BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies and kit thereof

Abstract

The invention discloses a BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies and a kit thereof, an expression strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on July 24, 2024, and the preservation number is CGMCC NO: 31440; the monoclonal antibody hybridoma cell strain E2mAb is preserved in the China General Microbiological Culture Collection Center on October 9, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.46055. The monoclonal antibody hybridoma cell strain E2mAb is a monoclonal antibody hybridoma cell strain E2mAb. According to the present invention, the BVDV receptor rCD46 protein is adopted as the coating antigen, the HRP labeled specific rE2 monoclonal antibody is adopted as the detection antibody, the condition optimization is performed to establish the BVDV antigen capture ELISA detection method, and the BVDV antigen capture ELISA detection kit based on the receptor rCD46 and the BVDV rE2 monoclonal antibody is firstly assembled in China;

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a method and kit for BVDV antigen capture ELISA based on rCD46 and rE2 monoclonal antibodies. Background Art

[0002] Bovine viral diarrhea (BVD), also known as bovine viral diarrhea-mucosal disease, is a disease widely prevalent worldwide caused by bovine viral diarrhea virus. BVD mainly causes clinical symptoms such as fever, mucosal erosion and ulceration, leukopenia, calf diarrhea, and cow abortion in cattle, bringing great harm to the global cattle industry. Since BVD has no specific symptoms clinically and usually occurs with immunosuppression, it has brought great difficulties to the diagnosis and prevention and control of BVD and posed a great threat to the livestock industry.

[0003] Currently, the detection of BVD pathogens is mainly carried out by methods such as qPCR, RT-PCR, and ELISA. Accurate and rapid detection of pathogens is crucial for the prevention of BVD. However, all current detection methods have defects. Traditional detection methods such as cell culture have a long detection cycle and cumbersome operations and are not suitable for large-scale detection; the PCR method has high sensitivity and specificity, but the operation requires specialized equipment and skilled personnel, increasing the detection cost, and it can only detect nucleic acids and cannot detect live pathogens. Therefore, there is an urgent need for a method and kit for BVDV antigen capture ELISA based on rCD46 and rE2 monoclonal antibodies. Summary of the Invention

[0004] The purpose of the present invention is to provide a method and kit for BVDV antigen capture ELISA based on rCD46 and rE2 monoclonal antibodies.

[0005] On the one hand, an rCD46 expression strain rbCD46 of BVDV receptor, the expression strain was deposited at the China General Microbiological Culture Collection Center on July 24, 2024, with the deposit number CGMCC NO: 31440.

[0006] In another aspect, a monoclonal antibody hybridoma cell line E2mAb strain, the cell line E2mAb strain was deposited at the China General Microbiological Culture Collection Center on October 9, 2024, with the deposit number CGMCC NO.: 46055.

[0007] In another aspect, a monoclonal antibody of a positive hybridoma cell line, the monoclonal antibody of the positive hybridoma cell line is a monoclonal antibody secreted by the above-mentioned monoclonal antibody hybridoma cell line.

[0008] Application of the monoclonal antibody secreted by the monoclonal antibody hybridoma cell line in a BVDV capture ELISA kit.

[0009] A kit for BVDV antigen capture ELISA based on rCD46 and rE2 monoclonal antibodies, the kit includes an enzyme-labeled plate and the monoclonal antibody; the enzyme-labeled plate is a solid-phase carrier coated with the BVDV receptor rCD46.

[0010] A method for BVDV antigen capture ELISA based on rCD46 and rE2 monoclonal antibodies, including antigen coating at a concentration of 0.5 μg / mL of BVDV receptor rCD46 protein, 100 μL per well, coating at 4°C for 12 h, and using HRP-labeled specific rE2 monoclonal antibody as the detection antibody.

[0011] Furthermore, it includes antigen coating at a concentration of 0.5 μg / mL of rCD46 protein, 100 μL per well, coating at 4°C for 12 h; washing once with PBST, selecting 5% fish gelatin as the blocking solution, blocking at 37°C for 2 h with 200 μL per well; washing 3 times with PBST, diluting positive serum and negative serum at a ratio of 1:1, adding 100 μL per well, reacting at 37°C for 1 h; washing 3 times with PBST, diluting the enzyme-labeled monoclonal antibody at a ratio of 1:1000, adding 100 μL per well, reacting at 37°C for 1 h; washing 3 times with PBST, patting dry, adding 100 μL of TMB per well, reacting at room temperature for 10 min; after color development, adding 50 μL per well of the termination solution and then measuring the OD450nm value with an enzyme-labeled instrument.

