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 invention belongs to the field of genetic engineering, and in particular relates to a BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies and a kit thereof. Background Art

[0002] Bovine viral diarrhea (BVD), also known as bovine viral diarrhea-mucosal disease, is a disease caused by the bovine viral diarrhea virus and is widely prevalent worldwide. BVD mainly causes clinical symptoms such as fever, mucosal erosion and ulceration, leukopenia, calf diarrhea, and cow abortion in cattle, which has brought great harm to the world's cattle industry. Since BVD has no specific clinical symptoms and is usually accompanied by immunosuppression, it brings great difficulties to the diagnosis and prevention of BVD, posing a huge threat to animal husbandry.

[0003] At present, the detection of BVD pathogens is mainly carried out by qPCR, RT-PCR and ELISA. Accurate and rapid detection of pathogens is crucial to the prevention of BVD, but all current detection methods have defects. Traditional detection methods such as cell culture have long detection cycles and cumbersome operations, which are not suitable for large-scale detection; PCR methods have high sensitivity and specificity, but the operation requires special equipment and skilled personnel, which increases the detection cost, and can only detect nucleic acids, not live pathogens. Therefore, a BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies and its kit are urgently needed. Summary of the invention

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

[0005] On the one hand, a BVDV receptor rCD46 expressing strain rbCD46 is provided, wherein the expressing strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 24, 2024, with the deposit number CGMCC NO: 31440.

[0006] On the other hand, the monoclonal antibody hybridoma cell line E2mAb strain was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 9, 2024, with the deposit number CGMCC NO.: 46055.

[0007] In another aspect, a positive hybridoma cell line monoclonal antibody is provided, wherein the positive hybridoma cell line monoclonal antibody is a monoclonal antibody secreted by the monoclonal antibody hybridoma cell line.

[0008] The monoclonal antibody secreted by the monoclonal antibody hybridoma cell line is used in a BVDV capture ELISA kit.

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

[0010] The invention discloses a BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies, comprising: using 0.5 μg / mL BVDV receptor rCD46 protein concentration, 100 μL / well for antigen coating, coating at 4°C for 12 hours, and using HRP-labeled specific rE2 monoclonal antibody as detection antibody.

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

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

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

[0014] The invention uses BVDV receptor rCD46 protein as coating antigen and HRP-labeled specific rE2 monoclonal antibody as detection antibody, and establishes a BVDV antigen capture ELISA detection method through condition optimization. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0017] Figure 3 It is the SDS-PAGE picture of the purified BVDV receptor rCD46 and BVDV rE2 protein;

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

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

[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 DESCRIPTION

[0021] The technical scheme of the present invention is further described below in conjunction with embodiments and comparative examples, but they should not be construed as limiting 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. BVDV invades cells after binding to CD46 and completes viral replication and proliferation after invasion. The E2 protein is an important structural protein of BVDV, and is divided into four functional regions, A, B, C and D, from the N-terminus to the C-terminus. The A and B regions (located at 4-87 nt and 88-164 nt, respectively) are the farthest from the membrane, are the most exposed regions on the virus surface, and are the most important parts of the E2 protein for producing immunogenicity. The present invention is based on the functions of rCD46 (recombinantly expressed CD46) and rE2 (recombinantly expressed E2) antigens, uses rCD46 receptor as a ligand for capturing antigens, uses HRP-labeled highly sensitive monoclonal antibody against rE2 antigen as a detection antibody, expresses rCD46 protein with 293 cells, prepares rE2 protein and monoclonal antibody with 293 cells, and labels the monoclonal antibody with HRP (horseradish peroxidase); uses rCD46 as a coating antigen, uses HRP-labeled rE2 monoclonal antibody as a detection antibody, establishes an antigen capture ELISA method for detecting BVDV virus, and develops an antigen capture ELISA kit for detecting BVDV virus.

[0023] Example 1. Expression of soluble rCD46 protein

[0024] 1.1 Vector construction and preliminary expression identification

[0025] like Figure 1As shown, the pTT5-rCD46 eukaryotic expression plasmid was constructed (the expression strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee on July 24, 2024, with a deposit number of CGMCC NO: 31440, named cell strain rbCD46, and the classification number was 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 100r / min for 5min, the culture medium was collected, and the cells were resuspended with an appropriate amount of PBS and ultrasonically broken; centrifuged at 4°C 12 000r / min for 20min, 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] 1L of 293F cells was used to express pTT5-rCD46 protein in large quantities and purified using Ni-NTA column. After purification, SDS-PAGE was used to verify the purification results 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 in the figure, the pTT5-rE2 eukaryotic expression plasmid was constructed, and the recombinant plasmid pTT5-rE2 was transfected into 293F cells using the transfection reagent PEI. After 5 days, the cells were collected and centrifuged at 100 r / min for 5 min, the culture medium was collected, and the cells were resuspended in 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] Results: WB confirmed that rE2 protein was successfully expressed.

