A kit for detecting gastric cancer markers and its application

Abstract

The present invention discloses a kit for detecting gastric cancer markers and its application, which belongs to the field of in vitro diagnosis. The kit for detecting gastric cancer markers comprises an enzyme label plate coated with a capture antibody, an HRP-labeled antibody working solution, an antigen standard, a washing solution, a color developing solution and a stop solution; the capture antibody G1EA and the HRP-labeled antibody G2EA are both antibodies that specifically bind to the GRK3 antigen. The kit for detecting gastric cancer markers of the present invention has strong specificity, can specifically detect the GRK3 protein content in serum, and is used as an auxiliary diagnostic method for gastric cancer with high expression of GRK3 protein, and has good stability and good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnosis, and in particular to a kit for detecting gastric cancer markers and an application thereof. Background Art

[0002] Gastric cancer is a tumor originating from the gastric mucosal epithelium. It is one of the malignant tumors with high incidence and mortality. Due to changes in people's lifestyles and dietary structure, the incidence of gastric cancer has become younger. Since the pathogenesis of gastric cancer is relatively complex, there are no obvious symptoms in the early stage, the diagnosis rate is extremely low, and the mortality rate is very high. The key to reducing the mortality rate of gastric cancer patients is early diagnosis and early treatment. Therefore, it is particularly important to find new markers and targets for gastric cancer. At present, gastroscopy is used as a tool for diagnosing gastric cancer. It is placed into the stomach cavity through a special tube from the mouth to observe the changes in the gastric mucosa with the naked eye. However, gastroscopy is somewhat invasive and can cause physical discomfort to patients, so people are not very accepting of gastroscopy. Therefore, it is urgent to develop a highly specific detection kit for early cancer screening using serum as a test sample to benefit more patients.

[0003] At present, serum antigen levels have been used for the preliminary assessment of cancer. Common serum markers for gastric cancer include CEA and CA19-9, but the specificity of these conventional serum markers is not high and cannot be used as a diagnostic standard for gastric cancer. Therefore, it is urgent to find a tumor marker to solve the current international problem, improve the early diagnosis rate of gastric cancer, and further improve the treatment effect of gastric cancer. Studies have shown that GRK3 is overexpressed in gastric cancer cells, which contributes to the migration and invasion of gastric cancer cells. Compared with the healthy control serum group, GRK3 showed significantly high expression in serum specimens of gastric cancer patients. Functionally, increased GRK3 expression promotes gastric cancer cell migration and invasion by regulating the transcription factor YAP1. These studies have shown that GRK3 protein is related to the occurrence and progression of gastric cancer, is a potential therapeutic target for gastric cancer, and can become a new tumor marker. Therefore, a kit for the specific detection of GRK3 protein can achieve simple, rapid, and sensitive detection of gastric cancer as a supplementary means for early detection of cancer in medicine. There are currently no products on the market for the detection of gastric cancer with GRK3 protein. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, one of the purposes of the present invention is to provide a kit for detecting gastric cancer markers, which can detect the GRK3 protein content in sample serum with strong specificity and good stability.

[0005] A second object of the present invention is to provide a kit for detecting gastric cancer markers.

[0006] One of the purposes of the present invention is achieved by the following technical solution:

[0007] A kit for detecting gastric cancer markers, the kit comprising an enzyme-labeled plate coated with a capture antibody, an HRP-labeled antibody working solution, an antigen standard, a washing solution, a color developing solution, and a stop solution;

[0008] The capture antibody is the monoclonal antibody G1EA against GRK3 protein, the heavy chain amino acid sequence of the monoclonal antibody G1EA is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2;

[0009] The antibody in the HRP-labeled antibody working solution is the anti-GRK3 protein monoclonal antibody G2EA, the heavy chain amino acid sequence of the monoclonal antibody G2EA is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0010] Furthermore, the antigen standard is a recombinant GRK3 protein antigen standard.

[0011] Furthermore, the washing solution is PBST buffer; the color developing solution is TMB color developing solution; and the stop solution is sulfuric acid.

[0012] Furthermore, the concentration of the color developing solution is 2 mg / L-4 mg / L.

[0013] Furthermore, the concentration of the stop solution is 2M-4M.