[0012] Furthermore, the OD cut-off value of the kit is 0.23.

[0013] Compared with the prior art, the biggest feature of the present invention adopting the above technical solution is:

[0014] The present invention uses the BVDV receptor rCD46 protein as the coating antigen and HRP-labeled specific rE2 monoclonal antibody as the detection antibody, and through condition optimization, a BVDV antigen capture ELISA detection method is established. Description of the Drawings

[0015] Figure 1 Schematic diagram of the pTT5-rCD46 plasmid map expressing the BVDV receptor rCD46 protein;

[0016] Figure 2 Schematic diagram of the pTT5-rE2 plasmid map expressing the BVDV rE2 protein;

[0017] Figure 3 SDS-PAGE diagram of purified BVDV receptor rCD46 and BVDV rE2 proteins;

[0018] Figure 4 Figure for measuring the antibody titer in mice immunized with BVDV rE2 protein;

[0019] Figure 5 Figure for the blocking result of BVDV rE2 monoclonal antibody with BVDV antibody-positive serum;

[0020] Figure 6 Figure for the critical value analysis of the BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies of the present invention. Detailed implementation manners

[0021] The technical solution of the present invention will be further described below in conjunction with examples and comparative examples, but it should not be construed as a limitation to the present invention:

[0022] Cell surface receptors are important mechanisms for viruses to bind and invade cells. CD46 is an important receptor for BVDV to invade cells. After BVDV binds to CD46, it invades cells and completes virus replication and proliferation after invasion. E2 protein is an important structural protein of BVDV. From the N-terminus to the C-terminus, it is divided into four functional regions A, B, C, and D. Regions A and B (located at 4-87 nt and 88-164 nt respectively) are the farthest from the membrane and are the most exposed regions on the virus surface. They are the most important parts for the E2 protein to generate immunogenicity. Based on the functions of rCD46 (recombinant expressed CD46) and rE2 (recombinant expressed E2) antigens of the present invention, using the rCD46 receptor as a ligand for capturing antigens, and using a highly sensitive monoclonal antibody against rE2 antigen labeled with HRP as a detection antibody, by expressing rCD46 protein with 293 cells, expressing and preparing rE2 protein with 293 cells and preparing monoclonal antibodies, labeling the monoclonal antibody with HRP (horseradish peroxidase); using rCD46 as the coating antigen and HRP-labeled rE2 monoclonal antibody as the detection antibody, an antigen capture ELISA method for detecting BVDV virus is established, and an antigen capture ELISA kit for detecting BVDV virus is developed.

[0023] Example 1. Expression of soluble rCD46 protein

[0024] 1.1 Vector construction and preliminary expression identification

[0025] As Figure 1As shown in the figure, the eukaryotic expression plasmid pTT5-rCD46 was constructed (the expression strain was deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on July 24, 2024, with the deposit number CGMCC NO: 31440, named cell line rbCD46, and the classification number Escherichia coli). The recombinant plasmid pTT5-rCD46 was transfected into 293F cells using the transfection reagent PEI. After 5 days, the cells were collected, centrifuged at 100 r / min for 5 min, the culture medium was collected, and the cells were resuspended with an appropriate amount of PBS and ultrasonically disrupted; centrifuged at 12,000 r / min at 4 °C for 20 min, and the supernatant was collected. Protein expression was verified by Western Blot. The results verified that the rCD46 protein was successfully expressed.

[0026] 1.2 Large-scale expression and purification

[0027] The pTT5-rCD46 protein was expressed in large quantities using 1 L of 293F cells and purified using a Ni-NTA column. After purification, the purification results were verified by SDS-PAGE, and the purified protein was collected.

[0028] Example 2. Expression of soluble rE2 protein and preparation of monoclonal antibodies

[0029] 2.1 Vector construction and preliminary expression identification

[0030] As Figure 2 shown in the figure, the eukaryotic expression plasmid pTT5-rE2 was constructed. The recombinant plasmid pTT5-rE2 was transfected into 293F cells using the transfection reagent PEI. After 5 days, the cells were collected, centrifuged at 100 r / min for 5 min, the culture medium was collected, and the cells were resuspended with an appropriate amount of PBS and ultrasonically disrupted; centrifuged at 12,000 r / min at 4 °C for 20 min, and the supernatant was collected. Protein expression was verified by Western Blot.

[0031] Result: After verification by WB, the rE2 protein was successfully expressed.

