A method of bvdv antigen capture elisa based on rcd46 and re2 mab and its kit

Abstract

The application discloses a BVDV antigen capture ELISA method and kit based on rCD46 and rE2 monoclonal antibodies, and the expression strain is preserved in the China General Microbiological Culture Collection Center on July 24, 2024, with a preservation number of CGMCC NO: 31440; the monoclonal antibody hybridoma cell strain E2mAb strain is preserved in the China General Microbiological Culture Collection Center on October 09, 2024, with a preservation number of CGMCC NO: 46055. The application establishes a BVDV antigen capture ELISA detection method by using a BVDV receptor rCD46 protein as a coating antigen and using HRP-labeled specific rE2 monoclonal antibodies as detection antibodies, and domestic BVDV antigen capture ELISA detection kits based on the receptor rCD46 and the BVDV rE2 monoclonal antibodies are assembled for the first time.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a method and kit for BVDV antigen capture ELISA based on rCD46 and rE2 monoclonal antibodies. Background Technology

[0002] Bovine viral diarrhea (BVD), also known as bovine viral diarrhea-mucosal disease, is a worldwide epidemic caused by the bovine viral diarrhea virus. BVD primarily causes clinical symptoms in cattle such as fever, mucosal erosion and ulceration, leukopenia, diarrhea in calves, and abortion in cows, posing a significant threat to the global cattle industry. Because BVD lacks specific clinical symptoms and is often accompanied by immunosuppression, its diagnosis and prevention are extremely difficult, posing a substantial threat to livestock farming.

[0003] Currently, the detection of BVD pathogens is mainly carried out using qPCR, RT-PCR, and ELISA methods. Accurate and rapid detection of the pathogen is crucial for BVD prevention. However, all current detection methods have limitations. Traditional methods, such as cell culture, have long detection cycles and are cumbersome to operate, making them unsuitable for large-scale detection. PCR methods have high sensitivity and specificity, but require specialized equipment and skilled personnel, increasing detection costs, and can only detect nucleic acids, not live pathogens. Therefore, there is an urgent need for a BVDV antigen capture ELISA method and kit based on rCD46 and rE2 monoclonal antibodies. Summary of the Invention

[0004] The purpose of this 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, there is a BVDV receptor rCD46 expressing strain rbCD46, which was deposited on July 24, 2024 at the China General Microbiological Culture Collection Center, with accession number CGMCC NO: 31440.

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

[0007] In another aspect, a positive hybridoma cell line monoclonal antibody, said positive hybridoma cell line monoclonal antibody being a monoclonal antibody secreted by said monoclonal antibody hybridoma cell line.

[0008] The application of monoclonal antibodies secreted by the monoclonal antibody hybridoma cell line in the BVDV capture ELISA kit.

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

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

[0011] Further, the process included: antigen coating with 100 μL / well of 0.5 μg / mL rCD46 protein at 4°C for 12 h; washing once with PBST; blocking with 200 μL / well of 5% fish gelatin at 37°C for 2 h; washing three times with PBST; adding 100 μL of positive and negative sera at a 1:1 ratio to each well and incubating at 37°C for 1 h; washing three times with PBST; adding 100 μL / well of enzyme-labeled monoclonal antibody at a 1:1000 ratio to each well and incubating at 37°C for 1 h; washing three times with PBST; patting dry; adding 100 μL of TMB to each well and incubating at room temperature for 10 min; after color development, adding 50 μL / well of stop solution and measuring the OD450nm value using a microplate reader.

[0012] Furthermore, the OD critical value of the kit is 0.23.

[0013] The most significant feature of this invention compared to existing technologies is that the above-described technical solution is:

[0014] This invention establishes a BVDV antigen capture ELISA detection method using BVDV receptor rCD46 protein as the coating antigen and HRP-labeled specific rE2 monoclonal antibody as the detection antibody through condition optimization. Attached Figure Description

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

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

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

[0018] Figure 4 This is a graph showing the antibody titer determination after mice were immunized with BVDV rE2 protein.

