Hybridoma cell strain secreting monoclonal antibody against bovine viral diarrhea virus NS3 protein and application of hybridoma cell strain

Abstract

The invention provides a hybridoma cell strain secreting an anti-bovine viral diarrhea virus NS3 protein monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of biology. The preservation number of the hybridoma cell strain 4B3 is CCTCC (China Center For Type Culture Collection) NO: C2025217. The invention also provides an anti-bovine viral diarrhea virus NS3 protein monoclonal antibody secreted by the hybridoma cell strain 4B3 and a sandwich ELISA kit for detecting bovine viral diarrhea virus. The hybridoma cell strain 4B3 obtained through screening can be specifically combined with recombinant NS3 protein and NS3 protein in cells infected with BVDV-1 and BVDV-2 type strains, so that the BVDV-1 and BVDV-2 type strains can be detected with high sensitivity and high specificity, and the hybridoma cell strain 4B3 can be used for Western blot detection and immunofluorescence detection of BVDV.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to hybridoma cell lines that secrete monoclonal antibodies against bovine viral diarrhea virus NS3 protein and their applications. Background Technology

[0002] Ruminant disease viruses, including bovine viral diarrhea virus (BVDV) and border disease virus (BDV), typically cause acute infectious diseases in cattle and sheep, posing a significant threat to livestock farming. BVDV belongs to the Flaviviridae family and the Pestivirus genus. This genus has numerous members and can infect ruminants such as cattle and sheep. Clinical symptoms of BVDV infection in cattle are complex and diverse, primarily characterized by diarrhea, fever, mucosal erosion and ulceration, abortion in pregnant cows, stillbirths, or birth defects. Furthermore, the virus can be transmitted through the placenta, producing persistently infected cattle (PI cattle). PI cattle are carriers and shed the virus year-round, becoming a major source of infection in livestock herds. Besides cattle, BVDV can also infect pigs, goats, sheep, and many other livestock, with widespread prevalence and significant harm. BVDV includes two types, BVDV-1 and BVDV-2. Analysis based on the viral 5′UTR sequence and comprehensive analysis of other genomic regions, such as N... pro Or E2, currently 21 BVDV-1 subtypes (au) and 4 BVDV-2 subtypes (ad) have been identified.

[0003] Vaccination, identification, and culling of infected cattle and PI cattle are crucial means of controlling BVDV infection and transmission. Currently, there are various detection methods for this virus, including virus isolation, indirect immunofluorescence, RT-PCR, quantitative RT-PCR, and ELISA. However, the sensitivity of existing BVDV detection methods needs further improvement. Summary of the Invention

[0004] The purpose of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody against the NS3 protein of bovine viral diarrhea virus (BVDV), which enables high-sensitivity detection of BVDV.

[0005] Another object of the present invention is to provide a monoclonal antibody against the NS3 protein of bovine viral diarrhea virus secreted by the hybridoma cell line.

[0006] Another object of the present invention is to provide a sandwich ELISA kit for detecting bovine viral diarrhea virus (BVDV) with high sensitivity and high specificity.

[0007] The objective of this invention is achieved through the following technical solution.

[0008] Hybridoma cell line 4B3, which secretes monoclonal antibodies against bovine viral diarrhea virus NS3 protein, has the accession number CCTCCNO: C2025217.

[0009] The present invention also provides a monoclonal antibody against bovine viral diarrhea virus NS3 protein secreted by the hybridoma cell line 4B3.

[0010] In this invention, the method for preparing the monoclonal antibody against bovine viral diarrhea virus NS3 protein includes the following steps: injecting the hybridoma cells 4B3 into the peritoneal cavity of BALB / c mice to prepare ascites, centrifuging and collecting the ascites supernatant, and purifying it to obtain the antibody.

[0011] The present invention also provides a sandwich ELISA kit for detecting bovine viral diarrhea virus, wherein the detection antibody is a horseradish peroxidase-labeled monoclonal antibody against bovine viral diarrhea virus NS3 protein as described in claim 2.

[0012] In this invention, the kit also includes an enzyme-labeled plate coated with an anti-NS3 protein polyclonal antibody, which is obtained by immunizing New Zealand white rabbits with NS3 protein, taking serum, and purifying it.

[0013] The present invention also provides the application of the monoclonal antibody to Western blot detection or indirect immunofluorescence detection of bovine viral diarrhea virus NS3 antigen for non-diagnostic purposes.

[0014] Beneficial Effects: The hybridoma cell line 4B3 obtained by screening in this invention can secrete a highly sensitive and specific monoclonal antibody for detecting BVDV. This monoclonal antibody can specifically bind to recombinant NS3 protein and NS3 protein in cells infected with BVDV-1 and BVDV-2 strains, thus effectively detecting BVDV-1 and BVDV-2 strains. Its recognition site differs from that used in previously reported monoclonal antibodies and commercial kits. The sandwich ELISA kit provided by this invention has good sensitivity, specificity, and reproducibility, providing technical support for monitoring BVDV infection and rapid identification of infected animals, especially PI individuals. In addition, the monoclonal antibody 4B3 has good reactivity and can be used for Western blot detection and immunofluorescence detection of BVDV. Attached Figure Description

[0015] Figure 1These are electrophoresis images for the expression and identification of recombinant NS3 protein. A is an SDS-PAGE electrophoresis image, and B is a Western blot result (His antibody detection). In the image, M represents the protein molecular weight standard; 1 represents the supernatant of the lysate after pET-28a-NS3-1 (BL21) induction; 2 represents the precipitate of the lysate after pET-28a-NS3-1 (BL21) induction; 3 represents the negative control protein; and 4 represents the purified recombinant NS3 protein.