[0032] 2.2 Large-scale expression and purification

[0033] The pTT5-rE2 protein was expressed in large quantities using 1 L of 293F cells and purified using a Ni-NTA column. After purification, the purification results were verified by SDS-PAGE, and the purified protein was collected.

[0034] 3. Preparation, purification and identification of rE2 monoclonal antibodies

[0035] The purified rE2 was emulsified with complete Freund's adjuvant and subcutaneously immunized for the first time in 4 SPF-grade female BALB / c mice at a dose of 60 μg protein / mouse, numbered: 1, 2, 3, 4.

[0036] Subcutaneous booster immunizations were performed 2 weeks, 4 weeks, and 6 weeks after the primary immunization, respectively, with a dose of 50 μg protein per mouse each time. Blood was collected from the orbital sinus 10 days after the third booster immunization to measure the serum titer. If the titer reached the standard, the mice were challenged intraperitoneally with 50 μg of the immunogen on day 14 after the third immunization.

[0037] Immune titer detection: Coated with "rE2" at 2 μg / ml overnight at 4°C; blocked with 5% skim milk for 2 h at 37°C; the serum was serially diluted 2-fold starting from 200-fold, with the blank control (blank) being 1*PBS and the negative control (negative) being the negative serum diluted 200-fold. Results: The mice challenged with #4 were selected for cell fusion experiments as Figure 4 shown.

[0038] 2.2 Cell fusion and monoclonal antibody cell line cloning and screening

[0039] The spleen cells of mice immunized with rE2 were fused with myeloma cells SP2 / 0, and subcloned 3 times. Screening was performed using the indirect ELISA method, and 5 positive hybridoma cell lines were obtained. Monoclonal antibody subclass identification was performed on them, and the results are shown in the following table.

[0040]

[0041] 2.3 Verification of the specificity of rE2 monoclonal antibody

[0042] Coated with "rE2" at 0.4 μg / ml overnight at 4°C; blocked with 3% fish gelatin for 2 h at 37°C; the BVDV antibody-positive serum was diluted 5-fold with 3% fish gelatin, and 100 μl was added to each well as the blocked well, and 100 μl of 3% fish gelatin was added to the wells without serum as the unblocked well, and incubated at 37°C for 1 h. Washed 3 times with PBST, 3 wells each of the blocked and unblocked wells were taken, and 100 μl of the supernatant of the same monoclonal antibody was added to each well, and incubated at 37°C for 1 h. Washed 3 times with PBST, 100 μl of HRP-labeled goat anti-mouse IgG (diluted 10,000-fold) diluted with 3% fish gelatin was added to each well, and incubated at 37°C for 1 h. Washed 3 times with PBST, and then 100 μl of TMB chromogenic solution was added to each well and allowed to act at room temperature for 10 min; after the chromogenic reaction was completed, 50 μL / well of the termination solution was added. The OD450nm value was measured with an enzyme-linked immunosorbent assay reader.

[0043] As Figure 5 a result, the responses of all 5 monoclonal antibodies decreased significantly after blocking with the BVDV antibody-positive serum, indicating that all 5 monoclonal antibodies are BVDV-specific monoclonal antibodies.

[0044] Example 3 Establishment of an antigen capture ELISA detection method based on rCD46 protein and HRP-labeled rE2 monoclonal antibody

[0045] 3.1 Monoclonal antibody preparation and labeling

[0046] Select the positive hybridoma cell line with the highest blocking rate. The hybridoma cell line, E2mAb strain, was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on October 9, 2024, with the deposit number CGMCC NO: 46055, named cell line BVDV E2mAb, and the classification number being mouse myeloma cell line). Expand the culture of the cells and prepare ascites. Purify the collected ascites and label it with HRP enzyme.

[0047] 3.2 Optimization of the optimal reaction conditions for the BVDV antigen capture ELISA antibody detection method

[0048] According to the checkerboard titration method, coat the antigen at protein concentrations of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL, 100 μL per well, and coat at 4°C for 12 h; wash 3 times with PBST, select 3% BSA, 5% skim milk, and 3% fish gelatin as the blocking solution, and block at 37°C for 3 h with 200 μL per well; wash 1 time with PBST, dilute the BVDV antigen positive control and negative control according to 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, add 100 μL to each well, and incubate at 37°C for 1 h; wash 3 times with PBST, dilute the enzyme-labeled monoclonal antibody according to 1:1000, 1:2000, 1:4000, 1:8000, add 100 μL to each well, and incubate at 37°C for 1 h; wash 3 times with PBST, pat dry, add 100 μL of TMB to each well, and incubate at room temperature for 10 min; after the color development is completed, add 50 μL of the termination solution per well. Measure the OD450nm value with an enzyme-labeled instrument, and determine the best combination conditions as the one with the highest P / N (Note: P / N = positive control OD450nm / negative control OD450nm × 100%).