[0032] 2.2 Large-scale expression and purification

[0033] 1L of 293F cells was used to express pTT5-rE2 protein in large quantities and purified using Ni-NTA column. After purification, SDS-PAGE was used to verify the purification results and the purified protein was collected.

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

[0035] The purified rE2 was emulsified with complete Freund's adjuvant and 60 ug protein / mouse was used for subcutaneous primary immunization of 4 SPF-grade BALB / c female mice, numbered 1, 2, 3, and 4.

[0036] Subcutaneous booster immunization was performed 2, 4, and 6 weeks after the initial immunization, with each immunization dose of 50ug protein / mouse. Blood was collected from the eye sockets 10 days after the third immunization to measure the serum titer. If the titer reached the standard, 50ug of the immunogen was used to impact the mice intraperitoneally 14 days after the third immunization.

[0037] Immunoassay: Use "rE2", 2ug / ml, 4℃ overnight coating; 5% skim milk, 37℃ blocking for 2h; serum is diluted 2 times from 200 times, blank control is 1*PBS, negative control is 200 times dilution of negative serum. Results: Select shock #4 mice for cell fusion experiment. Figure 4 shown.

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

[0039] The spleen cells of mice immunized with rE2 were fused with myeloma cells SP2 / 0, and subcloned three times. The indirect ELISA method was used for screening to obtain 5 positive hybridoma cell lines. The monoclonal antibody subclasses were identified, and the results are shown in the following table.

[0040]

[0041] 2.3 rE2 monoclonal antibody specificity verification

[0042] Use "rE2", 0.4ug / ml, 4℃ overnight coating; 3% fish gelatin, 37℃ blocking for 2h; dilute BVDV antibody positive serum with 3% fish gelatin 5 times, add 100ul to each well as blocking well, add 100ul of 3% fish gelatin to the well without serum as unblocked well, and incubate at 37℃ for 1h. Wash with PBST 3 times, take 3 wells of blocked wells and unblocked wells, add 100ul of the same monoclonal antibody supernatant, and incubate at 37℃ for 1h. Wash with PBST 3 times, add 100ul of HRP-labeled goat anti-mouse IgG (10000× dilution) diluted with 3% fish gelatin to each well, and incubate at 37℃ for 1h. Wash with PBST 3 times, then add 100ul TMB colorimetric solution to each well, and act at room temperature for 10min; after color development, add 50μL / well stop solution. Measure OD450nm value with an enzyme reader.

[0043] like Figure 5 The results showed that the reaction of the five monoclonal antibodies decreased significantly after adding BVDV antibody positive serum to block, indicating that the five monoclonal antibodies were all BVDV specific monoclonal antibodies.

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

[0045] 3.1 Monoclonal antibody preparation and labeling

[0046] A positive hybridoma cell line with the highest blocking rate is selected. The hybridoma cell line E2mAb strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 9, 2024, with a deposit number of CGMCC NO: 46055, named as cell line BVDV E2mAb, and classified as mouse myeloma cell line). The cells are expanded and cultured to prepare ascites, and the collected ascites is purified and labeled with HRP enzyme.

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

[0048] According to the square array titration method, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL protein concentrations were used for antigen coating at 100 μL / well and coated at 4°C for 12 h; PBST was washed three times, and the blocking solution was 3% BSA, 5% skim milk, and 3% fish gelatin, 200 μL / well, and blocked at 37°C for 3 h; PBST was washed once, and the BVDV antigen positive control and negative control Dilute 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 act at 37°C for 1 hour; 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 act at 37°C for 1 hour; wash 3 times with PBST, pat dry, add 100 μL TMB to each well, and act at room temperature for 10 minutes; after color development, add 50 μL / well stop solution. Measure the OD450nm value with an enzyme-labeled instrument, and determine the best combination condition 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: antigen coating at 0.5 μg / mL protein concentration, 100 μL / well, 4°C for 12 h; washed once with PBST, 5% fish gelatin was selected as the blocking solution, 200 μL / well was blocked at 37°C for 2 h; washed 3 times with PBST, BVDV antigen positive control and BVDV antigen negative control were diluted 1:1, 100 μL was added to each well, and 37°C was used for 1 h; washed 3 times with PBST, enzyme-labeled monoclonal antibody was diluted 1:1000, 100 μL was added to each well, and 37°C was used for 1 h; washed 3 times with PBST, patted dry, 100 μL TMB was added to each well, and 10 min was used at room temperature; after color development, 50 μL / well stop solution was added. OD450nm value was measured with an enzyme reader.