[0014] The second object of the present invention is achieved by adopting the following technical solution:

[0015] A kit for detecting gastric cancer markers includes the following steps:

[0016] (1) Add the sample to be tested to the ELISA plate coated with the capture antibody for incubation and rinse with washing solution;

[0017] (2) Adding the HRP-labeled monoclonal antibody working solution to the system of step (1) for incubation, and washing with washing solution;

[0018] (3) adding a color developing solution to the system of step (2) for color development;

[0019] (4) adding a stop solution to the system of step (3) to terminate the reaction;

[0020] (5) Perform dual-wavelength detection on the system of step (4), respectively detecting the OD values ​​at 450 nm and 630 nm, and calculating the results based on the standard curve.

[0021] Furthermore, the sample to be tested in step (1) is serum.

[0022] Compared with the prior art, the beneficial effect of the present invention is that the present invention provides a kit for detecting gastric cancer markers, which can specifically detect the GRK3 protein content in serum, and serve as an auxiliary diagnostic method for gastric cancer with high GRK3 protein expression, and has good stability, can effectively judge the risk of cancer in the population to be tested, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The standard curve of the kit for specific detection of recombinant GRK3 protein;

[0024] Figure 2 This is a diagram showing the specific identification results of G1EA and G2EA monoclonal antibodies. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0026] Example 1

[0027] The preparation of GRK3 protein monoclonal antibody comprises the following steps:

[0028] (1) Immunization of experimental animals: Human recombinant GRK3 protein was used as an antigen, and 8 healthy female mice were selected for subcutaneous multi-point injection. The serum of healthy female mice was used as a control. For the first immunization, 100 μL of 0.1 mg / mL recombinant human GRK3 protein was mixed with 100 μL of Freund's complete adjuvant in equal volumes. For the second immunization, 100 μL of 0.1 mg / mL recombinant human GRK3 protein was mixed with 100 μL of Freund's incomplete adjuvant in equal volumes. Immunization was performed once every 2 weeks for 3 consecutive times. The antibody titer of mouse serum was determined by indirect ELISA, and the mouse with the highest antibody titer was selected for subsequent experiments.

[0029] (2) Fusion of hybridoma cells: The mouse with the highest antibody titer obtained in step (1) was killed, and the mouse spleen was removed in a clean bench to prepare a spleen cell suspension. The spleen cells and myeloma cells were fused at a ratio of 5:1 under the action of 50% polyethylene glycol. The fused cells were cultured in HAT selection medium, and the cell culture supernatant was collected after one week. The positive hybridoma cells were screened by ELISA, and the screened positive hybridoma cells were subcloned three times by limiting dilution method, and the monoclonal cells with the strongest positive reaction were selected for culture.

[0030] (3) Preparation of monoclonal antibodies: The hybridoma cells obtained by subcloning were used to prepare mouse monoclonal antibodies. Six healthy female mice about 10 weeks old were injected with 0.5 mL of liquid paraffin per mouse. Two weeks later, 2×10 7 The subcloned cells were injected into the mouse peritoneum, and the injection status of the mouse was observed. If the mouse abdomen was obviously bulging, the mouse ascites was collected and filtered with a 0.2μm filter. The collected ascites of different mice were purified by ion exchange chromatography. The activity of the antibody was accurately measured using Biacore T200, and the two monoclonal antibodies with the highest activity were named G1EA and G2EA. The protein concentration was determined by the BCA method, and the antibody concentration was adjusted to 5mg / mL for standby use and stored at -20°C.

[0031] (4) Analysis of monoclonal antibody sequence structure: The obtained monoclonal antibodies G1EA and G2EA were further sequenced and identified to determine the heavy chain and light chain variable region sequences. The heavy chain amino acid sequence of monoclonal antibody G1EA is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of monoclonal antibody G2EA is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0032] SEQ ID NO.1:

[0033] QVQLDQTGAELVNPAGSLRISMVVSGFTFTTGNIYWAKTSTPTLEGIDDAGGCGGNMYNDPSIVTLESVISKEQSKTTYQMLTSNGPAEDNGIYYCHLAGNYGDYWGQGTGLLVSS;

[0034] SEQ ID NO.2:

[0035] DIVMTQSALLTTTCGTVTRTIASILTCWVQNDKANWYQQWYLHLFVGSFMDPKDQAYNRFARFSGAGSGTSYSLTITSMEAEDAATYYCAQGSTSPLAFGAGTHLTLK;

[0036] SEQ ID NO.3:

[0037] EVQFLESGGMLVRPGASVEMSCEGSGFHYNSYWGWWKQKPGKKGLQVGGPIEPGYDTKYAFGVETNKATLTAASSGFGLYLSAISLASEDVSYCIAIEHDYFPVSWGLGTTLAVSS;

[0038] SEQ ID NO.4:

[0039] QAVFTQSPRMMSSPGGQPLTCSCRAGGDVSQEKGKTVQEQRPISPLLISTGLIYTRAGVPARFGSGAVASDKAALTITGDRDEDEAIYFCGWLYSNNWIFTKLTIK.

