CN112415198B - Application of GP1BB detection reagent in preparation of lung cancer screening kit - Google Patents

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

Use of GP1BB detection reagent in the preparation of lung cancer screening kit

technical field

The invention relates to the field of in vitro diagnostic reagents, in particular to the use of GP1BB detection reagents in the preparation of lung cancer screening kits.

Background technique

Lung cancer is one of the most common malignant tumors in the world. Its morbidity and mortality are increasing year by year. Currently, the incidence rate ranks first in the world, which seriously threatens human health and life.

Lung cancer is a disease that is good at concealment. It often shows clinical symptoms when the disease develops to an advanced stage. 70-80% of lung cancer patients are already in the middle or late stage when they are diagnosed with lung cancer symptoms. The best cure time, five-year survival rate is low. For patients with early lung cancer, timely treatment can greatly improve the 5-year survival rate and quality of life of the patients. Therefore, early diagnosis and effective screening of lung cancer are very important.

Screening for lung cancer refers to routine physical examinations for those without lung cancer-related symptoms to detect lung cancer in time before symptoms appear. If the molecular markers of lung cancer in plasma or exosomes can be found, it is of great significance to prompt clinicians to take relevant treatment measures or decision-making for patients in the early stage.

GP1BB protein (UniProtKB number: P13224), there is no prior art related to GP1BB protein and lung cancer.

Contents of the invention

The purpose of the present invention is to provide a new lung cancer marker and the use of the detection reagent of the marker in the preparation of a lung cancer screening kit.

Technical scheme of the present invention comprises:

Use of a reagent for detecting GP1BB protein in the preparation of a lung cancer screening kit.

As mentioned above, the reagent for detecting GP1BB protein is an enzyme-linked immunoassay reagent, preferably an enzyme-linked immunosorbent assay reagent.

As mentioned above, the reagent for detecting GP1BB protein is a western blot reagent.

As mentioned above, the reagent for detecting GP1BB protein is a reagent for protein chip detection method.

As mentioned above, the reagent for detecting GP1BB protein is a reagent for detecting GP1BB protein in human plasma exosomes.

A lung cancer screening kit includes reagents for detecting GP1BB protein.

As in the aforementioned kit, the reagent for detecting GP1BB protein is an enzyme-linked immunoassay reagent, preferably an enzyme-linked immunosorbent assay reagent.

As in the aforementioned kit, the reagent for detecting GP1BB protein is a western blot reagent.

As in the aforementioned kit, the reagent for detecting GP1BB protein is a reagent for protein chip detection method.

As in the aforementioned kit, the reagent for detecting GP1BB protein is a reagent for detecting GP1BB protein in human plasma exosomes.

The key point of the present invention is to determine that the content of GP1BB in human plasma exosomes is significantly related to the risk of lung cancer. Therefore, the risk of lung cancer can be judged by detecting the content of GP1BB in human plasma exosomes. As for the specific detection of human plasma The method of GP1BB in exosomes can use various methods disclosed in the prior art. The embodiment of the present invention specifically uses the enzyme-linked immunoassay method (protein chip) for detection, but it is not limited to this method, any method that can detect the content of GP1BB Can be used for lung cancer screening.

The present invention provides a new lung cancer screening marker and a new lung cancer screening kit, which can realize effective screening of lung cancer; and plasma exosomes can be used as detection samples, and the harm to patients is very low. The invention has good application prospect.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through specific embodiments. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All 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".

Description of drawings

Figure 1: Comparison of GP1BB levels in plasma exosomes of lung cancer patients (LC), benign lung diseases (DC), and healthy controls (NC).

Figure 2: ROC analysis of patients with lung cancer (LC) and benign lung disease (DC).

Figure 3: ROC analysis of lung cancer patients (LC) and healthy controls (NC).

Detailed ways

Example 1 Relationship between GP1BB in plasma exosomes and lung cancer

1. Clinical data

Select 40 cases of lung cancer patients, 40 cases of benign lung diseases (non-malignant tumors such as tuberculosis and hamartoma), and 40 cases of healthy controls. The basic information is as follows:

2. Detection principle

The GP1BB protein GP1BB antibody (specifically recognizing the amino acid sequence of LLPYLAEDELR (SEQ ID NO.1) of the GP1BB protein) is immobilized on the protein chip. After incubation with the plasma exosome protein, the GP1BB protein in the plasma exosome protein will bind to it and wash Remove unbound antibodies and other proteins, and then use fluorescently labeled GP1BB antibody to bind to GP1BB protein for detection, and read the signal through a fluorescent scanner. The strength of the signal is positively correlated with the affinity and quantity of the antibody.

