CN110632306B - Application of ENO2 autoantibody detection reagent in preparation of lung cancer screening kit - Google Patents
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- CN110632306B CN110632306B CN201911025239.3A CN201911025239A CN110632306B CN 110632306 B CN110632306 B CN 110632306B CN 201911025239 A CN201911025239 A CN 201911025239A CN 110632306 B CN110632306 B CN 110632306B
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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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
- G01N33/57488—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 involving compounds identifable in body fluids
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6884—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from lung
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4746—Cancer-associated SCM-recognition factor, CRISPP
Abstract
The invention relates to the field of in-vitro diagnostic reagents, in particular to an application of an ENO2 autoantibody detection reagent in preparing a lung cancer screening kit. The invention discovers for the first time that the autoantibody level of the ENO2 protein in the serum of a lung cancer patient is obviously lower than that of a healthy patient. According to the invention, the reagent for detecting the ENO2 protein autoantibody is used for preparing the lung cancer screening kit, so that effective screening of lung cancer can be realized.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to an application of an ENO2 autoantibody detection reagent in preparing a lung cancer screening kit.
Background
Lung cancer is one of the most common malignant tumors in the world, the morbidity and mortality of the lung cancer are on the rising trend year by year, the morbidity is at the top of the world at present, and the health and the life of human beings are seriously threatened.
The lung cancer is a disease good in occult, clinical symptoms are often shown only when the disease develops to the advanced stage, 70-80% of lung cancer patients are already at the middle and advanced stages when the lung cancer symptoms are diagnosed, cancer cells are diffused, the best curing time is missed, and the five-year survival rate is low. For early-stage lung cancer patients, the survival rate and the survival quality of the patients can be greatly improved by 5 years and more through timely treatment. Early diagnosis of lung cancer and effective screening are therefore of paramount importance.
The screening of the lung cancer refers to that the conventional physical examination is carried out on people without lung cancer related symptoms, and the lung cancer is found in time before the symptoms appear. If the lung cancer molecular marker in the plasma can be found, the molecular marker has important significance for prompting a clinician to take relevant treatment measures or decisions for a patient at an early stage.
Autoantibodies are antibodies produced by the body to self-organs, cells or cellular components. At present, autoantibodies to certain proteins have become markers for lung cancer, such as: p53, NY-ESO-1, CYFRA, etc. (Tang Z-M, Link Z-G, Wang C-M, Wu Y-B, Kong J-L (2017) Serum tune-associated autoimmune polymers for lung cancer: A systematic review and meta-analysis. PLoS ONE 12(7): e 0182117).
ENO2(Ensembl: ENSG00000111674) encodes one of the three enolase isozymes found in mammals, which is a homodimer, and is present in mature neurons and cells of neuronal origin, with a switch from alpha to gamma enolase occurring in neural tissue during the development of rats and primates. At present, reports related to ENO2 protein autoantibodies are not found, and the prior art related to lung cancer is not found.
Disclosure of Invention
The invention aims to provide a novel autoantibody lung cancer marker and application of a detection reagent of the marker in preparation of a lung cancer screening kit.
The technical scheme of the invention comprises the following steps:
application of a reagent for detecting an ENO2 protein autoantibody in preparation of a lung cancer screening kit.
As the application, the reagent for detecting the ENO2 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or a combined immunoassay reagent.
As for the application, the reagent for detecting the ENO2 protein autoantibody is a western blot reagent.
As mentioned above, the reagent for detecting the ENO2 protein autoantibody is a reagent for a protein chip detection method.
As for the aforementioned use, the reagent for detecting the ENO2 protein autoantibody is a reagent for detecting an ENO2 protein autoantibody in human serum.
A lung cancer screening kit comprises a reagent for detecting an ENO2 protein autoantibody.
As the kit, the reagent for detecting the ENO2 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunoassay reagent.
As for the kit, the reagent for detecting the ENO2 protein autoantibody is a western blot reagent.
As the kit, the reagent for detecting the ENO2 protein autoantibody is a reagent for a protein chip detection method.
As the kit, the reagent for detecting the ENO2 protein autoantibody is a reagent for detecting the ENO2 protein autoantibody in human serum.
The key point of the invention is that the content of the ENO2 autoantibody in human blood is determined to be obviously related to the risk of lung cancer, so that the risk of lung cancer can be judged by detecting the content of the ENO2 autoantibody in the human blood, and as for a means for specifically detecting the ENO2 autoantibody in the human blood, various means disclosed in the prior art can be adopted.
The invention provides a new lung cancer screening marker and a new lung cancer screening kit, which can realize effective screening of lung cancer; and the serum can be used as a detection sample, so that the harm to a patient 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.
Hereinafter, "ENO 2 autoantibody" refers to "ENO 2 protein autoantibody".
Drawings
FIG. 1: lung cancer patients (LC), healthy control (NC) plasma levels of ENO2 autoantibodies were compared.
FIG. 2: lung cancer patients (LC) were analyzed by ROC with healthy controls (NC).
Detailed Description
Example 1 correlation of ENO2 autoantibodies in plasma with Lung cancer
First, clinical data
59 lung cancer patients and 29 healthy controls were selected, and the basic information was as follows:
| basic information | Patients with lung cancer | Healthy controls |
| Number of people | 59 | 29 |
| Age (age) | 54.5±7.6 | 42±8.9 |
| Proportion of male | 37(62.7％) | 13(46.7％) |
Second, detection principle
HuProtTMThe human protein customizing chip is fixed with ENO2 protein (the adopted ENO2 protein is full-length protein, the protein serial number is ENSP00000229277), after the incubation by adding serum, ENO2 autoantibodies (mainly including IgG and IgM antibodies and also some other antibodies) in the serum can be combined, the unbound antibodies and other proteins are removed by cleaning, and then anti-human IgM fluorescent mark is usedDetecting a secondary antibody (cy5 labeled and showing red) and an anti-human IgG fluorescent secondary antibody (cy3 labeled and showing green), reading signals by a fluorescence scanner, wherein the strength of the signals is in positive correlation with the affinity and the quantity of the antibodies.