[0016] Figure 2 Western blot was used to identify the differences in recognition sites between commercially available antibodies and 4B3. A is the anti-NS3 protein monoclonal antibody 4B3, B is a mixture of monoclonal antibodies WB112 and WB103, and C is the detection antibody in the commercially available BVDVNS3 antigen detection kit. Lane 1 is the protein marker, and lanes 2-6 contain protein samples of NS3, NS3-A, NS3-B, NS3-B1, and NS3-B2, respectively.

[0017] Figure 3 The reactivity characteristics of monoclonal antibody 4B3 were detected by indirect immunofluorescence, where A: MDBK cells infected with BVDV-1; B: MDBK cells infected with BVDV-2; and C: normal MDBK cells.

[0018] Figure 4 The results of optimized reaction conditions are shown. A: D corresponding to different dilutions of the sample to be tested. 450 Value and P / N value; B: D corresponding to different incubation times of the sample to be tested. 450 Values ​​and P / N values; C: D corresponding to different antibody incubation times. 450 Value and P / N value; D: D corresponding to different color development times 450 Values ​​and P / N values. Where, D... 450 Value refers to OD 450 value.

[0019] Figure 5 This demonstrates the specificity of the kit of the present invention.

[0020] Figure 6 A comparison of sensitivity test results between the kit of this invention and a commercially available kit. Figure (A) shows the sensitivity of the two kits to BVDV-1-infected MDBK cell lysates, and Figure (B) shows the sensitivity of the two kits to recombinant NS3 protein. "Sandwich ELISA" refers to the kit of this invention. The commercially available kit refers to the antigen detection kit from ID Vet. Detailed Implementation

[0021] The PBST in this invention is a PBS buffer solution with pH 7.4 containing 0.5% (w / w) Tween-20 and a concentration of 0.01 mol / L.

[0022] In this invention, the overnight wrapping time is 12-16 hours.

[0023] Example 1: Preparation of recombinant NS3 protein

[0024] 1. Construction of recombinant plasmids and protein expression

[0025] Based on the BVDVNS3 protein sequence published in GenBank (GenBank accession number: AGM75780), the coding gene for the major antigenic region (amino acids 195-683) was selected. Codon optimization yielded the sequence shown in SEQ ID NO:1, named NS3-1, whose encoded protein sequence is shown in SEQ ID NO:2. The sequence shown in SEQ ID NO:1 was synthesized and cloned into the pET-28a(+)BamHI and XhoI sites to obtain the recombinant plasmid pET-28a-NS3-1. The recombinant plasmid pET-28a-NS3-1 was transformed into *E. coli* competent cells (BL21). Single colonies were picked, identified by sequencing, and named pET-28a-NS3-1(BL21). pET-28a-NS3-1(BL21) was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C until OD500. 600 When the concentration reached 0.6, IPTG was added to a final concentration of 0.5 mmol / L, and expression was induced at 37℃ for 6 h. After collecting the bacterial cells, they were lysed by sonication, and the supernatant and precipitate of the lysate were collected separately. The expression of the recombinant protein was identified by SDS-PAGE gel electrophoresis. pET-28a(+) was transformed into competent *E. coli* cells to obtain the control strain. The control strain was cultured using the above method, and the lysate was used as a negative control protein for subsequent experiments. Figure 1 As shown in A, the recombinant NS3 protein (56kD) was successfully expressed and mainly existed in the supernatant form.

[0026] 2. Purification and identification of recombinant NS3 protein

[0027] The recombinant strain pET-28a-NS3-1(BL21) was inoculated into 200 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C until OD500. 600When the concentration reaches 0.6, add IPTG to a final concentration of 0.5 mmol / L, induce at 37℃ for 5 h, collect bacteria in 50 mL centrifuge tubes, centrifuge at 8000 r / min, 4℃ for 10 min, discard the supernatant, and resuspend the bacteria in 40 mL of PBS buffer (pH = 7.4, 0.01 mol / L). Sonicate the bacterial cells, centrifuge again, collect the supernatant, and follow the HisTrap procedure. TM The recombinant protein was purified using the HP (GE) purification instructions to obtain purified recombinant NS3 protein. The concentration of the purified recombinant NS3 protein was determined, and the protein was aliquoted and stored at -20°C.

[0028] 3. Western blot identification of recombinant NS3 protein

[0029] The purified recombinant NS3 protein was mixed with loading buffer, boiled for 10 min, and then subjected to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Afterward, the protein was transferred to a nitrocellulose membrane using a wet transfer method and blocked with PBST containing 5% skim milk at 37°C for 2 h. The membrane was washed three times with PBST (PBS buffer containing 0.5% (w / v) Tween-20, pH 7.4, 0.01 mol / L, hereinafter the same), and then incubated 1:1000 diluted His-tagged monoclonal antibody (Beijing TransGen Biotech Co., Ltd.) at room temperature for 1 h. After washing three times with PBST, the membrane was incubated 1:5000 diluted HRP-labeled goat anti-mouse IgG secondary antibody (Beijing TransGen Biotech Co., Ltd., hereinafter the same) at room temperature for 1 h. Finally, the membrane was washed three times with PBST, and color development was performed according to the DAB chromogenic reagent kit instructions (Tiangen Biotech (Beijing) Co., Ltd.). Figure 1 As shown in Figure B, the purified recombinant NS3 protein exhibits a specific band at 56 kDa in the swimming lane. The purified recombinant NS3 protein can react with the His-tagged monoclonal antibody, indicating that it is correctly expressed and has good reactivity.

