CN110412272B - Application of ALKBH3 autoantibody detection reagent in preparation of lung cancer screening kit - Google Patents
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- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 46
- 201000005202 lung cancer Diseases 0.000 title claims abstract description 46
- 208000020816 lung neoplasm Diseases 0.000 title claims abstract description 46
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 40
- 102100039086 Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3 Human genes 0.000 title claims abstract description 36
- 101000959152 Homo sapiens Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3 Proteins 0.000 title claims abstract description 36
- 238000012216 screening Methods 0.000 title claims abstract description 16
- 238000001514 detection method Methods 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 210000002966 serum Anatomy 0.000 claims abstract description 20
- 102000004169 proteins and genes Human genes 0.000 claims description 7
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- 238000002965 ELISA Methods 0.000 claims description 6
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- 238000000338 in vitro Methods 0.000 abstract description 2
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- 208000019693 Lung disease Diseases 0.000 description 5
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- 102100027051 Nucleic acid dioxygenase ALKBH1 Human genes 0.000 description 3
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- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 1
- 101000836620 Homo sapiens Nucleic acid dioxygenase ALKBH1 Proteins 0.000 description 1
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- 102000003839 Human Proteins Human genes 0.000 description 1
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- G—PHYSICS
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- 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 application of an ALKBH3 autoantibody detection reagent in preparation of a lung cancer screening kit. The invention discovers for the first time that the autoantibody level of the ALKBH3 protein in the serum of a lung cancer patient is obviously higher than that of a healthy patient. According to the invention, the reagent for detecting the ALKBH3 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 application of an ALKBH3 autoantibody detection reagent in preparation of 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).
ALKBH3 (Ensembl: ENSG00000166199 MIM: 610603) is an alkB homolog 3, α -oxoglutarate dioxygenase, which is commonly expressed in testis (RPKM 6.9), prostate (RPKM 6.8) and 25 other tissues.
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 an ALKBH3 protein autoantibody in preparing a lung cancer screening kit.
As the application, the reagent for detecting the ALKBH3 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or a combined immunoassay reagent.
As for the application, the reagent for detecting the ALKBH3 protein autoantibody is a western blot reagent.
As the application, the reagent for detecting the ALKBH3 protein autoantibody is a reagent for a protein chip detection method.
As for the application, the reagent for detecting the ALKBH3 protein autoantibody is a reagent for detecting the ALKBH3 protein autoantibody in human serum.
A lung cancer screening kit comprises a reagent for detecting an ALKBH3 protein autoantibody.
As the kit, the reagent for detecting the ALKBH3 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunoassay reagent.
According to the kit, the reagent for detecting the ALKBH3 protein autoantibody is a western blot reagent.
As the kit, the reagent for detecting the ALKBH3 protein autoantibody is a reagent for a protein chip detection method.
As the kit, the reagent for detecting the ALKBH3 protein autoantibody is a reagent for detecting the ALKBH3 protein autoantibody in human serum.
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, "ALKBH 3 autoantibody" refers to "ALKBH 3 protein autoantibody".
Drawings
FIG. 1: lung cancer patients (LC), benign lung Disease (DC), and healthy control (NC) plasma levels of akbh 3 autoantibodies were compared.
FIG. 2: ROC analysis of lung cancer patients (LC) and benign lung Disease (DC).
FIG. 3: lung cancer patients (LC) were analyzed by ROC with healthy controls (NC).
Detailed Description
Example 1 correlation of ALKBH3 autoantibodies in plasma with Lung cancer
First, clinical data
30 lung cancer patients, 29 lung benign diseases (non-malignant tumors such as tuberculosis and hamartoma) and 30 healthy controls are selected, and the basic information is as follows:
basic information | Patients with lung cancer | Benign lung disease | Healthy controls |
Number of |
30 | 29 | 30 |
Age (age) | 58±10.5 | 46.5±10.0 | 42±8.9 |
Proportion of male | 20(66.7) | 12(41.4%) | 13(46.7%) |
Second, detection principle
HuProtTMThe ALKBH3 protein is fixed on a human protein customizing chip, after serum is added for incubation, ALKBH3 autoantibodies (mainly including IgG and IgM antibodies and other types of antibodies) in the serum are combined, the unbound antibodies and other proteins are removed by cleaning, an anti-human IgM fluorescent labeled secondary antibody (cy5 labeled and red) and an anti-human IgG fluorescent secondary antibody (cy3 labeled and green) are used for detection, signals are read through a fluorescence scanner, and the strength of the signals is positively correlated with the affinity and the quantity of the antibodies.
Third, method
The reagents used in this section were as follows:
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 the ALKBH3 autoantibody in the plasma of the lung cancer patient was 39.9SNR (fluorescence signal relative quantitative ratio), the mean expression level of the ALKBH3 autoantibody in the plasma of the benign disease in the lung was 20.8SNR, and the mean expression level of the ALKBH3 autoantibody in the plasma of the healthy control was 27.8 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 <0.01) (fig. 1). The specificity of ROC analysis of lung cancer group and benign disease is 93.1%, and the sensitivity is 58.6% (FIG. 2); the results of ROC analysis of lung cancer and healthy controls showed specificity of 96.6% and sensitivity of 17.2% (FIG. 3), indicating that the ALKBH3 autoantibody specifically distinguishes lung cancer from benign lung disease and healthy controls.
The results show that the level difference of the ALKBH3 autoantibody in the serum of the lung cancer patient and the serum of the non-lung cancer patient is obvious, and the aim of screening the lung cancer can be achieved by detecting the level of the ALKBH3 autoantibody 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 ALKBH3 autoantibodies in serum".
The kit can screen the risk of lung cancer of a to-be-detected person by detecting the level of the ALKBH3 autoantibody in serum: if the level of akbh 3 autoantibodies is high (relative to healthy humans), the risk for lung cancer is high, and if the level of akbh 3 autoantibodies is low, the risk for 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 (5)
1. The application of a reagent for detecting an ALKBH3 protein autoantibody in preparing a lung cancer screening kit; the reagent for detecting the ALKBH3 protein autoantibody is a reagent for detecting an ALKBH3 protein autoantibody in human serum.
2. The use of claim 1, wherein the reagent for detecting the ALKBH3 protein autoantibody is an enzyme-linked immunoassay reagent.
3. The use of claim 2, wherein the enzyme-linked immunoassay reagent is an enzyme-linked immunosorbent assay reagent.
4. The use according to claim 1, wherein the reagent for detecting the ALKBH3 protein autoantibody is a western blot reagent.
5. The use according to claim 1, wherein the reagent for detecting the ALKBH3 protein autoantibody is a reagent for a protein chip detection method.
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EP1862804A1 (en) * | 2005-03-14 | 2007-12-05 | Link Genomics, Inc. | Method for diagnosis of prostate cancer |
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EP1862804A1 (en) * | 2005-03-14 | 2007-12-05 | Link Genomics, Inc. | Method for diagnosis of prostate cancer |
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Title |
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ALKBH3, a human AlkB homologue, contributes to cell survival in human non-small-cell lung cancer;Tasaki M et al;《Br J Cancer》;20110201;第104卷(第4期);第700-706页 * |
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