Third, method
The reagents used in this section were as follows:
the method comprises the following specific steps:
1) rewarming: taking out the chip from a refrigerator at-80 deg.C, putting in a refrigerator at 4 deg.C for rewarming for half an hour, and then putting in room temperature for rewarming for 15 min;
2) and (3) sealing: fixing 14 blocks in the rewarming chip, adding sealing liquid into each block after fixing, placing on a side swing bed, and sealing at room temperature for 3 hr;
3) incubation of serum samples: after sealing is finished, pouring the sealing liquid completely, then quickly adding a serum incubation liquid prepared in advance, wherein each chip can incubate 14 serum samples, the sample loading volume of each serum sample is 200 mu L, and the shaking table is laterally swung at 20rpm and incubated overnight at 4 ℃ (the serum samples are frozen and thawed in a chromatography cabinet at 4 ℃, and the incubation liquid is added to dilute in a ratio of 1: 50 to obtain the serum incubation liquid);
4) cleaning: the chip and the chip fence are taken out together, the sample is sucked, then the PBST with the same volume is added rapidly, and the cycle is repeated for a plurality of times, so that no cross contamination exists among the serum samples when the chip fence is detached. After the chip fence is removed, the chip is placed in a chip cleaning box with cleaning solution, and is cleaned for 3 times (10 min each time) by a horizontal shaking table at room temperature of 80 rpm;
5) and (3) secondary antibody incubation: transferring the chip into an incubation box added with 3mL of secondary antibody incubation liquid, laterally swinging a shaker at 40rpm, keeping out of the sun, and keeping at room temperature for 1 hr;
6) cleaning: the chip was removed (note that the upper surface of the chip was not touched or scratched), and placed in a chip washing cassette containing a washing solution, and washed 3 times 10min each time, on a horizontal shaker at room temperature and 80 rpm. After the completion, the mixture is washed for 2 times for 10min by ddH 2O;
7) drying;
8) scanning: scanning by using a crystal core LuxScan 10K microarray chip scanner;
9) data extraction: opening the corresponding GAL file (recording the position of protein in the chip), aligning the chip image and each array of the GAL file integrally, pressing an automatic alignment button, extracting data and storing.
Fourthly, the result
The mean expression level of ENO2 autoantibodies in the plasma of lung cancer patients was 79.0SNR (fluorescence signal to quantitative ratio), and the mean expression level of ENO2 autoantibodies in healthy control plasma was 110.1 SNR. The lung cancer group was statistically significant compared to healthy controls (p <0.05) (fig. 1). The results of ROC analysis of lung cancer groups versus healthy controls gave specificity of 96.6% and sensitivity of 17.0% (FIG. 2), indicating that ENO2 autoantibodies can specifically distinguish lung cancer from benign lung disease versus healthy controls.
The results show that the differences of the levels of the ENO2 autoantibodies in the serum of the lung cancer patients and the serum of the non-lung cancer patients are obvious, and the purpose of screening the lung cancer can be achieved by detecting the level of the ENO2 autoantibodies in the serum.
EXAMPLE 2 composition of the detection kit of the invention and method of use thereof
Kit composition
Detection kit (14 persons):
second, kit using method
Same as example 1, third part- "detection of ENO2 autoantibodies in serum".
The kit can screen the risk of lung cancer of a to-be-detected person by detecting the level of the ENO2 autoantibody in serum: if the level of ENO2 autoantibodies is low (relative to healthy people), the risk of lung cancer is high, and if the level of ENO2 autoantibodies is high, the risk of lung cancer is low. The method can be used for the auxiliary diagnosis of clinical lung cancer, provides effective basis for patients to take relevant treatment measures or decisions, and has good clinical application prospect.
Claims (4)
1. The application of a reagent for detecting an ENO2 protein autoantibody in human serum in preparing a lung cancer screening kit.
2. The use of claim 1, wherein the reagent for detecting the autoantibody to the ENO2 protein in human serum is a reagent for enzyme-linked immunosorbent assay.
3. The use according to claim 1, wherein the reagent for detecting autoantibodies to the ENO2 protein in human serum is a western blot reagent.
4. The use of claim 1, wherein the reagent for detecting the autoantibody to the ENO2 protein in human serum is a reagent for a protein chip detection method.
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Citations (1)
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| CN109073660A (en) * | 2016-02-29 | 2018-12-21 | 麦恩泰科特有限公司 | It can be used for treating the predictive marker of wet age related macular degeneration |
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| US20050130245A1 (en) * | 2001-09-17 | 2005-06-16 | Syn X Pharma, Inc. | Diagnosis and treatment of early pre-type-1 diabetes utilizing neuronal proteins |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109073660A (en) * | 2016-02-29 | 2018-12-21 | 麦恩泰科特有限公司 | It can be used for treating the predictive marker of wet age related macular degeneration |
Non-Patent Citations (2)
| Title |
|---|
| Diagnostic value of α-enolase expression and serum α-enolase autoantibody levels in lung cancer;Lihong Zhang,et al;《J Bras Pneumol.》;20181231;第44卷(第1期);18-23页 * |
| Serum tumor-associated autoantibodies as diagnostic biomarkers for lung cancer：A systematic review and meta-analysis;Zhen-Ming Tang, et al;《Zhen-Ming Tang, et al》;20170727;第12卷(第7期);摘要，第4页倒数第3行至第5页第4行，第10页，表1 * |
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