An ELISA kit for detecting gastric cancer markers and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an ELISA kit for detecting gastric cancer markers and its application. The kit includes an enzyme labeling plate coated with a capture antibody and an HRP-labeled antibody working solution; the heavy chain amino acid sequence of the capture antibody RAD54L-1 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 the HRP-labeled antibody RAD54L-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4. The ELISA kit for detecting gastric cancer markers of the present invention has strong specificity, can specifically detect the RAD54L protein content in serum, and is used as an auxiliary diagnostic method for gastric cancer with high expression of RAD54L protein, and has good stability and good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an ELISA kit for detecting gastric cancer markers and an application thereof. Background Art

[0002] Gastric cancer is a tumor that originates from the gastric mucosal epithelium. Due to unreasonable diet structure, excessive life pressure, infection by pathogenic microorganisms and other reasons, the incidence of gastric cancer is also getting younger. 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.

[0003] At present, the diagnosis of gastric cancer mainly relies on gastroscopy and biopsy, while auxiliary detection methods also include the immunological reaction of gastric cancer, the determination of chemical components in the body and enzyme reaction. Gastroscopy is a common tool for diagnosing gastric cancer. It observes the changes of gastric mucosa with the naked eye through a special tube, but gastroscopy is invasive and can cause physical discomfort to patients. In recent years, more and more disease markers have been used for the census and diagnosis of clinical diseases, but their application effect is still obviously insufficient. For example, carcinoembryonic antigens such as CEA, CA19-9, and CA125 in serum can be used as disease markers for gastric cancer, but the sensitivity and specificity of these markers are relatively low and cannot be used as indicators for disease diagnosis. Therefore, it is of great significance to find a tumor marker to solve the current problem and improve the early diagnosis rate of gastric cancer.

[0004] Related studies have shown that RAD54L protein is related to the occurrence and progression of gastric cancer and can be a tumor marker for gastric cancer. At the same time, the double sandwich ELISA detection method has the advantages of simple operation, high efficiency, reliability, and suitability for rapid screening of large numbers of samples. Based on this, the ELISA detection kit is more stable and specific in detecting samples. Therefore, the ELISA kit that specifically detects RAD54L protein can achieve simple, rapid, and sensitive detection of gastric cancer as a supplementary means of early gastric cancer detection in medicine. However, there are currently no products on the market that detect gastric cancer for RAD54L protein. Summary of the invention

[0005] The first purpose of the present invention is to provide an ELISA kit for detecting gastric cancer markers, which can specifically detect the content of RAD54L protein in serum. As an auxiliary diagnostic method for gastric cancer with high expression of RAD54L protein, it has the advantages of simple detection method and high accuracy.

[0006] The second object of the present invention is to provide an application of an ELISA kit for detecting gastric cancer markers.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] An ELISA kit for detecting gastric cancer markers, the ELISA kit comprising an enzyme-labeled plate coated with a capture antibody and an HRP-labeled antibody working solution;

[0009] The capture antibody is the monoclonal antibody RAD54L-1 against the RAD54L protein; the HRP-labeled antibody is the monoclonal antibody RAD54L-2 against the RAD54L protein;

[0010] The heavy chain amino acid sequence of the monoclonal antibody RAD54L-1 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 the monoclonal antibody RAD54L-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0011] Furthermore, the concentration of the coated capture antibody is 5 μg / mL-20 μg / mL; the dilution multiple of the HRP labeled antibody working solution is 1 / 1000-1 / 8000.

[0012] Furthermore, the ELISA kit also includes an antigen standard, a washing solution, a color developing solution and a stop solution.

[0013] Furthermore, the antigen standard is a recombinant RAD54L protein antigen standard.

[0014] Furthermore, the washing solution is PBST; the color developing solution is TMB color developing solution; and the stop solution is sulfuric acid.

[0015] An application of an ELISA kit for detecting gastric cancer markers, wherein the ELISA kit is used to detect the content of RAD54L protein in serum.