[0030] Example 2: Preparation of monoclonal antibody hybridoma cell lines

[0031] 1. Immunize BALB / c mice

[0032] Purified recombinant NS3 protein was emulsified with ISA201 adjuvant at a volume ratio of 1:1 and then injected subcutaneously at multiple sites to immunize BALB / c mice (immunization dose: 50 μg recombinant NS3 protein / mouse, 0.2 mL). Two booster immunizations were then performed, each two weeks apart, for a total of three immunizations. The immunization dose and method were the same as the first immunization. Two weeks after the third immunization, blood was collected, and the serum titer of the immunized mice was detected using an indirect ELISA method. The indirect ELISA method is as follows: purified recombinant NS3 protein was diluted to a concentration of 1 μg / mL with 0.05 mol / L, pH 9.6 carbonate buffer, coated onto 96-well microplates, 100 μL / well, and incubated overnight at 4°C. Wash three times with PBST and blot dry. Add 200 μL of PBST containing 0.5% (w / w) BSA to each well and block at 37°C for 2 h. Wash three times with PBST and blot dry. Add serially diluted mouse serum (before and after immunization) and incubate at 37°C for 1 h. Wash three times with PBST and blot dry. Add 100 μL of HRP-labeled goat anti-mouse IgG (Beijing TransGen Biotech Co., Ltd.) diluted 1:4000 to each well and incubate at 37°C for 1 h. Wash three times with PBST and blot dry. Add TMB substrate chromogenic solution (Huzhou Yingchuang Biotechnology Co., Ltd., the same below) and incubate at room temperature in the dark for 10 min. Stop the reaction by adding 50 μL of 2 mol / L sulfuric acid aqueous solution to each well. Measure the OD of each well using a microplate reader. 450nm Value, P is the OD of each detection well. 450nm Value, N is the OD value of negative serum (pre-immunized mouse serum). 450nm The serum titer was determined by the maximum serum dilution with a P / N ratio ≥ 2.1. Mice with a serum titer > 51200 were selected and given a booster immunization with 50 μg of purified recombinant NS3 protein via intraperitoneal injection 4 days before cell fusion.

[0033] 2. Cell fusion

[0034] The PEG cell fusion method was adopted, and the specific operation was as follows: mouse myeloma cells (SP2 / 0) and spleen cells from immunized BALB / c mice (mice with serum titers >51200 selected in Title 1 of this embodiment) were thoroughly mixed at a ratio of 1:5, centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and an appropriate amount of serum-free RPMI-1640 medium (purchased from Hyclone) was added for resuspending. The cells were centrifuged at 2000 rpm for 5 min to wash them, the supernatant was discarded, and the bottom of the tube was gently tapped to loosen and evenly disperse the cells. The tube was preheated in a 37°C water bath, and 0.8 mL of PEG2000 preheated in a 37°C water bath was added within 90 s while shaking. After adding the contents, let stand for 1 min. Then, within 5 min, add 1 mL, 2 mL, 3 mL, 3 mL, and 3 mL of serum-free RPMI-1640 medium (purchased from Hyclone) preheated to 37°C in sequence at rates of 1 mL / min, 2 mL / min, 3 mL / min, 3 mL / min, and 3 mL / min. Let stand at 37°C for 10 min. Then, centrifuge at 2000 rpm and 25°C for 5 min, discard the supernatant, and resuspend the contents in RPMI-1640 medium containing 20% ​​FBS (fetal bovine serum), 20% HAT, and 10% hybridoma cell-adding factor (Biolong Technology Co., Ltd.). Aliquot the contents into 96-well plates, 200 μL per well, and incubate in a 5% CO2 incubator. During this period, the condition of the cells in the wells was observed. After 5 days of fusion, the nutrient solution was replaced and cultured again. When the cells grew to 1 / 10 to 1 / 5 of the bottom area of ​​the 96-well plate, the supernatant of the fusion cells was taken and the indirect ELISA antibody detection was performed using the following method: the purified recombinant NS3 protein was diluted with 0.05 mol / L, pH 9.6 carbonate buffer to a concentration of 1 μg / mL, coated on a 96-well microplate, 100 μL / well, and incubated overnight at 4°C. Wash three times with PBST and blot dry. Add 200 μL of PBST containing 0.5% (w / w) BSA to each well and block at 37°C for 2 h. Wash three times with PBST and blot dry. Add 100 μL of fusion cell supernatant or SP2 / 0 cell culture supernatant to the corresponding wells and incubate at 37°C for 1 h. Wash three times with PBST and blot dry. Add 100 μL of 1:4000 diluted HRP-labeled goat anti-mouse IgG (Beijing TransGen Biotech Co., Ltd.) to each well and incubate at 37°C for 1 h. Wash three times with PBST and blot dry. Add 100 μL of TMB substrate chromogenic solution to each well and incubate at room temperature in the dark for 10 min. Stop the reaction by adding 50 μL of 2 mol / L sulfuric acid aqueous solution to each well. Measure the OD of each well using a microplate reader. 450nm Value, P is the OD of each detection well. 450nm Value, N is the OD of SP2 / 0 cell culture supernatant. 450nm The P / N ratio was used as the criterion for judging positive wells. Hybridoma cell lines with positive test results, high P / N values ​​and good growth status were selected for subcloning.