[0019] Figure 5 The image shows the results of blocking BVDV rE2 monoclonal antibody with positive serum of BVDV antibody;

[0020] Figure 6 This is a critical value analysis diagram of the BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies of the present invention. Detailed Implementation

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

[0022] Cell surface receptors are an important mechanism for viruses to bind to and invade cells. CD46 is an important receptor for BVDV to invade cells. After binding to CD46, BVDV invades cells and completes viral replication and proliferation after invasion. The E2 protein is an important structural protein of BVDV, divided into four functional regions (A, B, C, and D) from the N-terminus to the C-terminus. Regions A and B (located at 4-87 nt and 88-164 nt, respectively) are furthest from the membrane and are the most exposed areas on the viral surface, making them the most important parts for the immunogenicity of the E2 protein. This invention is based on the functions of rCD46 (recombinantly expressed CD46) and rE2 (recombinantly expressed E2) antigens. It utilizes the rCD46 receptor as a ligand for capturing the antigen and a highly sensitive monoclonal antibody labeled with HRP against the rE2 antigen as the detection antibody. The invention establishes an antigen capture ELISA method for detecting BVDV virus by expressing rCD46 protein in 293 cells, preparing rE2 protein and a monoclonal antibody using 293 cells, and labeling the monoclonal antibody with HRP (horseradish peroxidase). Using rCD46 as the coating antigen and the HRP-labeled rE2 monoclonal antibody as the detection antibody, a BVDV virus antigen capture ELISA kit is developed.

[0023] Example 1. Expression of soluble rCD46 protein

[0024] 1.1 Vector Construction and Preliminary Expression Identification

[0025] like Figure 1As shown, a pTT5-rCD46 eukaryotic expression plasmid was constructed (the expression strain was deposited on July 24, 2024, at the China General Microbiological Culture Collection Center, accession number CGMCC NO: 31440, named cell line rbCD46, classification number Escherichia coli). The recombinant plasmid pTT5-rCD46 was transfected into 293F cells using PEI transfection reagent. Cells were collected after 5 days, centrifuged at 100 rpm for 5 min, the culture medium was collected, and the cells were resuspended in an appropriate amount of PBS and sonicated. The cells were then centrifuged at 12,000 rpm for 20 min at 4℃, and the supernatant was collected. Protein expression was verified by Western blotting. The results confirmed successful expression of rCD46 protein.

[0026] 1.2 Large-scale expression and purification

[0027] pTT5-rCD46 protein was expressed in large quantities using 1L 293F cells and purified using a Ni-NTA column. The purification results were verified using 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] like Figure 2 As shown, a eukaryotic expression plasmid pTT5-rE2 was constructed. The recombinant plasmid pTT5-rE2 was transfected into 293F cells using the transfection reagent PEI. Cells were collected after 5 days, centrifuged at 100 rpm for 5 min, the culture medium was collected, and the cells were resuspended in an appropriate amount of PBS and sonicated. The cells were then centrifuged at 12,000 rpm for 20 min at 4℃, and the supernatant was collected. Protein expression was verified by Western blotting.

[0031] Result: Western blot analysis confirmed successful expression of rE2 protein.

[0032] 2.2 Large-scale expression and purification

[0033] pTT5-rE2 protein was expressed in large quantities using 1L 293F cells and purified using a Ni-NTA column. 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 four SPF-grade BALB / c female mice at a dose of 60 μg protein per mouse, numbered 1, 2, 3, and 4.

[0036] Mice were subcutaneously boosted with 50 μg of protein per mouse at 2, 4, and 6 weeks after the initial immunization. Blood was collected from the orbital sinus 10 days after the third booster immunization to measure serum titer. If the titer met the target, mice were administered an intraperitoneal shock dose of 50 μg of immunogen 14 days after the third immunization.

[0037] Immunotiter assay: "rE2", 2ug / ml, was used for overnight coating at 4℃; 5% skim milk was used for blocking at 37℃ for 2 hours; serum was serially diluted 2-fold starting from 200-fold; the blank control was 1*PBS, and the negative control was a 200-fold dilution of negative serum. Results: Cell fusion experiments were performed using shock #4 mice as follows... Figure 4 As shown.

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

[0039] Spleen cells from mice immunized with rE2 were fused with myeloma cells SP2 / 0, and subcloning was performed three times. Five positive hybridoma cell lines were obtained through indirect ELISA screening. Monoclonal antibody subclass identification was then performed, and the results are shown in the table below.

