CN112415198B - Application of GP1BB detection reagent in preparation of lung cancer screening kit - Google Patents
Application of GP1BB detection reagent in preparation of lung cancer screening kit Download PDFInfo
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- CN112415198B CN112415198B CN202011314966.4A CN202011314966A CN112415198B CN 112415198 B CN112415198 B CN 112415198B CN 202011314966 A CN202011314966 A CN 202011314966A CN 112415198 B CN112415198 B CN 112415198B
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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
- G01N33/57496—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
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 patient 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 hiding, clinical symptoms are usually shown only when the disease is developed to the advanced stage, 70-80% of lung cancer patients are in the middle and advanced stages when being diagnosed with the lung cancer symptoms, 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 No. P13224), the prior art of the GP1BB protein related to lung cancer is not found 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 an enzyme-linked immunosorbent assay reagent, preferably a reagent for enzyme-linked immunosorbent assay.
As the application, the reagent for detecting 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 aforementioned application, the reagent for detecting GP1BB protein is a reagent for detecting GP1BB protein in human plasma exosome.
A lung cancer screening kit comprises a reagent for detecting GP1BB protein.
As the kit, the reagent for detecting GP1BB protein is an enzyme-linked immunoassay reagent, and preferably a reagent for enzyme-linked immunosorbent assay.
As the kit, the reagent for detecting GP1BB protein is a western blot reagent.
As the kit, the reagent for detecting GP1BB protein is a reagent for a protein chip detection method.
As the kit, the reagent for detecting GP1BB protein is a reagent for detecting GP1BB protein in human plasma exosome.
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 lung cancer, so that the risk of lung cancer can be judged by detecting the content of GP1BB in human plasma exosomes, and as for a means for specifically detecting GP1BB in human plasma exosomes, 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 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 "GP1BB" refers to "GP1BB protein".
Drawings
FIG. 1: GP1BB levels in lung cancer patients (LC), benign lung Disease (DC), healthy control (NC) plasma exosomes 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 relationship of GP1BB in plasma exosomes to lung cancer
1. 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:
2. principle of detection
The GP1BB protein GP1BB antibody (LLPYLAEDELR (SEQ ID NO. 1) amino acid sequence for specifically recognizing the GP1BB protein) is fixed on the protein chip, after the incubation by adding the plasma exosome protein, the GP1BB protein in the plasma exosome protein is combined, the unbound antibody and other proteins are removed by washing, the GP1BB antibody marked by fluorescence is combined on the GP1BB protein for detection, signals are read by a fluorescence scanner, and the strength of the signals is positively correlated with the affinity and the quantity of the antibody.
3. Method of producing a composite material
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 15min;
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 3hr;
3) Incubation of plasma exosome protein samples: after sealing is finished, pouring the sealing liquid completely, then quickly adding the 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, laterally shaking the bed at 20rpm, and incubating overnight at 4 ℃ (the plasma exosome protein samples are frozen and thawed in a chromatography cabinet at 4 ℃, and the plasma exosome protein incubation liquid is obtained by diluting the incubation liquid in a proportion of 1;
4) Cleaning: taking out the chip and the chip fence together, sucking out the sample, then quickly adding the PBST with the same volume, and circulating for several times so as to ensure that no cross contamination exists among the plasma exosome protein samples when the chip fence is detached. After removing the chip fence, putting the chip into a chip cleaning box with cleaning solution, horizontally shaking at room temperature of 80rpm, and cleaning for 10min for 3 times;
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 1hr;
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 for 10min each time, on a horizontal shaker at room temperature and 80 rpm. Go toAfter the formation, use ddH 2 O 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.
4. Results
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 distinguishes lung cancer patients from benign lung disease and healthy controls.
The results show that the level difference of GP1BB in plasma exosomes of lung cancer patients and non-lung cancer patients is obvious, and the aim of screening 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
1. Kit composition
Detection kit (14 persons):
2. method for using kit
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 persons 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 (5)
1. The application of a reagent for detecting GP1BB protein in human plasma exosomes in preparing a lung cancer screening kit.
2. The use of claim 1, wherein the reagent for detecting GP1BB protein in human plasma exosomes is an enzyme-linked immunoassay reagent.
3. The use according to claim 1, wherein the reagent for detecting GP1BB protein in human plasma exosomes is a reagent for enzyme-linked immunosorbent assay.
4. The use according to claim 1, wherein the reagent for detecting GP1BB protein in human plasma exosomes is a western blot reagent.
5. The use according to claim 1, wherein the reagent for detecting GP1BB protein in human plasma exosomes is a reagent for a protein chip detection method.
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