[0035] 3. Cloning of hybridoma cells

[0036] The selected positive cell lines were stained with trypan blue, counted, and diluted to 200 cells / 10 mL with RPMI-1640 medium containing 20% ​​FBS (fetal bovine serum) and 10% hybridoma cell-adding factor (Bio-Long Technology Co., Ltd.). The diluted cell suspension was added to 100 μL per well of a 96-well plate and incubated at 37°C in a 5% CO2 incubator. Cell lines were observed during incubation. When the cell lines grew to 1 / 10 to 1 / 5 of the bottom area of ​​each well, ELISA was performed promptly according to the indirect ELISA method described in Title 2 of this embodiment. Positive wells with single-cloned cells were recorded, and the same subcloning was performed at least three times until all cloned cell lines showed positive results in supernatant detection and the OD values ​​of each well were [data missing]. 450nm The values ​​were relatively close. Hybridoma cell line 4B3 was ultimately selected. Hybridoma cell line 4B3 was expanded through culture, the culture supernatant was collected and stored at -20°C, and the cells were stored in liquid nitrogen.

[0037] 4. Stability determination of antibodies secreted by hybridoma cell lines

[0038] The obtained hybridoma cell line 4B3 was continuously cultured and passaged 20 times, and subjected to liquid nitrogen cryopreservation and thawing experiments. The antibody titers of the culture supernatant from different passages of the hybridoma cell line, as well as the antibody titers of the culture supernatant after liquid nitrogen cryopreservation and thawing, were detected using the indirect ELISA method described in Title 2 of this embodiment. The titers were found to be consistently between 32,000 and 64,000, demonstrating that this hybridoma cell line can continuously and stably secrete monoclonal antibodies against BVDVNS3 protein. The monoclonal antibody secreted by hybridoma cell line 4B3 was named anti-BVDVNS3 protein monoclonal antibody 4B3, abbreviated as anti-NS3 protein monoclonal antibody 4B3.

[0039] The preservation information for hybridoma cell line 4B3 is as follows:

[0040] Classification and nomenclature: Hybridoma cell line 4B3

[0041] Hybridoma cell line 4B3,

[0042] The deposit date is July 22, 2025.

[0043] The full name of the depository is the China Center for Type Culture Collection, abbreviated as CCTCC.

[0044] The address of the depositary institution is Wuhan University, Wuhan, China.

[0045] The accession number is CCTCC NO: C2025217.

[0046] Example 3: Preparation of anti-NS3 protein monoclonal antibody 4B3 and HRP-labeled anti-NS3 protein monoclonal antibody 4B3 1. Preparation of anti-NS3 protein monoclonal antibody 4B3

[0047] Sterile liquid paraffin was injected intraperitoneally into 10-12 week old BALB / c mice (purchased from the Comparative Medicine Experimental Center of Yangzhou University), 0.5 mL per mouse. Seven days later, each mouse was injected intraperitoneally with 0.2 mL of hybridoma cell line 4B3 suspension (containing 2 × 10⁻⁶ cells). 6 (Hybridoma cells). After 7-10 days, ascites fluid was collected from mice with significantly distended abdomens. The fluid was centrifuged at 8000 rpm for 20 min, and the supernatant was collected. Monoclonal antibody 4B3 in the ascites fluid was purified using a Protein A / G agarose gel chromatography column (Shanghai Beyotime Biotechnology Co., Ltd.) according to the manufacturer's instructions. The purified anti-NS3 protein monoclonal antibody 4B3 was obtained by dialyzing with PBS buffer at pH 7.4 for 12 h. After determining the concentration, the antibody was aliquoted and stored at -20℃.

[0048] 2. Preparation of HRP-labeled anti-NS3 protein monoclonal antibody 4B3

[0049] The purified monoclonal antibody 4B3 was HRP-labeled using a horseradish peroxidase labeling kit (including pre-activated HRP, labeling start solution, and stop solution, purchased from Bio-Long Technology Co., Ltd.): 100 μL of pre-activated HRP was added to 0.5 mL of 2 mg / mL monoclonal antibody 4B3, and after thorough mixing, 108 μL of labeling start solution was added, mixed, and incubated at 37°C in the dark for 2.5 h. Then, 50 μL of stop solution was added, and the reaction was terminated by incubation at room temperature for 1 h, yielding HRP-labeled anti-NS3 protein monoclonal antibody 4B3 (abbreviated as HRP-4B3). The concentration of HRP-4B3 was adjusted to 0.5 mg / mL with PBS buffer (pH 7.4), aliquoted, and stored at -20°C.

[0050] Example 4: Characterization of anti-NS3 protein monoclonal antibody 4B3

[0051] This embodiment identifies the characteristics of the anti-NS3 protein monoclonal antibody 4B3.

[0052] 1. Isotype determination of anti-NS3 protein monoclonal antibody 4B3

[0053] Hybridoma cell line 4B3 was expanded and cultured in RPMI-1640 medium containing 20% ​​FBS (fetal bovine serum) and incubated at 37°C in a 5% CO2 incubator for 3 days. The culture supernatant was then collected.

