CN110596387A - Application of COX10 autoantibody detection reagent in preparation of lung cancer screening kit - Google Patents
Application of COX10 autoantibody detection reagent in preparation of lung cancer screening kit Download PDFInfo
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- CN110596387A CN110596387A CN201910894448.5A CN201910894448A CN110596387A CN 110596387 A CN110596387 A CN 110596387A CN 201910894448 A CN201910894448 A CN 201910894448A CN 110596387 A CN110596387 A CN 110596387A
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- 101000919980 Homo sapiens Protoheme IX farnesyltransferase, mitochondrial Proteins 0.000 title claims abstract description 50
- 102100030729 Protoheme IX farnesyltransferase, mitochondrial Human genes 0.000 title claims abstract description 50
- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 49
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 49
- 201000005202 lung cancer Diseases 0.000 title claims abstract description 49
- 208000020816 lung neoplasm Diseases 0.000 title claims abstract description 49
- 238000012216 screening Methods 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title description 3
- 210000002966 serum Anatomy 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 241000282414 Homo sapiens Species 0.000 claims description 8
- 238000002965 ELISA Methods 0.000 claims description 6
- 238000001262 western blot Methods 0.000 claims description 4
- 238000003018 immunoassay Methods 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 238000011534 incubation Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 208000019693 Lung disease Diseases 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 description 1
- 101150115542 Cox10 gene Proteins 0.000 description 1
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000013075 data extraction Methods 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000010197 meta-analysis Methods 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
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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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Abstract
The invention relates to the field of in-vitro diagnostic reagents, in particular to an application of a COX10 autoantibody detection reagent in preparing a lung cancer screening kit. The invention discovers for the first time that the autoantibody level of COX10 protein in the serum of a lung cancer patient is significantly lower than that of a healthy patient. According to the invention, the reagent for detecting the COX10 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 a COX10 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, WangC-M, Wu Y-B, Kong J-L (2017) Serum tune-associated autoimmune agents as diagnostic biologics for lung cancer: A systematic review and meta-analysis. PLoS ONE 12(7): e 0182117).
COX10 gene (gene sequence number Ensembl: ENSG00000006695) is widely distributed in testis, heart and other tissues. At present, no report related to COX10 protein autoantibody exists, and no prior art related to lung cancer exists.
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:
the application of a reagent for detecting COX10 protein autoantibody in preparing a lung cancer screening kit.
As the application, the reagent for detecting the COX10 protein autoantibody is a reagent for an enzyme-linked immunosorbent assay or a combined immunoassay reagent.
As for the above-mentioned application, the reagent for detecting the COX10 protein autoantibody is a western blot reagent.
As mentioned above, the reagent for detecting COX10 protein autoantibody is a reagent for protein chip detection method.
As for the aforementioned use, the reagent for detecting the COX10 protein autoantibody is a reagent for detecting the COX10 protein autoantibody in human serum.
A lung cancer screening kit comprises a reagent for detecting an autoantibody of COX10 protein.
As the kit, the reagent for detecting the COX10 protein autoantibody is a reagent for an enzyme-linked immunosorbent assay or an enzyme-linked immunoassay reagent.
As in the kit, the reagent for detecting the COX10 protein autoantibody is a western blot reagent.
As the kit, the reagent for detecting the COX10 protein autoantibody is a reagent for a protein chip detection method.
As with the aforementioned kit, the reagent for detecting COX10 protein autoantibodies is a reagent for detecting COX10 protein autoantibodies in human serum.
The key point of the invention is that the content of COX10 autoantibody in human blood is determined to be obviously related to the risk of lung cancer, so the risk of lung cancer can be judged by detecting the content of COX10 autoantibody in human blood, and as for the specific means for detecting the COX10 autoantibody in 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.
"COX 10 autoantibody" herein refers to "COX 10 protein autoantibody".
Drawings
FIG. 1: lung cancer patients (LC), benign lung disease patients (DC), healthy control (NC) plasma COX10 autoantibody levels were compared.
FIG. 2: ROC analysis of lung cancer patients (LC) and benign lung disease patients (DC)
FIG. 3: lung cancer patients (LC) were analyzed by ROC with healthy controls (NC).
Detailed Description
Example 1 correlation of COX10 autoantibodies in plasma with lung cancer
First, clinical data
30 lung cancer patients, 29 lung benign disease patients and 29 healthy controls are selected, and the basic information is as follows:
| basic information | Patients with lung cancer | Benign lung disease | Healthy controls |
| Number of people | 30 | 29 | 29 |
| Age (age) | 49.5±5.7 | 46.5±10 | 42.0±8.9 |
| Proportion of male | 20(66.7％) | 12(41.4％) | 13(44.8％) |
Second, detection principle
HuProtTMThe human protein custom chip is fixed with COX10 protein (the adopted COX10 protein is full-length protein, the protein serial number is Ensembl: ENSP00000462190), after being incubated by adding serum, COX10 autoantibodies (mainly comprising IgG and IgM antibodies and also other types of antibodies) in the serum can be combined, the unbound antibodies and other proteins are cleaned and removed, anti-human IgM fluorescent labeled secondary antibody (cy5 labeled and shown in red) and anti-human IgG fluorescent secondary antibody (cy3 labeled and shown in green) are used for detection, a fluorescence scanner is used for reading signals, and the strength of the signals is positively correlated with the affinity and the quantity of the antibodies.
