, ETHE1, GAGE 7; FIG. 3E shows ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53, ETHE 1; FIG. 3F shows CTAG1A, MAGEA4, ELAVL4, UCHL1, GAGE7, p53, ETHE 1; FIG. 3G is CTAG1A, ANXA1, ELAVL4, UCHL1, GAGE7, p53, ETHE 1; FIG. 3H is CTAG1A, ANXA1, MAGEA4, UCHL1, GAGE7, p53, ETHE 1; FIG. 3I is CTAG1A, ANXA1, MAGEA4, ELAVL4, GAGE7, p53, ETHE 1; FIG. 3J shows CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, p53, ETHE 1; FIG. 3K is CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, ETHE 1; FIG. 3L shows CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p 53; FIG. 3M shows CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53, ETHE 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 recombinant antigenic protein expression and purification
The cloning plasmid containing the target gene is used as a template, and the required gene segment is obtained by PCR amplification. The amino acid sequence of the CTAG1A can be shown as SEQ ID NO. 1; the amino acid sequence of the ANXA1 can be shown as SEQ ID NO. 2; the amino acid sequence of the MAGEA4 can be shown as SEQ ID NO. 3; the amino acid sequence of ELAVL4 can be shown as SEQ ID NO. 4; the amino acid sequence of UCHL1 can be shown as SEQ ID NO. 5; the amino acid sequence of the GAGE7 can be shown as SEQ ID NO. 6; the amino acid sequence of the p53 can be shown as SEQ ID NO. 7; the amino acid sequence of the ETHE1 can be shown as SEQ ID NO. 8.
The plasmid vector pET-21a (+) is cut by BamHI and EcoRI for double digestion, and after agarose electrophoresis of the cut product, the large fragment of the vector is recovered by using gel recovery Kit. The PCR product was similarly recovered after double digestion with BamHI and EcoRI, and ligated into a vector under the action of a recombinase. And transforming the ligation product into escherichia coli DH5 alpha, screening according to the marker (ampicillin resistance) of the recombinant vector, picking out single spots, and identifying and confirming that the obtained recombinant plasmid contains a correct exogenous fragment by colony PCR and sequencing.
The recombinant plasmid containing the antigen fragment obtained in the above is transformed into competent cells of escherichia coli BL21(DE3), the induction expression is carried out through isopropyl thio-beta-D-galactoside (IPTG), the obtained recombinant antigen protein is purified through two steps of Ni-NTA column and molecular sieve, then the quantification is carried out through Bradford method, the expression, purification and quantification results of the antigen protein are identified and confirmed through SDS-PAGE, and the obtained antigen protein is stored for standby.
Example 2 Combined detection of multiple autoantibodies
First, clinical sample
160 lung cancer patients and 107 healthy controls were selected, and the basic information is shown in table 1:
TABLE 1
Figure BDA0002399682620000101
Second, detection principle
The antigen proteins of CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p53 and ETHE1 prepared in the example 1 are fixed on an enzyme-labeled pore plate, after serum is added for incubation, each antigen protein in the serum is combined with autoantibodies (mainly comprising IgG and IgA antibodies and also other types of antibodies) corresponding to the autoantibodies, the unbound antibodies and other proteins are washed away, an anti-human IgG horseradish peroxidase labeled secondary antibody is added for incubation, the unbound secondary antibody is washed away, a horseradish peroxidase substrate is added, after the substrate is developed, the substrate is detected by an enzyme reader, and the strength of the signal is in positive correlation with the number of the autoantibodies.
Third, method
The reagents used in this section are shown in table 2:
TABLE 2
Figure BDA0002399682620000102
Figure BDA0002399682620000111
1) Coating antigen protein: diluting the protein by using a coating buffer solution, respectively adding 50 ng/hole of the diluted protein into reaction holes of a polystyrene plate, and standing overnight at 4 ℃;
2) rewarming: and taking the enzyme label plate out of a refrigerator at 4 ℃, putting the enzyme label plate at room temperature for rewarming for half an hour, and washing for four times.