[0054] The subtype of monoclonal antibody 4B3 was determined using a monoclonal antibody subtype identification kit (purchased from Thermo Fisher Scientific). The results showed that the heavy chain of this monoclonal antibody was of the IgG1 subtype and the light chain was of the κ type.

[0055] 2. Western blot analysis to identify differences between commercially available antibodies and the 4B3 recognition site.

[0056] Based on the protein sequence of BVDVNS3 published in GenBank (GenBank accession number: AGM75780), different fragment regions were selected: 195-371aa (amino acids 195-371, named NS3-A protein), 367-683aa (amino acids 367-683, named NS3-B protein), 367-549aa (amino acids 367-549, named NS3-B1 protein), and 536-683aa (amino acids 536-683, named NS3-B2 protein). According to SEQ ID... The sequence shown in NO:1 was synthesized into the corresponding fragment, cloned into pET-28a(+) to obtain recombinant plasmids, resulting in recombinant vectors pET-28a-NS3-A expressing NS3-A, pET-28a-NS3-B expressing NS3-B, pET-28a-NS3-B1 expressing NS3-B1, and pET-28a-NS3-B2 expressing NS3-B2, respectively. Following the method in Example 1, *E. coli* competent cells were transformed to prepare recombinant proteins NS3-A, NS3-B, NS3-B1, and NS3-B2, respectively. Western blot analysis was used to identify differences in the recognition sites of the commercially available antibodies and 4B3. The commercially available antibodies included a mixture of anti-BVDV NS3 protein antibodies WB112 and WB103 (purchased from APHA, UK) and a commercially available BVDV NS3 antigen detection kit (ID).

[0057] The detection antibody was found in BVD P80 antigen capture (ID.Vet, France). Results are as follows: Figure 2 As shown, a mixture of commercially available anti-BVDVNS3 protein antibodies WB112 and WB103 (purchased from APHA, UK) and a commercially available BVDVNS3 antigen detection kit (ID) are also included. The detection antibody in BVD P80 antigen capture (ID. Vet, France) reacts with recombinant proteins NS3, NS3-B, and NS3-B2, while 4B3 reacts with NS3, NS3-B, and BS3-B1. This indicates that the 4B3 recognition site is located in NS3 protein 367-549aa, while the commercially available antibody recognition sites are located in 195-371aa and 536-683aa.

[0058] 3. Indirect immunofluorescence assay to detect the reaction characteristics of anti-NS3 protein monoclonal antibody 4B3

[0059] The reaction characteristics of the anti-NS3 protein monoclonal antibody 4B3 with the NS3 protein of BVDV in infected cells were detected by indirect immunofluorescence assay. BVDV-1JS2201 (Genbank accession number: OP856581) and BVDV-2C201602 (Genbank accession number: MG420995) strains were seeded at MOI = 0.5 in 24-well cell culture plates with a confluent MDBK cell line. Uninoculated MDBK cells were used as a blank control. Cells were cultured at 37°C for 3 days, the culture medium was discarded, and immunostaining fixation solution (Shanghai Beyotime Biotechnology Co., Ltd.) was added for 20 minutes at room temperature to fix the cells. n. Wash three times with PBST, add immunostaining blocking solution (Shanghai Beyotime Biotechnology Co., Ltd.), block at 37℃ for 1 h, wash three times with PBST, add 100 μL / well of culture supernatant of hybridoma cell line 4B3, incubate at 37℃ for 1 h, wash three times with PBST, and pat dry; add 100 μL / well of FITC-labeled goat anti-mouse IgG (Shanghai Beyotime Biotechnology Co., Ltd.) diluted 1:500, incubate at 37℃ for 1 h, wash three times with PBST, pat dry, and observe under a fluorescence microscope. Results are as follows. Figure 3 As shown, the monoclonal antibody 4B3 produced by the hybridoma cell line 4B3 specifically reacts with cells infected with BVDV-1 and BVDV-2, exhibiting green fluorescence mainly located in the cytoplasm, while showing no specific reaction with blank control cells. This indicates that monoclonal antibody 4B3 can be used for immunofluorescence detection of BVDV-1 and BVDV-2 strains.

[0060] 4. Monoclonal antibody 4B3 blocking ELISA detection

[0061] To determine whether monoclonal antibody 4B3 could be used to block ELISA detection, BVDV-1 positive serum, BVDV-2 positive serum, BDV positive serum, and BVDV negative serum were used for the experiment. The purified recombinant NS3 protein was diluted to 0.5 μg / mL with 0.05 mol / L, pH 9.6 carbonate buffer and coated onto 96-well microplates at 100 μL / well, incubated overnight at 4°C. The plates were washed three times with PBST and blotted dry. PBST containing 0.5% (w / v) BSA was added at 200 μL / well, and the plates were blocked at 37°C for 2 h. The plates were washed three times with PBST and blotted dry. Samples diluted with PBST at a 1:5 volume ratio (100 μL / well) were added to the sample detection wells and incubated at 37°C for 1 h. A blank control well was also included, containing only 100 μL of PBST. Wash three times with PBST and blot dry. Add 100 μL of HRP-4B3 diluted 1:1000 (previous concentration: 0.5 mg / mL) to each well and incubate at 37°C for 1 h. Wash three times with PBST and blot dry. Add 100 μL of TMB substrate chromogenic solution to each well and incubate at room temperature in the dark for 10 min. Terminate the reaction by adding 50 μL of 2 mol / L sulfuric acid aqueous solution to each well. After terminating the reaction, measure the OD of each well using a microplate reader. 450nm The blocking rate PI (%) is calculated using the following formula: PI = 100% × (OD of blank control wells) 450nm Value - OD of sample detection well 450nm Value) / Blank control well OD 450nm The results are shown in Table 1. Positive sera of BVDV-1, BVDV-2, and BDV can all block the reaction between 4B3 and recombinant NS3 protein, while negative sera cannot. This indicates that the monoclonal antibody can be used to establish a blocking ELISA method to detect positive samples of BVDV-1, BVDV-2, and BDV. Since BDV and BVDV NS3 antigens have high homology, the monoclonal antibody 4B3 of this invention recognizes the common antigenic epitope of BVDV and BDV and can also be used to detect positive samples of BDV. This is advantageous for actual quarantine processes, as BDV and BVDV both belong to the ruminant virus family and usually cause acute infectious diseases, posing a significant threat to herbivorous livestock farming.