[0040] Example 2

[0041] A method for preparing a kit for detecting gastric cancer markers comprises the following steps:

[0042] (1) Preparation of ELISA plate coated with capture antibody:

[0043] Select the monoclonal antibody G1EA prepared in Example 1 as the capture antibody. Use an enzyme-labeled 96-well plate as a solid phase carrier, dilute the capture antibody to 2 μg / mL with a coating buffer (50 mM carbonate buffer) to obtain a coating solution. Add 100 μL of the coating solution to the ELISA plate, seal the plate, and coat overnight at 4°C. Discard the coating solution, wash 3 times with a washing solution, add 300 μL / well of the blocking solution, block at room temperature for 4 hours, discard the liquid in the plate, dry it, and store it at 4°C.

[0044] (2) Preparation of HRP-labeled antibody working solution:

[0045] The monoclonal antibody G2EA prepared in Example 1 was selected as the labeled antibody. The monoclonal antibody G2EA was dialyzed in 50mM carbonate buffer for 24h and its concentration was adjusted to 2mg / mL. 5mg HRP was weighed and dissolved in 1mL distilled water. 10.5mg NaIO4 was weighed and dissolved in 1mL distilled water to prepare a 0.1M NaIO4 solution. 0.2mL NaIO4 solution was mixed with 1mL HRP solution, stirred at room temperature in the dark for 40min, and dialyzed in 10 mM NaAc buffer at 4℃ overnight. Carbonate buffer was added to the above-mentioned aldehyded HRP solution to raise its pH to 9.0-9.5, and then 5mL of 2mg / mL monoclonal antibody G2EA was added and gently stirred at room temperature in the dark for 2 hours. Measure 0.1 mL of 4 mg / L NaBH4, add it all to the reaction solution in the previous step, mix well, and react at 4°C for 2 hours. Put the above sample into a dialysis bag and dialyze it overnight at 4°C. Centrifuge at 10,000 r / min for 30 min to remove the precipitate. The supernatant is the enzyme conjugate. Add an equal amount of 60% glycerol and divide it into aliquots. Store at 4°C away from light.

[0046] (3) Kit standard curve determination:

[0047] Remove the ELISA plate coated with the capture antibody, return to room temperature, and wash the plate three times with PBST buffer. Add 100 μL of recombinant GRK3 protein antigen of different dilutions (25 pg / mL, 50 pg / mL, 75 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL), incubate at 37°C for 1 h, wash three times with PBST buffer and spin dry. Add 100 μL of HRP-labeled antibody working solution to the reaction well, incubate at 37°C for 1 h, wash five times with PBST buffer, add 100 μL of 2 mg / L TMB color development solution to the reaction well, color at room temperature in the dark for 15 min, and add 100 μL of 2M sulfuric acid to terminate the reaction. Use an ELISA reader for dual wavelength detection, measure the OD value at a reference wavelength of 450 nm and the OD value at a reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value at 450 nm. Draw a standard curve with the concentration of the standard as the horizontal axis (pg / mL) and OD 450 - OD 630 as the vertical axis. Draw a standard curve based on the results, such as Figure 1 shown.

[0048] Test Example 1

[0049] Specificity identification of G1EA and G2EA monoclonal antibodies:

[0050] BSA protein and recombinant GRK3 protein were mixed with loading buffer (2×) containing SDS at a volume ratio of 2:1, the sample was subjected to SDS-PAGE, and the protein was transferred to a nitrocellulose membrane using a semi-dry gel transfer instrument. The blocking solution was blocked at 37°C for 1h and washed 3 times with PBST buffer. The monoclonal antibodies for G1EA and G2EA proteins prepared in Example 2 were diluted to a concentration of 1μg / mL, and the diluted monoclonal antibodies G1EA and G2EA were added respectively, and incubated at 37°C for 1 h. The membrane was washed to remove unbound antibodies. Anti-mouse IgG secondary antibody labeled with alkaline phosphatase was added and incubated at 37°C for 1 h to further amplify the signal. Wash five times with PBST buffer, immerse the membrane in a mixed 2mg / L TMB colorimetric solution for color development, and add 100 μL 2M sulfuric acid to terminate the reaction. Observe the experimental results to determine whether the monoclonal antibody has the ability to specifically recognize GRK3 protein.