[0050] 3.3 Determination of critical value of antigen capture ELISA antibody detection method

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

[0052] 3.4 Preparation and shelf life test of BVDV antigen capture ELISA test kit

[0053] Antigen coating was performed at 100 μL / well with 0.5 μg / mL rCD46 protein concentration at 4°C for 12 h; PBST was used for washing 3 times, and 5% fish gelatin + 10% sucrose was used as the blocking solution at 200 μL / well at 37°C for 2 h. After blotting and drying, the cells were placed in a sealed bag, and vacuum-sealed after adding a desiccant;

[0054] Add 1‰ proclin to the BVDV antigen positive control and the BVDV antigen negative control and dispense them into 1 ml / tube;

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

[0056] TMB colorimetric solution, 22 ml / bottle;

[0057] Dispense the stop solution (2 m HCl) into 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 colorimetric solution, and 1 bottle of stop solution, assemble into a test kit, store in a 2-8℃ cold storage, and take out a test kit every 3 months for verification. During verification, use the frozen BVDV virus culture fluid as the standard sample, and set 3 replicates.

[0059] The process is as follows: take out the ELISA plate, wash once with PBST, dilute BVDV virus culture fluid, BVDV antigen positive control and BVDV antigen negative control at 1:1, add 100 μL to each well, and act at 37°C for 1 hour; wash 3 times with PBST, dilute enzyme-labeled monoclonal antibody at 1:1000, add 100 μL to each well, and act at 37°C for 1 hour; wash 3 times with PBST, pat dry, add 100 μL TMB to each well, and act at room temperature for 10 minutes; after color development, add 50 μL / well stop solution. OD450nm value is measured by microplate reader.

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

[0061] 3.5 Specificity test of BVDV antigen capture ELISA test kit

[0062] Infectious bovine rhinotracheitis virus culture fluid, bovine rotavirus culture fluid, bovine transmissible gastroenteritis virus culture fluid, etc. were tested using the antigen capture ELISA method with optimized conditions, and the test results were all negative.

[0063] 3.6BVDV antigen capture ELISA test kit sensitivity test

[0064] One portion of BVD virus solution was diluted 2-fold in series and tested using the established antigen capture ELISA method to assess the sensitivity of the method.

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

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

[0067] The BVDV virus culture fluid was detected using the same batch of coated ELISA plates, repeated 3 times, and the intra-batch coefficient of variation was calculated.

[0068] Three different batches of ELISA plates were coated and BVDV virus culture fluid was tested. The results were repeated three times to obtain the average value and calculate the inter-batch coefficient of variation. Based on the above results, the repeatability of the method was evaluated.

[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-assay reproducibility results

[0071]

[0072]

[0073] Batch-to-batch reproducibility results

[0074] Sample No. 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 test kit

[0076] 360 samples of sera from non-immunized cattle in the field were tested using BVDV antigen capture ELISA detection reagent. The results 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, obtained a monoclonal antibody of specific rE2 protein and carried out HRP labeling through immunization of mice and clone screening, took rCD46 protein as coating antigen and HRP-labeled specific rE2 monoclonal antibody as detection antibody, established a BVDV antigen capture ELISA detection method through condition optimization, and assembled the first BVDV antigen capture ELISA detection kit in China. It has been verified that the kit has good specificity and sensitivity and a shelf life of at least 9 months.

[0078] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A BVDV receptor rCD46 expressing strain rbCD46, characterized in that: The expression strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 24, 2024, 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 in the General Microbiology Center of China Microorganism Culture Collection Administration on October 9, 2024, with the deposit number CGMCC NO.: 46055.

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

4. Use of the monoclonal antibody secreted by the monoclonal antibody hybridoma cell line according to claim 2 in preparing a capture ELISA kit for detecting BVDV antigen.

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

6. A BVDV antigen capture ELISA method based on rCD46 and rE2 monoclonal antibodies, characterized in that: The method includes coating the antigen with 0.5 μg / mL BVDV receptor rCD46 protein at a concentration of 100 μL / well, coating at 4° C. for 12 hours, and using 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 was performed at a protein concentration of 0.5 μg / mL at 100 μL / well and coated at 4°C for 12 h; washed once with PBST, and 5% fish gelatin was selected as the blocking solution, 200 μL / well was blocked at 37°C for 2 h; washed three times with PBST, positive serum and negative serum were diluted 1:1, 100 μL was added to each well, and incubated at 37°C for 1 h; washed three times with PBST, enzyme-labeled monoclonal antibody was diluted 1:1000, 100 μL was added to each well, and incubated at 37°C for 1 h; washed three times with PBST, patted dry, 100 μL TMB was added to each well, and incubated at room temperature for 10 min; after color development, 50 μL / well stop solution was added and the OD450nm value was measured with an enzyme reader.

8. The 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 was 0.23.

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