3) And (3) sealing: adding a confining liquid, incubating for one hour at 37 ℃, and washing twice;
4) adding a serum sample: adding a 1:200 diluted serum sample, placing the serum sample in a side-swinging shaking table at 12rpm, incubating for 2.5h at room temperature, washing for four times (the serum needs to be placed in a4 ℃ freeze-thaw in advance, and the diluted serum is vibrated and mixed uniformly and then added with the sample);
5) adding an enzyme-labeled antibody: to each reaction well, horseradish peroxidase-labeled anti-human IgG (4. mu.g/ml) was added. Placing the mixture on a side-swinging shaking table at 12rpm, incubating the mixture for 1 hour at room temperature in a dark place, and washing the mixture twice;
6) adding a chromogenic substrate: adding 50 μ l of temporarily prepared TMB substrate solution into each reaction well;
7) and (4) terminating: after the liquid in the wells turned blue, 25. mu.l of 2M H was added to each well2SO4；
8) And (4) computer reading: readings of 0D 450 were measured in a TECAN F50 microplate reader and the data were saved.
Different antigen combinations were chosen as follows:
1.CTAG1A，ELAVL4，UCHL1，p53，ETHE1。
2.CTAG1A，ELAVL4，UCHL1，p53，ETHE1，ANXA1。
3.CTAG1A，ELAVL4，UCHL1，p53，ETHE1，MAGEA4。
4.CTAG1A，ELAVL4，UCHL1，p53，ETHE1，GAGE7。
5.ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1。
6.CTAG1A，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1。
7.CTAG1A，ANXA1，ELAVL4，UCHL1，GAGE7，p53，ETHE1。
8.CTAG1A，ANXA1，MAGEA4，UCHL1，GAGE7，p53，ETHE1。
9.CTAG1A，ANXA1，MAGEA4，ELAVL4，GAGE7，p53，ETHE1。
10.CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，p53，ETHE1。
11.CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，ETHE1。
12.CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，p53。
13.CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1。
the specificity and sensitivity of the different antigen combinations are shown in table 3:
TABLE 3
Figure BDA0002399682620000121
Fourth, result analysis
1. Based on the current sample analysis, the expression levels of CTAG1A (fig. 1A), ELAVL4 (fig. 1D), UCHL1 (fig. 1E), p53 (fig. 1G), and ete 1 (fig. 1H) autoantibodies in the plasma of lung cancer patients were shown to be statistically significantly different from healthy controls.
2. In the single-index autoantibody ROC analysis (fig. 2) of the lung cancer group and the healthy group, when the specificity was 90%, the sensitivities of CTAG1A (fig. 2A), ELAVL4 (fig. 2D), and p53 (fig. 2G) were better, 35.4%, 23.3%, and 32.3%, respectively.
3. In ROC analysis (fig. 3) of different antigen combinations to identify lung cancer and healthy people, sensitivity was seen to be significantly improved by combining different antigens when the specificity was 90% compared to a single index. Wherein the combination 7, 8, 9, 12 and 13 has a sensitivity of 51.7%, 52.4%, 51.7%, 52.0% and 51.7%, respectively, of 50% or more at a specificity of 90%.
From the above results, it can be seen that lung cancer and healthy persons can be identified by measuring the autoantibody level in a serum sample by the present method. Different autoantibody indexes are combined, which is helpful for improving the detection rate and accuracy of cancer diagnosis. The method has good application prospect in clinical auxiliary diagnosis.

Claims (5)

1. The use of an autoantibody or a detection reagent for the autoantibody in the manufacture of a product for diagnosing lung cancer, wherein the autoantibody is selected from the group consisting of autoantibodies derived from any one of the following combinations of antigenic proteins:
(1)CTAG1A，ANXA1，ELAVL4，UCHL1，GAGE7，p53，ETHE1；
(2)CTAG1A，ANXA1，MAGEA4，UCHL1，GAGE7，p53，ETHE1；
(3)CTAG1A，ANXA1，MAGEA4，ELAVL4，GAGE7，p53，ETHE1；
(4) CTAG1A, ANXA1, MAGEA4, ELAVL4, UCHL1, GAGE7, p 53; or
(5)CTAG1A，ANXA1，MAGEA4，ELAVL4，UCHL1，GAGE7，p53，ETHE1。
2. The use of claim 1, wherein the autoantibody is an IgG and/or IgA.
3. The use of claim 1, wherein the autoantibody comprises IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgE or IgD.
4. The use of claim 1, wherein the lung cancer is selected from the group consisting of small cell lung cancer and non-small cell lung cancer.
5. The use of claim 1, wherein the lung cancer is selected from lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer or small cell lung cancer.
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