[0049] After optimization, the reaction conditions were determined as follows: coat the antigen at a protein concentration of 0.5 μg / mL, 100 μL per well, and coat at 4°C for 12 h; wash 1 time with PBST, select 5% fish gelatin as the blocking solution, and block at 37°C for 2 h with 200 μL per well; wash 3 times with PBST, dilute the BVDV antigen positive control and the BVDV antigen negative control according to 1:1, add 100 μL to each well, and incubate at 37°C for 1 h; wash 3 times with PBST, dilute the enzyme-labeled monoclonal antibody according to 1:1000, add 100 μL to each well, and incubate at 37°C for 1 h; wash 3 times with PBST, pat dry, add 100 μL of TMB to each well, and incubate at room temperature for 10 min; after the color development is completed, add 50 μL of the termination solution per well. Measure the OD450nm value with an enzyme-labeled instrument.

[0050] 3.3 Determination of the cut-off value for the antigen capture ELISA antibody detection method

[0051] As shown in the figure, 15 bovine sera positive for BVDV antigen and 14 bovine sera negative for BVDV antigen were detected by the established antigen capture ELISA method. The cut-off value was calculated by the method of negative mean + 3×standard deviation, and the cut-off value was determined to be 0.23.

[0052] 3.4 Preparation and Shelf-life Test of BVDV Antigen Capture ELISA Kit

[0053] Antigen coating was performed at a concentration of 0.5 μg / mL rCD46 protein, 100 μL / well, and coated at 4 °C for 12 h; washed 3 times with PBST, and the blocking solution was selected as 5% fish gelatin + 10% sucrose, 200 μL / well, blocked at 37 °C for 2 h, dried after aspiration and then air-dried, placed in a sealed bag, and vacuum-sealed after adding desiccant;

[0054] Add BVDV antigen positive control and BVDV antigen negative control to 1‰ procl in, dispense, 1 ml / tube;

[0055] Add 10×PBST to 1‰ proclin, dispense, 50 ml / bottle;

[0056] TMB chromogenic solution was dispensed, 22 ml / bottle;

[0057] Dispense the stop solution (2 m HCl), 15 ml / bottle.

[0058] Take 2 96-well antigen-coated plates, 1 tube of BVDV antigen positive control, 1 tube of BVDV antigen negative control, 1 bottle of 10×PBST, 1 bottle of TMB chromogenic solution, and 1 bottle of stop solution, assemble into 1 kit, store in a cold storage at 2 - 8 °C, take out 1 kit every 3 months for verification. When verifying, use the BVDV virus culture solution stored frozen as the standard sample, and set 3 replicates.

[0059] The procedure is as follows: Take out the ELISA plate, wash it once with PBST, dilute the BVDV virus culture solution, BVDV antigen positive control, and BVDV antigen negative control at a ratio of 1:1, add 100 μL to each well, and incubate at 37 °C for 1 h; wash 3 times with PBST, dilute the enzyme-labeled monoclonal antibody at a ratio of 1:1000, add 100 μL to each well, and incubate at 37 °C for 1 h; wash 3 times with PBST, pat dry, add 100 μL of TMB to each well, and incubate at room temperature for 10 min; after the color development is completed, add 50 μL / well of stop solution. Measure the OD450nm value with an enzyme-labeled instrument.

[0060] Results: After the kit was stored for 9 months, the OD value did not show a significant decrease. It shows that the kit is stable.

[0061] 3.5 Specificity Test of BVDV Antigen Capture ELISA Kit

[0062] The antigen capture ELISA method with optimized conditions was used to detect the culture fluids of infectious bovine rhinotracheitis virus, bovine rotavirus, bovine infectious gastroenteritis virus, etc., and the test results were all negative.

[0063] 3.6 Sensitivity test of BVDV antigen capture ELISA kit

[0064] One portion of bovine viral diarrhea virus solution was serially diluted 2-fold, and detected by the established antigen capture ELISA method to evaluate the sensitivity of this method.

[0065] The results showed that the bovine viral diarrhea virus solution could still be detected after being diluted to 6×10 3 TCID 50 / mL.