[0016] Further, the following steps are included:

[0017] (1) Take out the ELISA plate coated with the capture antibody, wash the plate with washing solution, add the sample to be tested, incubate at room temperature, and then wash with washing solution;

[0018] (2) Add the HRP-labeled antibody working solution to the system in step (1), incubate at room temperature for 1-3 hours, and then wash with washing solution;

[0019] (3) Adding a color developing solution to the system after the reaction in step (2), developing the color at room temperature in the dark for 5-10 min, and then adding a stop solution to terminate the reaction;

[0020] (4) Use an ELISA reader to measure the OD value at an absorption wavelength of 450 nm.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0022] The present invention provides an ELISA kit for detecting gastric cancer markers. The kit can specifically detect the content of RAD54L protein in human serum samples, has strong specificity and high accuracy, is simple to operate, and is easy to use. It greatly improves the detection efficiency and diagnosis efficiency of gastric cancer and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Optimize the concentration of coated antibodies for ELISA kits;

[0024] Figure 2 Optimize the best dilution factor of labeled antibodies for ELISA kits;

[0025] Figure 3 The ELISA kit was used to detect the relative expression of RAD54L protein in the serum of healthy people and gastric cancer patients;

[0026] Figure 4 ROC curve was used to analyze the diagnostic value of serum marker RAD54L protein in gastric cancer. DETAILED DESCRIPTION

[0027] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0028] Example 1

[0029] The preparation of monoclonal antibodies against RAD54L protein specifically comprises the following steps:

[0030] (1) Immunization of experimental animals: Recombinant RAD54L protein was used as an immunogen. The recombinant RAD54L protein was diluted to 2 mg / mL and immunized by multiple subcutaneous injections in the abdomen. A total of 8 female mice were injected. The first immunization was emulsified with an equal volume of complete Freund's adjuvant, and the subsequent immunizations were emulsified with incomplete Freund's adjuvant. The interval between each immunization was two weeks. The entire immunization process was performed three times, with 200 μg injected each time. After the third immunization injection, the mouse serum was collected and the mouse serum antibody titer was analyzed using ELISA. The mice with the highest titer were selected for subsequent experiments.

[0031] (2) Fusion and screening of hybridoma cells: The mouse with the highest antibody titer was killed, and the spleen of the mouse was taken to prepare a spleen cell suspension. The spleen cell suspension was mixed with myeloma cells at a cell number ratio of 5:1, polyethylene glycol was used as a fusion agent, and the cells were fused in a 37°C water bath for 2 minutes. The fused cells were cultured in HAT selective medium, and the cell culture supernatant was collected after one week. The positive hybridoma cells were screened by ELISA. 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 expansion culture.

[0032] (3) Preparation of monoclonal antibody against RAD54L protein: The hybridoma cells obtained in step (2) were inoculated into the peritoneal cavity of female mice sensitized with paraffin oil. The state of the mouse abdomen was observed. When the mouse abdomen was obviously bulging, the mouse ascites was collected. The collected ascites was centrifuged at 8000 rpm for 30 min, the supernatant was diluted and added with silica powder, incubated at room temperature for half an hour, and then centrifuged at 6000 rpm for 10 min to obtain the supernatant containing the monoclonal antibody.

[0033] (4) Purification of monoclonal antibody of RAD54L protein: The affinity chromatography column was balanced with buffer, and then the monoclonal antibody supernatant obtained in step (3) was loaded onto the affinity chromatography column. After loading, the column was washed twice with buffer B1 (Tris-HCL buffer with a pH of 7.1) and buffer B2 (acetate buffer with a pH of 5.0) until the UV absorption value was stable, and then the washing was stopped. After the washing, the affinity chromatography fluid was eluted with buffer B3 (acetate buffer with a pH of 3.6), and the protein solution was collected when the UV absorption value reached 100 mAU for later use. The protein concentration was determined by the BCA method, and the antibody concentration was adjusted to 5 mg / mL with PBS buffer.