[0051] The results are as follows Figure 2As shown, the monoclonal antibody of G1EA protein has no band in lane 1 (BSA protein) and obvious bands in lane 2 (recombinant GRK3 protein). The monoclonal antibody of G2EA protein has no band in lane 3 (BSA protein) and obvious bands in lane 4 (recombinant GRK3 protein). This shows that the monoclonal antibodies G1EA and G2EA can specifically bind to the recombinant GRK3 protein, but have no obvious binding characteristics with the BSA protein, indicating that the monoclonal antibodies of G1EA and G2EA proteins have good specificity.

[0052] Test Example 2

[0053] Blood sample testing:

[0054] The detection method is as follows: 10 blood samples from healthy people (numbered 1-10) and 10 blood samples from gastric cancer patients (numbered 11-20) were collected and placed at room temperature to coagulate, and then centrifuged at 6000rpm for 10min to obtain the supernatant. The serum samples No. 1-20 were added to the enzyme-labeled plate coated with the capture antibody and incubated for 1h, and washed 3 times with PBST buffer. 100μL of HRP-labeled antibody working solution was added to the reaction well, incubated at room temperature for 1h, and washed 5 times with PBST buffer. 100μL of 2mg / L TMB colorimetric solution was added to the reaction well, color developed at room temperature in the dark for 15 min, and 100μL of 2M sulfuric acid was added to terminate the reaction. The OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm was measured using an enzyme-labeled instrument for dual-wavelength detection. The content of GRK3 protein in the serum sample to be tested was calculated according to the standard curve, and the average was taken after three repetitions. The results are shown in Table 1.

[0055] Table 1 Clinical sample test results

[0056]

[0057] The results are shown in Table 1. The kit of the present invention can accurately detect the GRK3 protein content in the serum of gastric cancer patients, and the result is positive. The blood of healthy people contains trace amounts of GRK3 protein, and the detected concentration is not within the linear range of the kit of the present invention, and the result is negative. This shows that the kit of the present invention can use serum as a sample to complete the detection of gastric cancer markers, which can meet the needs of basic research and clinical diagnosis.

[0058] Test Example 3

[0059] Gastric cancer marker kit stability test:

[0060] The sera of volunteers No. 11, No. 15, and No. 18 in Experimental Example 2 were taken as samples, and 10 biological replicates were performed for each sample to test the stability of the kit. The results are shown in Table 2.

[0061] Table 2 Clinical sample test results

[0062]

[0063] As shown in Table 2, the kit of the present invention can quantitatively detect the content of human serum GRK3 protein, and has good stability, which can meet the needs of basic research and clinical diagnosis.

[0064] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A kit for detecting gastric cancer markers, characterized in that: The kit comprises an ELISA plate coated with a capture antibody, an HRP labeled antibody working solution, an antigen standard, a washing solution, a color developing solution and a stop solution; The capture antibody is the monoclonal antibody G1EA against GRK3 protein, the heavy chain amino acid sequence of the monoclonal antibody G1EA is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; The antibody in the HRP-labeled antibody working solution is the anti-GRK3 protein monoclonal antibody G2EA, the heavy chain amino acid sequence of the monoclonal antibody G2EA is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.

4.

2. A kit for detecting gastric cancer markers according to claim 1, characterized in that: The antigen standard is a recombinant GRK3 protein antigen standard.

3. A kit for detecting gastric cancer markers according to claim 1, characterized in that: The washing solution is PBST buffer; the color developing solution is TMB color developing solution; and the stop solution is sulfuric acid.

4. A kit for detecting gastric cancer markers according to claim 3, characterized in that: The concentration of the color developing solution is 2 mg / L-4 mg / L.

5. A kit for detecting gastric cancer markers according to claim 3, characterized in that: The concentration of the stop solution is 2M-4M.

6. A use of a kit for detecting gastric cancer markers according to any one of claims 1 to 5, characterized in that: The process includes: (1) Add the sample to be tested to the ELISA plate coated with the capture antibody for incubation and rinse with washing solution; (2) Add the HRP-labeled antibody working solution to the system of step (1) for incubation, and rinse with washing solution; (3) adding a color developing solution to the system of step (2) for color development; (4) adding a stop solution to the system of step (3) to terminate the reaction; (5) Perform dual-wavelength detection on the system of step (4), respectively detecting the OD values ​​at 450 nm and 630 nm, and calculating the results based on the standard curve.

7. Use of a kit for detecting gastric cancer markers as claimed in claim 6, characterized in that: The sample to be tested in step (1) is serum.

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