[0066] 3.7 Repeatability test of BVDV antigen capture ELISA kit

[0067] The enzyme-linked immunosorbent assay (ELISA) plates coated with the same batch were used to detect the BVDV virus culture fluid, repeated 3 times, and the within-batch coefficient of variation was calculated.

[0068] ELISA plates coated with 3 different batches were used to detect the BVDV virus culture fluid, repeated 3 times and the average value was taken, and the between-batch coefficient of variation was calculated. Based on the above results, the repeatability of this method was evaluated.

[0069] The results showed that the within-batch coefficient of variation was less than 1%, and the between-batch coefficient of variation was less than 3%, both within the acceptable range.

[0070] Within-batch repeatability results

[0071]

[0072]

[0073] Between-batch repeatability results

[0074] Sample number Replicate 1 Replicate 2 Replicate 3 Mean Standard deviation Coefficient of variation 1 1.852 1.795 1.781 1.809 0.031 1.70％ 2 1.218 1.152 1.128 1.166 0.038 3.26％ 3 1.681 1.628 1.599 1.636 0.034 2.08％

[0075] 3.8 Field application of BVDV antigen capture ELISA kit

[0076] Three hundred and sixty serum samples from non-immunized cattle in the field were taken and detected with the BVDV antigen capture ELISA kit. The result showed that the positive rate of BVDV antigen in non-immunized cattle in the field was 1.4% (5 / 360).

[0077] The present invention successfully prepared soluble bovine rCD46 and BVDV rE2 proteins with biological activity through a eukaryotic system. Through immunizing mice and clone screening, monoclonal antibodies specific to the rE2 protein were obtained and labeled with HRP. Using the rCD46 protein as the coating antigen and the HRP-labeled specific rE2 monoclonal antibody as the detection antibody, a BVDV antigen capture ELISA detection method was established through condition optimization. The BVDV antigen capture ELISA detection kit was assembled in China for the first time. After verification, it has good specificity and sensitivity, and the shelf life is at least more than 9 months.

[0078] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and all are covered by the protection scope of the present invention.

Claims

1. A BVDV receptor rCD46-expressing bacterial strain rbCD46, characterized in that, The described expression strain was deposited on July 24, 2024, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC NO: 31440.

2. Monoclonal antibody hybridoma cell line E2mAb strain, characterized in that, The cell line E2mAb strain was deposited on October 9, 2024, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC NO.: 46055.

3. A monoclonal antibody E2mAb of a monoclonal antibody hybridoma cell line, characterized in that: The monoclonal antibody of the monoclonal antibody hybridoma cell line is the monoclonal antibody secreted by the monoclonal antibody hybridoma cell line described in claim 2.

4. Use of the monoclonal antibody secreted by the monoclonal antibody hybridoma cell line described in claim 2 in the preparation of an antigen capture ELISA kit for detecting BVDV.

5. A kit for BVDV antigen capture ELISA based on rCD46 and rE2 monoclonal antibodies, characterized in that: The kit includes an enzyme-linked immunosorbent assay (ELISA) plate and the monoclonal antibody secreted by the monoclonal antibody hybridoma cell line described in claim 3; the ELISA plate is a solid-phase carrier coated with the BVDV receptor rCD46 as described in claim 1.

6. A BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies, characterized in that, It includes antigen coating at a concentration of 0.5 μg / mL BVDV receptor rCD46 protein at 100 μL / well for 12 h at 4°C, and HRP-labeled specific rE2 monoclonal antibody as the detection antibody.

7. A BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies according to claim 6, characterized in that, Antigen coating is carried out at a protein concentration of 0.5 μg / mL at 100 μL / well for 12 h at 4°C; washed once with PBST, 5% fish gelatin is selected as the blocking solution, and blocked at 200 μL / well for 2 h at 37°C; washed 3 times with PBST, positive serum and negative serum are diluted at a ratio of 1:1, 100 μL is added to each well, and incubated at 37°C for 1 h; washed 3 times with PBST, the enzyme-labeled monoclonal antibody is diluted at a ratio of 1:1000, 100 μL is added to each well, and incubated at 37°C for 1 h; washed 3 times with PBST, patted dry, 100 μL of TMB is added to each well, and incubated at room temperature for 10 min; after color development, 50 μL / well of the termination solution is added, and the OD450nm value is measured with an enzyme-linked immunosorbent assay (ELISA) reader.

8. A BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies according to claim 6, characterized in that, The OD cut-off value of the kit is 0.23.

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