[0062] Table 1. Results of ELISA detection using monoclonal antibody 4B3 blocking.

[0063]

[0064] Example 5: Establishment of a sandwich ELISA detection method

[0065] 1. Preparation of polyclonal antibodies

[0066] Purified recombinant NS3 protein was emulsified with ISA201 adjuvant at a volume ratio of 1:1 and then injected subcutaneously at multiple sites to immunize New Zealand white rabbits. The immunization dose was 250 μg of recombinant NS3 protein per rabbit, with an inoculation volume of 1 mL. Three booster immunizations were then performed, each two weeks apart from the previous immunization, using the same dosage and method as the first immunization. Two weeks after the fourth immunization, blood samples were collected, and serum titers were detected using an indirect ELISA method. The indirect ELISA method was as follows: purified recombinant NS3 protein was diluted to a concentration of 0.5 μg / mL with 0.05 mol / L, pH 9.6 carbonate buffer, coated onto 96-well microplates, 100 μL / well, and incubated overnight at 4°C. Wash three times with PBST and blot dry. Add 200 μL of PBST containing 0.5% (w / v) BSA to each well and block at 37°C for 2 h. Wash three times with PBST and blot dry. Add 100 μL of rabbit serum (2-fold diluted before and after immunization) to each well and incubate at 37°C for 1 h. Wash three times with PBST and blot dry. Add 100 μL of HRP-labeled goat anti-rabbit IgG (Beijing TransGen Biotech Co., Ltd.) diluted 1:4000 to each well and incubate at 37°C for 1 h. Wash three times with PBST and blot dry. Add 100 μL of TMB substrate chromogenic solution (Huzhou Yingchuang Biotechnology Co., Ltd., hereinafter the same) to each well and develop color at room temperature in the dark for 10 min. Stop the reaction by adding 50 μL of 2 mol / L sulfuric acid aqueous solution to each well. Measure the OD of each well using a microplate reader. 450nm Value, P is the OD of each detection well. 450nm Value, N is the OD of negative serum (pre-immune serum) 450nm The titer was determined using the highest serum dilution with a P / N ratio ≥ 2.1. The titer of the polyclonal antibody was 512,000. Anti-NS3 protein polyclonal antibody was purified from rabbit serum using a Protein A / G agarose gel chromatography column (Shanghai Beyotime Biotechnology Co., Ltd.), and the concentration was determined to be 2 mg / mL. The antibody titer was 128,000 as detected by ELISA. After aliquoting, the antibody was stored at -20℃.

[0067] 2. Establishment of BVDV sandwich ELISA detection method

[0068] (1) Determination of the optimal coating concentration of the capture antibody and the optimal dilution of the detection antibody

[0069] The optimal coating concentration of the anti-NS3 protein polyclonal antibody (prepared in Title 1 of this embodiment) and the optimal dilution of HRP-4B3 were determined using a square matrix titration method. The specific method is as follows: 2 mg / mL of purified anti-NS3 protein polyclonal antibody was diluted with antigen coating buffer (0.05 mol / L, pH 9.6 carbonate buffer) to concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, and added to 96-well microplates, 100 μL per well, and incubated overnight (12-16 h) at 4°C. After washing the microplate three times with PBST, 200 μL of PBST containing 0.5% (w / w) bovine serum albumin was added to each well, and the plate was blocked at 37°C for 2 h. The enzyme was then washed with PBST. The plate was labeled three times. MDBK cell lysate infected with BVDV served as a positive control, and MDBK cell lysate served as a negative control. Both positive and negative controls were diluted 1:10 with PBST, with 100 μL added to each well. The plates were incubated at 37°C for 1 h. After washing the plate three times with PBST, 100 μL of HRP-4B3 diluted with PBST at concentrations of 1:500, 1:1000, 1:2000, 1:4000, and 1:6000 (pre-dilution concentration 0.5 mg / mL) was added to each well, and the plates were incubated at 37°C for 1 h. After washing the plate three times with PBST, 100 μL of TMB substrate chromogenic solution was added to each well, and the plate was incubated for 10 min. The reaction was terminated by adding 50 μL of 2 mol / L sulfuric acid aqueous solution. The OD values ​​were then read using a microplate reader. 450 Value. Select the condition corresponding to the maximum P / N value as the optimal coating concentration for the capture antibody and the optimal dilution for the detection antibody.

[0070] The results are shown in Table 2. The final coating concentration of the anti-NS3 protein polyclonal antibody was determined to be 2 μg / mL, and the dilution of 0.5 mg / mL HRP-4B3 was 1:2000.

