CN112415198A - Application of GP1BB detection reagent in preparation of lung cancer screening kit - Google Patents
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- CN112415198A CN112415198A CN202011314966.4A CN202011314966A CN112415198A CN 112415198 A CN112415198 A CN 112415198A CN 202011314966 A CN202011314966 A CN 202011314966A CN 112415198 A CN112415198 A CN 112415198A
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- 101001070786 Homo sapiens Platelet glycoprotein Ib beta chain Proteins 0.000 title claims abstract description 56
- 102100034168 Platelet glycoprotein Ib beta chain Human genes 0.000 title claims abstract description 56
- 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 19
- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title description 5
- 210000001808 exosome Anatomy 0.000 claims abstract description 30
- 102000004169 proteins and genes Human genes 0.000 claims description 22
- 108090000623 proteins and genes Proteins 0.000 claims description 22
- 238000002965 ELISA Methods 0.000 claims description 9
- 241000282414 Homo sapiens Species 0.000 claims description 8
- 238000001262 western blot Methods 0.000 claims description 4
- 238000000338 in vitro Methods 0.000 abstract description 2
- 238000011534 incubation Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 238000000034 method Methods 0.000 description 8
- 238000004140 cleaning Methods 0.000 description 7
- 238000007789 sealing Methods 0.000 description 6
- 208000019693 Lung disease Diseases 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000000243 solution Substances 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 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
- 208000002927 Hamartoma Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction 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
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 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
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
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- 230000007017 scission Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
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- 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
- G01N33/57496—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 intracellular compounds
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Abstract
The invention relates to the field of in-vitro diagnostic reagents, in particular to application of a GP1BB detection reagent in preparing a lung cancer screening kit. The invention discovers for the first time that the level of GP1BB protein in plasma exosome of lung cancer patients is obviously higher than that of healthy people. According to the invention, the reagent for detecting GP1BB protein 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 a GP1BB 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 molecular marker of the lung cancer in the plasma or its exosome 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.
GP1BB protein (UniProtKB accession number: P13224), and the prior art of the GP1BB protein related to lung cancer is not seen at present.
Disclosure of Invention
The invention aims to provide a novel 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 GP1BB protein in preparing a lung cancer screening kit.
As mentioned above, the reagent for detecting GP1BB protein is enzyme-linked immunosorbent assay reagent, preferably reagent for enzyme-linked immunosorbent assay.
As for the application, the reagent for detecting the GP1BB protein is a western blot reagent.
As mentioned above, the reagent for detecting GP1BB protein is a reagent for a protein chip detection method.
As the aforementioned application, the reagent for detecting GP1BB protein is a reagent for detecting GP1BB protein in human plasma exosome.
A lung cancer screening kit, which comprises a reagent for detecting GP1BB protein.
As the kit, the reagent for detecting GP1BB protein is an enzyme-linked immunosorbent assay reagent, preferably a reagent for enzyme-linked immunosorbent assay.
As the kit, the reagent for detecting the GP1BB protein is a western blot reagent.
As the kit, the reagent for detecting GP1BB protein is used for a protein chip detection method.
As the kit, the reagent for detecting the GP1BB protein is a reagent for detecting the GP1BB protein in human plasma exosomes.
The key point of the invention is that the content of GP1BB in human plasma exosomes is determined to be obviously related to the risk of suffering from lung cancer, so that the risk of suffering from lung cancer can be judged by detecting the content of GP1BB in human plasma exosomes, as for a means for specifically detecting GP1BB in human plasma exosomes, various means disclosed in the prior art can be adopted, and the embodiment of the invention specifically adopts an enzyme-linked immunoassay method (protein chip) for detection, but not only is limited to the means, and any method capable of detecting the content of GP1BB can be used for screening lung cancer.
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 plasma exosome 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 "GP 1 BB" refers to "GP 1BB protein".
Drawings
FIG. 1: lung cancer patients (LC), benign lung Disease (DC), healthy control (NC) plasma exosomes were compared for GP1BB levels.
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 relationship of GP1BB with Lung cancer in plasma exosomes
First, clinical data
40 lung cancer patients, 40 lung benign diseases (non-malignant tumors such as tuberculosis and hamartoma) and 40 healthy controls are selected, and the basic information is as follows:
second, detection principle
The protein chip is fixed with GP1BB protein GP1BB antibody (LLPYLAEDELR (SEQ ID NO.1) amino acid sequence for specifically recognizing GP1BB protein), after the incubation by adding plasma exosome protein, GP1BB protein in the plasma exosome protein is combined, unbound antibody and other protein are removed by cleaning, then GP1BB antibody which is marked by fluorescence is combined on GP1BB protein for detection, the signal is read by a fluorescence scanner, and the strength of the signal is positively correlated with the affinity and the quantity of the antibody.