3. Method

1. Preparation of plasma exosomal protein:

The sample was taken out from -80°C, centrifuged at 12,000g for 15 minutes at 4°C, and the supernatant was transferred to a new centrifuge tube, filtered through a 0.22 μM microporous membrane, and then isolated exocrine using the PTM-EVs kit produced by PTM Company according to the instructions. body. Add Urea with a final concentration of 8M and protease inhibitors for ultrasonic lysis, and use the BCA kit to determine the protein concentration.

2. Detection of plasma exosome GP1BB protein

The reagents are as follows:

chip Protein chip immobilized with GP1BB protein antibody Secondary Antibody Incubation Solution Contains Cy5-labeled GP1BB antibody blocking solution 3 parts by volume 10% BSA, add 7 parts by volume 1×PBS solution Incubation solution 1 volume part of 10% BSA, add 9 volume parts of 1X PBST solution Washing fluid 1 x PBST

1) Rewarming: Take the chip out of the -80°C refrigerator, put it in a 4°C refrigerator for half an hour, and then place it at room temperature for 15 minutes;

2) Sealing: After rewarming the chip, fix 14 blocks on the fence. After fixing, add blocking solution to each block, place it on a side swing shaker, and seal it at room temperature for 3 hours;

3) Incubation of plasma exosome protein samples: After the sealing is completed, pour out the blocking solution, and then quickly add the pre-prepared plasma exosome protein incubation solution. Each chip can incubate 14 plasma exosome protein samples. The loading volume of the plasma exosomal protein sample is 200 μL, and the side-swing shaker is 20 rpm, and incubated overnight at 4°C (the plasma exosome protein sample is first frozen and thawed in a chromatography cabinet at 4°C, and the incubation solution is added to dilute it at a ratio of 1:50. , to obtain plasma exosome protein incubation solution);

4) Cleaning: Take out the chip and the chip fence together, suck off the sample, and then quickly add an equal volume of PBST, and repeat this cycle several times to ensure that there is no cross-contamination between plasma exosome protein samples when the chip fence is removed. After dismantling the chip fence, place the chip in a chip cleaning box with cleaning solution, on a horizontal shaker, at room temperature 80rpm, wash 3 times, 10min each time;

5) Secondary antibody incubation: transfer the chip to an incubation box added with 3mL of secondary antibody incubation solution, side-swing shaker at 40rpm, protected from light, room temperature for 1hr;

6) Cleaning: Take out the chip (be careful not to touch or scratch the upper surface of the chip), put it in a chip cleaning box with cleaning solution, on a horizontal shaker, room temperature 80rpm, wash 3 times, 10min each time. After completion, wash with ddH ₂ O twice for 10 minutes each time;

7) drying;

8) Scanning: scan with LuxScan 10K microarray chip scanner;

9) Data extraction: Open the corresponding GAL file (which records the position of the protein in the chip), align the chip image and each array of the GAL file as a whole, press the automatic alignment button, extract the data and save it.

4. Results

The average expression level of GP1BB in plasma exosomes of lung cancer patients was 2.84 (protein relative quantitative ratio), the average expression level of GP1BB in plasma exosomes of lung benign diseases was 0.77, and the average expression level of GP1BB in plasma exosomes of healthy controls The level is 0.19. The lung cancer group had statistical significance compared with the lung benign disease group (p<0.05) and the healthy control group (p<0.01) (Figure 1). The ROC analysis result specificity of lung cancer group and benign disease is 97.5%, and sensitivity is 20.0% (Fig. 2); ROC analysis result specificity of lung cancer group and healthy control is 97.5%, sensitivity is 45.0% (Fig. 3), It was shown that GP1BB can specifically distinguish lung cancer patients from benign lung diseases and healthy controls.

From the above results, it can be seen that the level of GP1BB in plasma exosomes of lung cancer patients and non-lung cancer patients is significantly different, and the purpose of lung cancer screening can be achieved by detecting the level of GP1BB in plasma exosomes.

Embodiment 2 The composition of detection kit of the present invention and its application method

1. Kit composition

Detection kit (14 persons):

2. How to use the kit

Same as the third part of embodiment 1——"method".

The kit of the present invention can screen the risk of lung cancer in the population to be tested by detecting the level of GP1BB in plasma exosomes: if the level of GP1BB is high (relative to healthy people and patients with benign lung diseases), the risk of lung cancer The risk is high, and if the level of GP1BB is low, the risk of lung cancer is low. It can be used for auxiliary diagnosis of clinical lung cancer, and provide effective basis for patients to take relevant treatment measures or decision-making, and has a good clinical application prospect.

SEQUENCE LISTING

<110> West China Hospital of Sichuan University

<120> Use of GP1BB detection reagent in the preparation of a 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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