[0040]

[0041] 2.3 Validation of rE2 monoclonal antibody specificity

[0042] Coating was performed overnight at 4°C using "rE2" at 0.4 μg / ml; blocking was done at 37°C for 2 hours with 3% fish gelatin; 100 μl of BVDV antibody-positive serum was diluted 5-fold with 3% fish gelatin and added to each well as a blocking well, while 100 μl of 3% fish gelatin was added to each well as an unblocked well, and incubated at 37°C for 1 hour. After washing three times with PBST, 100 μl of the same monoclonal antibody supernatant was added to each of the three blocking and three unblocked wells, and incubated at 37°C for 1 hour. After washing three times with PBST, 100 μl of HRP-labeled goat anti-mouse IgG (10000× dilution) diluted with 3% fish gelatin was added to each well, and incubated at 37°C for 1 hour. After washing three times with PBST, 100 μl of TMB chromogenic solution was added to each well, and incubated at room temperature for 10 minutes; after chromogenic development, 50 μL of stop solution was added to each well. The OD450nm value was measured using a microplate reader.

[0043] like Figure 5 As a result, the reactions of all five monoclonal antibodies decreased significantly after the addition of BVDV antibody-positive serum for blocking, indicating that all five monoclonal antibodies were 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] The positive hybridoma cell line with the highest blocking rate was selected. The hybridoma cell line E2mAb was deposited at the China General Microbiological Culture Collection Center on October 9, 2024, with accession number CGMCC NO: 46055, named cell line BVDV E2mAb (classification number: mouse myeloma cell line). The cells were expanded and cultured to prepare ascites fluid. The collected ascites fluid was purified and labeled with HRP enzyme.

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

[0048] According to the matrix titration method, antigens were coated at protein concentrations of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL (100 μL / well) and coated at 4°C for 12 h. After washing three times with PBST, blocking buffer of 3% BSA, 5% skim milk, and 3% fish gelatin (200 μL / well) was used for blocking at 37°C for 3 h. After washing once with PBST, BVDV antigen positive and negative controls were added. Dilute the enzyme-labeled monoclonal antibody at ratios of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128, adding 100 μL to each well and incubating at 37°C for 1 hour. Wash three times with PBST. Dilute the enzyme-labeled monoclonal antibody at ratios of 1:1000, 1:2000, 1:4000, and 1:8000, adding 100 μL to each well and incubating at 37°C for 1 hour. Wash three times with PBST, blot dry, add 100 μL of TMB to each well, and incubate at room temperature for 10 minutes. After color development, add 50 μL of stop solution per well. Measure the OD450nm value using a microplate reader, and determine the optimal combination of conditions based on the highest P / N ratio (Note: P / N = Positive control OD450nm / Negative control OD450nm × 100%).

[0049] After optimization, the reaction conditions were determined as follows: Antigen coating was performed at a concentration of 0.5 μg / mL, 100 μL / well, at 4℃ for 12 h; washing once with PBST, and blocking with 5% fish gelatin (200 μL / well) at 37℃ for 2 h; washing three times with PBST, and adding 100 μL of BVDV antigen positive and negative controls (diluted 1:1) to each well, incubating at 37℃ for 1 h; washing three times with PBST, and adding 100 μL of enzyme-labeled monoclonal antibody (diluted 1:1000) to each well, incubating at 37℃ for 1 h; washing three times with PBST, patting dry, and adding 100 μL of TMB to each well, incubating at room temperature for 10 min; after color development, adding 50 μL / well of stop solution. The OD value at 450 nm was measured using a microplate reader.

[0050] 3.3 Determination of Critical Values ​​for Antigen Capture ELISA Antibody Detection Method

[0051] The established antigen capture ELISA method was used to detect 15 BVDV antigen-positive bovine serum samples and 14 BVDV antigen-negative bovine serum samples. The cutoff value was calculated by adding 3 × standard deviation to the negative mean, and the cutoff value was determined to be 0.23.

[0052] 3.4 Preparation and Shelf Life Test of BVDV Antigen Capture ELISA Detection Kit

[0053] Antigen coating was performed at a concentration of 0.5 μg / mL rCD46 protein, 100 μL / well, at 4℃ for 12 h; washed 3 times with PBST; the blocking buffer was 5% fish gelatin + 10% sucrose, 200 μL / well, at 37℃ for 2 h; after aspirating and drying, the cells were placed in a sealed bag, desiccant was added, and then vacuum sealed.

[0054] Add 1‰ proclaz in BVDV antigen positive control and BVDV antigen negative control, aliquot, 1ml / tube;

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

[0056] TMB colorimetric solution is dispensed in 22ml bottles;

[0057] Dispense the stop solution (2m HCl) into 15ml vials.