[0071] Table 2. Determination of the optimal coating concentration of the capture antibody and the optimal dilution of the detection antibody.

[0072]

[0073] (2) Screening of the optimal dilution of the sample to be tested

[0074] Positive and negative controls were diluted with PBST at dilutions of 1:2, 1:4, 1:10, and 1:20, respectively, and tested according to the conditions determined in Title 2 (1) of this embodiment to examine the effect of sample dilution on the test results. The optimal dilution of the test sample was determined to be 1:2 based on the maximum P / N value. Figure 4 A).

[0075] (3) Screening of the optimal incubation time for the sample to be tested

[0076] The positive and negative controls were diluted at a ratio of 1:2, and then tested according to the conditions determined in heading 2 (2) of this embodiment. The only difference was the incubation time of the test samples at 37°C, which was changed to 30 min, 45 min, 60 min, 90 min, and 120 min, to investigate the effect of the incubation time on the test results. Based on the maximum P / N value, the optimal incubation condition for the test samples was determined to be 45 min at 37°C. Figure 4 B).

[0077] (4) Screening of the optimal incubation time for antibody detection

[0078] The detection was performed according to the conditions determined in heading 2 (3) of this embodiment. Only the incubation time at 37°C after the addition of HRP-4B3 was changed to 30 min, 45 min, 60 min, 90 min, and 120 min to examine the effect of antibody incubation time on the detection results. Based on the maximum P / N value, the optimal incubation condition for HRP-4B3 was determined to be 45 min at 37°C. Figure 4 C).

[0079] (5) Selection of the optimal color development time

[0080] The detection was performed according to the conditions determined in heading 2 (4) of this embodiment, with only the substrate color development reaction time changed to 5 min, 10 min, 15 min, and 20 min, to examine the effect of color development time on the detection results. The optimal substrate color development reaction time was determined to be 15 min based on the maximum P / N value. Figure 4 D).

[0081] 3. Sandwich ELISA kit for detecting bovine viral diarrhea virus, usage method, evaluation method and application.

[0082] (1) Sandwich ELISA kit for detecting bovine viral diarrhea virus

[0083] A sandwich ELISA kit for detecting bovine viral diarrhea virus (hereinafter referred to as the kit of this invention) comprises the following components: an enzyme-labeled plate coated with polyclonal antibody against NS3 protein, PBST, HRP-4B3, negative control, positive control, TMB substrate chromogenic solution, and stop solution.

[0084] ELISA plate coated with anti-NS3 protein polyclonal antibody: 2 mg / mL purified anti-NS3 protein polyclonal antibody (prepared in this example) was diluted to 2 μg / mL with antigen coating buffer (0.05 mol / L, pH 9.6 carbonate buffer), and added to a 96-well ELISA plate, 100 μL per well, and coated overnight at 4°C; washed 3 times with PBST; 200 μL of PBST containing 0.5% (w / w) BSA (bovine serum albumin) was added to each well, and blocked at 37°C for 2 h; washed 3 times with PBST to obtain the ELISA plate coated with anti-NS3 protein polyclonal antibody.

[0085] PBST: PBS buffer solution with pH 7.4 containing 0.5% (w / w) Tween-20 and a concentration of 0.01 mol / L.

[0086] HRP-4B3: The preparation method is described in Example 3, and the concentration is 0.5 mg / mL.

[0087] Negative control: MDBK cell lysis buffer. MDBK cells were cultured in DMEM medium containing 10% fetal bovine serum until a confluent monolayer was obtained, resulting in MDBK cell culture. The culture was subjected to three freeze-thaw cycles, centrifuged at 12,000 rpm and 4°C for 5 min, and the supernatant was collected as MDBK cell lysis buffer.

[0088] Positive control: MDBK cell lysate infected with BVDV-1JS2201 strain (Genbank accession number: OP856581). MDBK cell cultures infected with BVDV for 3 days were subjected to three freeze-thaw cycles, inactivated by a 56°C water bath for 30 min, and centrifuged at 12000 rpm at 4°C for 5 min. The supernatant was collected as the BVDV-infected MDBK cell lysate, with a viral titer of 10. 6 TCID 50 / mL.

[0089] TMB substrate developing solution: purchased from Huzhou Yingchuang Biotechnology Co., Ltd.

[0090] Termination solution: 2 mol / L sulfuric acid aqueous solution.

[0091] (2) Sandwich ELISA method for detecting bovine viral diarrhea virus

[0092] The sandwich ELISA method for detecting bovine viral diarrhea virus using the kit of this invention (hereinafter referred to as the method of this invention) includes the following steps: Purified anti-NS3 protein polyclonal antibody is diluted to 2 μg / mL with antigen coating buffer (0.05 mol / L, pH 9.6 carbonate buffer), and 100 μL is added to each well of a 96-well microplate. The plate is incubated overnight (12-16 h) at 4°C; the plate is washed three times with PBST; and 200 μL of PBST containing 0.5% BSA is added to each well. Block at 37℃ for 2 hours; wash 3 times with PBST; dilute the test sample, negative control, and positive control with PBST at a dilution of 1:2, add 100 μL to each well, and incubate at 37℃ for 45 min; wash 3 times with PBST; add 100 μL of HRP-4B3 diluted with PBST at a dilution of 1:2000 to each well, and incubate at 37℃ for 45 min; wash 3 times with PBST; add 100 μL of TMB substrate chromogenic solution to each well, and develop for 10 min. Stop the reaction by adding 50 μL of 2 mol / L sulfuric acid aqueous solution. Read the OD value using a microplate reader. 450nm value.