[0034] (5) Analysis of the monoclonal antibody sequence of RAD54L protein: The activity of the purified monoclonal antibodies was accurately measured, and the two monoclonal antibodies with the highest activity were named RAD54L-1 and RAD54L-2, and their variable region sequences were determined. Monoclonal antibody RAD54L-1 and monoclonal antibody RAD54L-2 include a light chain and a heavy chain, respectively; the heavy chain amino acid sequence of monoclonal antibody RAD54L-1 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 RAD54L-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0035] Table 1

[0036]

[0037] Example 2

[0038] Preparation of capture antibody-coated ELISA plate and HRP-labeled antibody working solution:

[0039] (1) The monoclonal antibody RAD54L-1 prepared in Example 1 was selected as the capture antibody, and an enzyme-labeled 96-well plate was used as a solid phase carrier. The capture antibody was diluted to a certain concentration with a carbonate buffer to obtain a coating solution. 100 μL of the coating solution was added to the enzyme-labeled plate, and the plate was sealed with a sealing film and placed at 4°C for overnight coating. The coating solution was discarded, and the plate was washed 3 times with a washing solution. 200 μL of BSA blocking solution was added to each well, and the plate was blocked at room temperature for 3 hours. The liquid in the plate wells was discarded, the plate was patted dry, sealed, and stored at 4°C to obtain an enzyme-labeled plate coated with the capture antibody.

[0040] (2) Select the monoclonal antibody RAD54L-2 prepared in Example 1 as the labeled antibody. Weigh 25 mg of HRP and dissolve it in 1.2% glutaraldehyde solution, and let it stand at room temperature overnight. Pass the reacted enzyme solution through a Sephadex G-25 chromatography column, elute with physiological saline, control the flow rate at 1 mL / min, collect the brown effluent, and if the volume is greater than 5 mL, concentrate it to 5 mL with PEG. Place the brown effluent in a 25 mL beaker and stir slowly. Weigh 12.5 mg of the RAD54L-2 antibody to be labeled and dilute it to 5 mL with physiological saline, and add it dropwise to the beaker containing the enzyme solution while stirring. Add 0.25 mL of 1 M PH9.5 carbonate buffer to the beaker, continue stirring for 3 hours, then add 0.25 mL of 0.2 M lysine, mix well, and let it stand at room temperature for 2 hours. Stir and add an equal volume of saturated ammonium sulfate dropwise to the beaker, place at 4°C for 1 hour, centrifuge the solution after the reaction at 3000rpm for half an hour, and discard the supernatant. Wash the precipitate twice with a semi-saturated ammonium sulfate solution, and finally dissolve the precipitate in a small amount of 0.15M, pH7.4 PBS. Put the above solution into a dialysis bag, dialyze against 0.15M, pH7.4 PBS buffered saline, remove ammonium ions, centrifuge at 10000rpm for 30min, and remove the precipitate. The supernatant is the enzyme conjugate, and the HRP labeled antibody working solution is obtained.

[0041] Example 3

[0042] Optimization and preparation of ELISA kits:

[0043] (1) Optimization of coating antibody concentration: The coating antibody selected was the monoclonal antibody RAD54L-1 targeting the RAD54L protein. The monoclonal antibody RAD54L-1 was coated according to the concentration gradient of 40μg / mL, 20μg / mL, 10μg / mL, 5μg / mL, and 2.5μg / mL. After blocking, 0.1μg / mL of RAD54L protein was added to react for 1h. The HRP-labeled antibody working solution was added to the reaction wells, incubated at 37°C for 1h, washed three times with PBST washing solution and dried. 100μL of 2mg / L TMB color development solution was added to the reaction wells, color was developed at room temperature in the dark for 10 min, and 100μL of 2M sulfuric acid was added to terminate the reaction. After the reaction, the OD value was measured at 450nm. The OD value minus the blank OD value was the largest, which was the optimal coating antibody concentration. The results are as follows Figure 1 As shown, 10 μg / mL is the optimal coating concentration.

[0044] (2) Optimization of HRP-labeled antibody working solution: The concentration of HRP-labeled antibody RAD54L-2 was optimized. The HRP-labeled antibody RAD54L-2 was diluted in a gradient of 1 / 250, 1 / 1000, 1 / 2000, 1 / 4000, 1 / 8000, 1 / 16000, and 1 / 32000, and the detection was performed according to the method in the above step (1). The optimal dilution concentration was selected when the maximum OD value was measured and the lowest antibody concentration was detected. The results are as follows: Figure 2 As shown, 1 / 4000 is the optimal dilution factor.

[0045] (3) Preparation of ELISA kit: The prepared enzyme labeling plate coated with capture antibody, HRP labeled antibody working solution, antigen standard RAD54L protein, washing solution PBST, color development solution TMB, and stop solution sulfuric acid are packaged separately to form the ELISA kit of the present invention.