Third, method (detection of COX10 autoantibody in serum)
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 completion with ddH2O cleaning for 2 times, 10min each time;
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 COX10 autoantibody in the plasma of lung cancer patients was 15.5SNR (fluorescence signal to quantitative ratio), the mean expression level of COX10 autoantibody in the plasma of lung benign disease patients was 20.8SNR, and the mean expression level of COX10 autoantibody in healthy control plasma was 22.4 SNR. The lung cancer group was statistically significant compared to both the benign lung disease group (p <0.05) and the healthy control group (p <0.05) (fig. 1). The specificity of the ROC analysis result of the lung cancer group and the benign lung disease group is 96.4 percent, the sensitivity is 6.7 percent (figure 2), the specificity of the ROC analysis result of the lung cancer group and the healthy control is 96.6 percent, the sensitivity is 20.0 percent (figure 3), and the result shows that the COX10 autoantibody can specifically distinguish the lung cancer from the healthy control.
From the above results, it is known that the difference between the levels of COX10 autoantibodies in the serum of lung cancer patients and non-lung cancer patients is significant, and the purpose of screening lung cancer can be achieved by detecting the level of COX10 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 COX10 autoantibodies in serum".
The kit can screen the risk of lung cancer of the people to be detected by detecting the level of COX10 autoantibody in serum: the risk of lung cancer is high if COX10 autoantibodies are low (relative to healthy people) and low if COX10 autoantibodies are high. 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 (10)
1. The application of a reagent for detecting COX10 protein autoantibody in preparing a lung cancer screening kit.
2. The use of claim 1, wherein the reagent for detecting the autoantibody to the COX10 protein is a reagent for an enzyme-linked immunosorbent assay or a reagent for a combined immunoassay.
3. The use of claim 1, wherein the reagent for detecting an autoantibody to a COX10 protein is a western blot reagent.
4. The use of claim 1, wherein said reagent for detecting COX10 protein autoantibody is a reagent for a protein chip detection method.
5. The use according to any one of claims 1 to 4, wherein the reagent for detecting the COX10 protein autoantibody is a reagent for detecting the COX10 protein autoantibody in human serum.
6. A lung cancer screening kit, which comprises a reagent for detecting an autoantibody to COX10 protein.
7. The kit of claim 6, wherein the reagent for detecting the COX10 protein autoantibody is an ELISA reagent or an ELISA reagent.
8. The kit of claim 6, wherein the reagent for detecting an autoantibody to COX10 protein is a western blot reagent.
9. The kit of claim 6, wherein the reagent for detecting COX10 protein autoantibody is a reagent for a protein chip detection method.
10. The kit according to any one of claims 6 to 9, wherein the reagent for detecting the COX10 protein autoantibody is a reagent for detecting a COX10 protein autoantibody in human serum.
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| CN201910894448.5A CN110596387B (en) | 2019-09-20 | 2019-09-20 | Application of COX10 autoantibody detection reagent in preparation of lung cancer screening kit |
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| CN201910894448.5A CN110596387B (en) | 2019-09-20 | 2019-09-20 | Application of COX10 autoantibody detection reagent in preparation of lung cancer screening kit |
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| CN110596387B CN110596387B (en) | 2020-06-12 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012064733A2 (en) * | 2010-11-09 | 2012-05-18 | Medimmune, Llc | Antibody scaffold for homogenous conjugation |
| CN107044979A (en) * | 2017-03-06 | 2017-08-15 | 温鹏 | Hemn and β glucuronidases combine application and kit in the heterogeneous hyperplasia detection of bronchial epithelial cell |
| CN107108708A (en) * | 2014-12-22 | 2017-08-29 | 加州大学评议会 | For generating antigen, the composition of antibody and method and immunotherapeutical compositions and method |
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- 2019-09-20 CN CN201910894448.5A patent/CN110596387B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012064733A2 (en) * | 2010-11-09 | 2012-05-18 | Medimmune, Llc | Antibody scaffold for homogenous conjugation |
| CN107108708A (en) * | 2014-12-22 | 2017-08-29 | 加州大学评议会 | For generating antigen, the composition of antibody and method and immunotherapeutical compositions and method |
| CN107044979A (en) * | 2017-03-06 | 2017-08-15 | 温鹏 | Hemn and β glucuronidases combine application and kit in the heterogeneous hyperplasia detection of bronchial epithelial cell |
Non-Patent Citations (2)
| Title |
|---|
| KEITAROU SAIKI 等: "HEME 0 BIOSYNTHESIS IN ESCHERZCHZA COLI: THE CYOE GENE IN THE CYTOCHROME BO OPERON ENCODES A PROTOHEME IX FARNESYLTRANSFERASE", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
| 杨波 等: "细胞色素氧化酶10在非梗阻性无精子症患者睾丸组织中的表达", 《中华男科学杂志》 * |
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