[0058] Assemble two 96-well antigen-coated plates, one tube of BVDV antigen positive control, one tube of BVDV antigen negative control, one bottle of 10×PBST, one bottle of TMB chromogenic solution, and one bottle of stop solution into one kit. Store the kit in a freezer at 2-8℃. Take out one kit every 3 months for validation. Use frozen BVDV virus culture medium as the standard sample for validation, with 3 replicates.

[0059] The procedure is as follows: Remove the ELISA plate, wash once with PBST, and dilute BVDV virus culture medium, BVDV antigen positive control, and BVDV antigen negative control 1:1, adding 100 μL to each well and incubating at 37°C for 1 h; wash three times with PBST, dilute the enzyme-labeled monoclonal antibody 1:1000, adding 100 μL to each well and incubating at 37°C for 1 h; wash three times with PBST, blot dry, add 100 μL TMB to each well, and incubate at room temperature for 10 min; after color development, add 50 μL / well stop solution. Measure the OD450nm value using a microplate reader.

[0060] As a result, the OD value did not decrease significantly after the kit was stored for 9 months, indicating that the kit is stable.

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

[0062] The antigen capture ELISA method, which has been optimized, was used to test cultures of infectious bovine rhinotracheitis virus, bovine rotavirus, and bovine transmissible gastroenteritis virus. All test results were negative.

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

[0064] One sample of bovine viral diarrhea virus was serially diluted twofold and detected using the established antigen capture ELISA method to evaluate the sensitivity of the method.

[0065] The results showed that diluting bovine viral diarrhea virus fluid to 6×10⁻⁶... 3 TCID 50 It can still be detected after / mL.

[0066] 3.7 Repeatability Test of BVDV Antigen Capture ELISA Detection Kit

[0067] BVDV virus culture medium was detected using ELISA plates coated from the same batch, repeated three times, and the intra-batch coefficient of variation was calculated.

[0068] Three different batches of ELISA plates were coated, and BVDV virus culture medium was detected. The results were repeated three times, and the average value was taken to calculate the inter-batch coefficient of variation. The reproducibility of the method was evaluated based on the above results.

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

[0070] Intra-batch repeatability results

[0071]

[0072]

[0073] Inter-batch repeatability results

[0074] Sample number Repeat 1 Repeat 2 Repeat 3 average value 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 Detection Kit

[0076] 360 serum samples from unimmunized cattle in the field were collected and tested using a BVDV antigen capture ELISA reagent. The results showed that the positive rate of BVDV antigen in unimmunized cattle in the field was 1.4% (5 / 360).

[0077] This invention successfully prepared biologically active soluble bovine rCD46 and BVDV rE2 proteins using a eukaryotic system. Through mouse immunization and clonal screening, a monoclonal antibody specific to the rE2 protein was obtained and HRP-labeled. Using 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. This invention is the first in China to assemble a BVDV antigen capture ELISA detection kit, which has been verified to have good specificity and sensitivity, and a shelf life of at least 9 months.

[0078] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies, characterized in that, The assay included antigen coating with 0.5 μg / mL BVDV receptor rCD46 protein at a concentration of 100 μL / well, coated at 4℃ for 12 h, and HRP-labeled specific rE2 monoclonal antibody as the detection antibody. The BVDV receptor rCD46 expressing bacterial strain rbCD46 was deposited at the China General Microbiological Culture Collection Center on July 24, 2024, with accession number CGMCC NO.31440. The rE2 monoclonal antibody was derived from the monoclonal antibody hybridoma cell line E2mAb, which was deposited at the China General Microbiological Culture Collection Center on October 9, 2024, with accession number CGMCC NO.46055.

2. The BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies according to claim 1, characterized in that, Antigen was coated with 100 μL / well of 0.5 μg / mL protein at 4°C for 12 h; washed once with PBST, and blocked with 200 μL / well of 5% fish gelatin at 37°C for 2 h; washed three times with PBST, and 100 μL of positive and negative sera were added to each well at a 1:1 ratio and incubated at 37°C for 1 h; washed three times with PBST, and 100 μL of enzyme-labeled monoclonal antibody was added to each well at a 1:1000 ratio and incubated at 37°C for 1 h; washed three times with PBST, dried, and 100 μL of TMB was added to each well and incubated at room temperature for 10 min; after color development, 50 μL / well of stop solution was added and the OD450nm value was measured using a microplate reader.

3. The BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies according to claim 2, characterized in that, The critical value for the OD450nm value is 0.23.

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