[0046] Test Example 1

[0047] Blood sample testing:

[0048] 20 blood samples from healthy people (numbers 1-20) and 20 blood samples from gastric cancer patients (numbers 21-40) were collected. After blood collection, they were placed at room temperature to coagulate, and then centrifuged at 6000rpm for 10 minutes to obtain the supernatant. 20 minutes before the experiment, the ELISA plate coated with the capture antibody was removed, returned to room temperature, washed three times with PBST washing solution and dried. 100 μL of the above-mentioned serum sample to be tested was added, incubated at 37°C for 1 hour, washed three times with PBST washing solution and dried. 100 μL of HRP-labeled antibody working solution was added to the reaction well, incubated at 37°C for 1 hour, washed three times with PBST washing solution and dried. 100 μL of 2mg / L TMB color development solution was added to the reaction well, color developed at room temperature in the dark for 10 minutes, and 100 μL of 2M sulfuric acid was added to terminate the reaction. After the reaction, the OD450 value was detected by an ELISA instrument, and the RAD54L protein content in the serum sample was calculated by the standard curve.

[0049] The results are as follows Figure 3 The following table shows the relative expression of RAD54L protein in healthy people and gastric cancer patients. Figure 3 It can be seen that the content of RAD54L protein in the serum of gastric cancer patients is significantly different from that in the serum of healthy people. By detecting the content of RAD54L protein in serum, the purpose of screening gastric cancer can be achieved.

[0050] Test Example 2

[0051] ROC curve evaluation of the diagnostic value of ELISA kit in gastric cancer:

[0052] 40 samples were collected, including 20 healthy serum samples as a control group and 20 early gastric cancer serum samples as an observation group. The RAD54L protein level was detected using the operating steps of Experimental Example 1. The ROC curve was plotted for the test results, and the AUC value was calculated to evaluate the diagnostic value in gastric cancer.

[0053] The results are as follows Figure 4 The figure shows the ROC curve of serum marker RAD54L for diagnosing gastric cancer. The AUC value of serum marker RAD54L is 0.819. The larger the ROC curve area, the greater the diagnostic value of the disease. When the AUC is above 0.7, it means that the model has a good classification ability. The above results show that serum marker RAD54L can specifically distinguish healthy people from gastric cancer patients, and the purpose of screening gastric cancer can be achieved by detecting the content of serum marker RAD54L protein.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. An ELISA kit for detecting gastric cancer markers, characterized in that: The ELISA kit comprises an enzyme labeling plate coated with a capture antibody and an HRP labeled antibody working solution; The capture antibody is the monoclonal antibody RAD54L-1 against the RAD54L protein; the HRP-labeled antibody is the monoclonal antibody RAD54L-2 against the RAD54L protein; The heavy chain amino acid sequence of the monoclonal antibody RAD54L-1 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 the monoclonal antibody RAD54L-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.

4.

2. An ELISA kit for detecting gastric cancer markers according to claim 1, characterized in that: The concentration of the coated capture antibody is 5 μg / mL-20 μg / mL; the dilution multiple of the HRP labeled antibody working solution is 1 / 1000-1 / 8000.

3. An ELISA kit for detecting gastric cancer markers according to claim 1, characterized in that: The ELISA kit also includes an antigen standard, a washing solution, a color developing solution and a stop solution.

4. An ELISA kit for detecting gastric cancer markers according to claim 3, characterized in that: The antigen standard is a recombinant RAD54L protein antigen standard.

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

6. An ELISA kit for detecting gastric cancer markers according to claim 3, characterized in that: The steps of the detection are: (1) Take out the ELISA plate coated with the capture antibody, wash the plate with washing solution, add the sample to be tested, incubate at room temperature, and then wash with washing solution; (2) Add the HRP-labeled antibody working solution to the system in step (1), incubate at room temperature for 1-3 hours, and then wash with washing solution; (3) Adding a color developing solution to the system after the reaction in step (2), developing the color at room temperature in the dark for 5-10 min, and then adding a stop solution to terminate the reaction; (4) Use an ELISA reader to measure the OD value at an absorption wavelength of 450 nm.

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