Third, method
1. Preparation of plasma exosome proteins:
the sample was removed from-80 ℃ and centrifuged at 12000g for 15 minutes at 4 ℃ and the supernatant was transferred to a new centrifuge tube and filtered through a 0.22. mu.M microporous membrane, and then the exosomes were isolated using PTM-EVs kit manufactured by PTM corporation according to the instructions. Adding Urea with a final concentration of 8M and protease inhibitor for ultrasonic cleavage, and using a BCA kit for protein concentration determination.
2. Plasma exosome GP1BB protein assay
The reagents were as follows:
| chip and method for manufacturing the same | Protein chip fixed with GP1BB protein antibody |
| Second antibody incubation liquid | Antibody containing Cy 5-labeled GP1BB |
| Sealing liquid | 3 parts by volume of 10% BSA, 7 parts by volume of 1 XPBS solution |
| Incubation liquid | 1 part by volume of 10% BSA, 9 parts by volume of 1 XPBST solution |
| Cleaning liquid | 1×PBST |
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 plasma exosome protein samples: after sealing is finished, pouring the sealing liquid, then quickly adding a prepared plasma exosome protein incubation liquid, wherein each chip can incubate 14 plasma exosome protein samples, the sample loading volume of each plasma exosome protein sample is 200 mu L, and the shaking table is laterally swung at 20rpm and incubated overnight at 4 ℃ (the plasma exosome protein samples are frozen and thawed in a chromatography cabinet at 4 ℃, and the incubation liquid is added for dilution in a ratio of 1: 50 to obtain the plasma exosome protein incubation liquid);
4) cleaning: taking out the chip and the chip fence together, sucking out the sample, then quickly adding PBST with the same volume, and circulating for several times to ensure that no cross contamination exists among plasma exosome protein 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 GP1BB in lung cancer patient plasma exosomes was 2.84 (protein relative quantitative ratio), the mean expression level of GP1BB in lung benign disease plasma exosomes was 0.77, and the mean expression level of GP1BB in healthy control plasma exosomes was 0.19. 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 the lung cancer group and benign diseases is 97.5%, and the sensitivity is 20.0% (figure 2); the ROC analysis of the lung cancer group and the healthy control showed 97.5% specificity and 45.0% sensitivity (FIG. 3), indicating that GP1BB specifically distinguished lung cancer patients from benign lung disease and healthy controls.
The results show that the level difference of GP1BB in the plasma exosomes of the lung cancer patients and the non-lung cancer patients is obvious, and the aim of screening the lung cancer can be fulfilled by detecting the level of GP1BB in the plasma exosomes.
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, part three- "method".
The kit can screen the risk of lung cancer of the people to be detected by detecting the level of GP1BB in the plasma exosome: if GP1BB levels are high (relative to healthy humans and benign lung disease patients), the risk of lung cancer is high, and if GP1BB levels are low, 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.
SEQUENCE LISTING
<110> Sichuan university Hospital in western China
Application of <120> GP1BB detection reagent in preparation of lung cancer screening kit
<130> GYKH1094-2020P0111923CC20JS036
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 11
<212> PRT
<213> Artificial sequence
<400> 1
Leu Leu Pro Tyr Leu Ala Glu Asp Glu Leu Arg
1 5 10
Claims (10)
1. Application of a reagent for detecting GP1BB protein in preparing a lung cancer screening kit.
2. The use according to claim 1, wherein the reagent for detecting GP1BB protein is an enzyme-linked immunosorbent assay reagent, preferably a reagent for enzyme-linked immunosorbent assay.
3. The use according to claim 1, wherein the reagent for detecting GP1BB protein is a western blot reagent.
4. The use according to claim 1, wherein the reagent for detecting GP1BB protein 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 GP1BB protein is a reagent for detecting GP1BB protein in human plasma exosomes.
6. A lung cancer screening kit, which is characterized by comprising a reagent for detecting GP1BB protein.
7. The kit according to claim 6, wherein the reagent for detecting GP1BB protein is an enzyme-linked immunosorbent assay reagent, preferably a reagent for enzyme-linked immunosorbent assay.
8. The kit of claim 6, wherein the reagent for detecting GP1BB protein is a western blot reagent.
9. The kit according to claim 6, wherein the reagent for detecting GP1BB protein 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 GP1BB protein is a reagent for detecting the GP1BB protein in human plasma exosomes.
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