[0093] (3) Determination of Cut-off value

[0094] The OD values ​​of 100 BVDV-negative bovine serum samples and 100 BVDV-negative bovine ear tissue samples were detected using the kit and method of this invention. 450 The average value is 0.1015, and the standard deviation is 0.0177. Therefore, when the OD of the sample to be tested... 450 A value ≥ 0.1546 is considered positive; when the OD value of the sample is ≥ 0.1546, it is considered positive. 450 A value ≤ 0.1369 is considered negative; when the OD value of the sample is ≤ 0.1369, it is considered negative. 450 A value between these two values ​​is considered suspicious. The bovine ear tissue samples were obtained using a specialized tool, with each sample having a diameter of 2-3 mm. After soaking each bovine ear tissue sample in 200 μL of PBST for 12 hours, the supernatant was collected and analyzed using the kit of this invention.

[0095] (4) Specificity test

[0096] The kit of this invention was used to detect positive samples of BVDV-1a, BVDV-1b, BVDV-2, BDV, CSFV, IBRV, BPIV-3, and M. bovis according to the method of this invention. Each sample was tested three times. The results are as follows: Figure 5 Except for BVDV-1a, BVDV-1b, BVDV-2, and BDV positive samples, all other sample test results were negative, proving that the kit of the present invention can specifically detect BVDV and BDV positive samples.

[0097] (5) Sensitivity test

[0098] The lysate of MDBK cells infected with BVDV-1JS2201 strain (virus titer 10) was separately processed. 6 TCID 50 The recombinant NS3 protein (4 μg / mL) and NS4 protein (4 μg / mL) were serially diluted 2-fold starting at a dilution of 1:16 using PBST. The kit of this invention (using the method of this invention) and the ID Vet antigen detection kit (ID Vet) were used simultaneously. The BVD P80 antigencapture assay was performed, with a sample volume of 100 μL, to assess the sensitivity of the kit. Results are as follows: Figure 6 When the dilution of BVDV-1b-infected MDBK cell lysate was 1:512, the detection result using the kit of this invention was still positive, meaning the limit of detection for viral fluid using the kit of this invention is 10. 2.29 TCID 50 When the recombinant NS3 protein was diluted to 1:256, the detection result using the kit of this invention was still positive, meaning the limit of detection (LOD) for NS3 protein using the kit of this invention was 1.56 ng. The experimental results indicate that the kit of this invention has good sensitivity. When the lysate of BVDV-1b-infected MDBK cells was diluted to 1:32, the detection result using the ID.Vet kit was still positive, meaning the limit of detection (LOD) for viral fluid using the ID.Vet antigen detection kit was 10 ng. 3.49 TCID 50 When the recombinant NS3 protein was diluted to 1:64, the ID Vet antigen detection kit still showed a positive result, indicating that the limit of detection for NS3 protein using the ID Vet antigen detection kit was 6.25 ng. These results demonstrate that the detection sensitivity of the kit is significantly higher than that of similar kits.

[0099] (6) Clinical sample testing

[0100] The antigen detection kits from ID Vet (ID Vet) were used respectively. The BVD P80 antigen capture kit and the kit of this invention (using the method of this invention) were used to detect 500 bovine serum and bovine ear tissue samples collected from different cattle farms in Shanghai, Jiangsu, Henan, and Ningxia Hui Autonomous Region. The detection results of the two methods were compared, and the concordance rate of the two detection methods was calculated. The results are shown in Table 3. The overall concordance rate between the kit of this invention and the ID Vet antigen detection kit was 97.4%, the negative concordance rate was 100%, and the positive concordance rate was 91.2%. The positive detection rate of the kit of this invention is higher than that of the ID Vet antigen detection kit, indicating that the kit of this invention can be applied to clinical testing.

[0101] Table 3. Comparison of consistency between the reagent kit of the present invention and commercially available reagent kits.

[0102]

Claims

1. Hybridoma cell strain 4B3 secreting monoclonal antibody against NS3 protein of bovine viral diarrhea virus, with the preservation number of CCTCC NO: C2025217.

2. Monoclonal antibody against NS3 protein of bovine viral diarrhea virus secreted by the hybridoma cell strain 4B3 of claim 1.

3. The method for preparing the monoclonal antibody against NS3 protein of bovine viral diarrhea virus according to claim 2, characterized in that The method comprises the following steps: injecting the hybridoma cell 4B3 into the abdominal cavity of a BALB / c mouse, preparing ascites, centrifuging and collecting the ascites supernatant, and obtaining the monoclonal antibody against NS3 protein of bovine viral diarrhea virus after purification.

4. A sandwich ELISA kit for the detection of bovine viral diarrhea virus, characterized in that The antibody is horseradish peroxidase-labeled monoclonal antibody against NS3 protein of bovine viral diarrhea virus of claim 2.

5. The kit of claim 4, wherein The kit further comprises an enzyme-labeled plate coated with anti-NS3 protein polyclonal antibody, which is obtained by immunizing a New Zealand white rabbit with NS3 protein, taking serum, and purifying the serum.

6. The monoclonal antibody of claim 2 is used for Western blot detection or indirect immunofluorescence detection of NS3 antigen of bovine viral diarrhea virus for non-diagnostic purposes.

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