A competitive monoclonal antibody against bovine ephemeral fever virus and its application in constructing a competitive ELISA kit

Abstract

This invention discloses a competitive monoclonal antibody against bovine ephemeral fever virus (BOF) and its application in constructing a competitive ELISA kit, belonging to the field of monoclonal antibody technology. The technical problem this invention aims to solve is how to accurately detect BEF. This invention provides a competitive monoclonal antibody against BEF, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.7. This enables rapid and accurate diagnosis of BEF, facilitating timely implementation of prevention and control measures and reducing the risk of epidemic transmission.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a competitive monoclonal antibody against bovine ephemeral fever virus and its application in the construction of a competitive ELISA kit. Background Technology

[0002] Bovine ephemeral fever (BEF) is an acute, febrile, vector-borne infectious disease caused by bovine ephemeral fever virus (BEFV). It primarily infects dairy and cattle, characterized by sudden onset of high fever, lacrimation, salivation, rapid breathing, lameness, and leukopenia. The disease spreads rapidly and has a high morbidity rate. Although the mortality rate is relatively low, infected cattle experience a significant decrease in milk production, and draft cattle lose their ability to work, causing severe economic losses to the cattle industry.

[0003] Currently, diagnostic methods for bovine ephemeral fever mainly include virus isolation and culture, serological testing, and molecular biological detection. However, these methods suffer from problems such as complex operation, long processing time, high requirements for experimental conditions, or insufficient sensitivity and specificity, making it difficult to meet the needs for rapid and accurate diagnosis. Therefore, developing efficient and specific diagnostic reagents and treatment methods is of great significance for the prevention and control of bovine ephemeral fever. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to accurately detect bovine ephemeral fever virus. The purpose of this invention is to provide an antibody for detecting bovine ephemeral fever virus.

[0005] This invention provides a competitive monoclonal antibody against bovine ephemeral fever virus. The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody is shown in SEQ ID NO.3; the amino acid sequence of CDR-H2 is shown in SEQ ID NO.4; and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.5. The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody is shown in SEQ ID NO.8; the amino acid sequence of CDR-H2 is shown in SEQ ID NO.9; and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.10.

[0006] Further specified, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.7.

[0007] This invention provides a nucleic acid molecule that encodes the above-mentioned monoclonal antibody.

[0008] Further specifying, the gene sequence encoding the heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.1; the gene sequence encoding the light chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.6.

[0009] This invention provides the application of the above-mentioned competitive monoclonal antibody against bovine ephemeral fever virus and the above-mentioned nucleic acid molecule in the preparation of a kit for detecting bovine ephemeral fever virus.

[0010] To further specify, the kit is a competing ELISA kit.

[0011] This invention provides a kit for detecting bovine ephemeral fever virus, the kit comprising the above-mentioned competitive monoclonal antibody against bovine ephemeral fever virus, HRP-labeled goat anti-mouse IgG antibody, and antigen; the antigen is bovine ephemeral fever virus.

[0012] Beneficial Effects: The competitive monoclonal antibody 33G2 against bovine ephemeral fever virus (BOFV) obtained in this invention exhibits high specificity and sensitivity, specifically recognizing key epitopes of BDFV antigens and showing no cross-reactivity with other common bovine pathogens, providing a powerful tool for the accurate detection of BDFV. This monoclonal antibody demonstrates significant competitive inhibitory ability, effectively blocking the binding of BDFV to host cell receptors, and can be used for the treatment of BDF, providing a new approach for clinical treatment. The competitive ELISA detection method established based on this monoclonal antibody has advantages such as high sensitivity, strong specificity, simple operation, and short detection time, enabling rapid and accurate diagnosis of BDFV, facilitating timely implementation of prevention and control measures, and reducing the risk of epidemic transmission. Attached Figure Description

[0013] Figure 1 Figure showing the purification and identification results of bovine ephemeral fever virus; Figure 2 The image shows the serum titer test results of immunized mice. Figure 3 This is a diagram illustrating the specificity identification of monoclonal antibodies. Figure 4 For the identification of target proteins of monoclonal antibodies; A: eukaryotic expression of N protein; B: eukaryotic expression of P protein; C: eukaryotic expression of G protein; D: positive control; E: negative control; Figure 5 This is a graph showing the results of monoclonal antibody competition. Figure 6 Figure showing the results of a specificity test for competing ELISA. Detailed Implementation

[0014] Example 1. Purification and Immunization of Bovine Epidemic Fever Virus I. Purification of Bovine Epidemic Fever Virus Bovine ephemeral fever virus (CGMCC NO.46379) cell culture medium was repeatedly freeze-thawed to release the virus. Cell debris and other impurities were removed by low-speed centrifugation. Protease inhibitors were added to prevent degradation. The virus was then loaded onto a continuous iodixanol density gradient of 15%-35% and centrifuged at 30,000 rpm and 4°C for 4 h. One tube was collected per milliliter from top to bottom, for a total of 13 tubes. TCID levels at different gradients were measured. 50 The peak positions of complete viral particles were determined, and the results are as follows: Figure 1 As shown, viral nucleic acid was measured using quantitative real-time PCR, and viral protein was detected by Western blot. The results showed that the viral content was highest in layer 5. Figure 1 The fifth layer, after being centrifuged at 30,000 rpm to remove alcohol, can be used as an immunogen, aliquoted and frozen, with low-temperature operation throughout to avoid virus inactivation.

[0015] II. Immunizing mice Five 4-5 week old female Balb / c mice were selected and immunized with bovine eperythritis virus (BEV) antigen. The initial immunization dose was 30 μg / mouse, emulsified with an equal volume of Freund's complete adjuvant and BEV antigen, and injected intramuscularly into the leg, 0.2 mL at each site. A second immunization was administered 2 weeks later, with the same dose, emulsified with an equal volume of Freund's incomplete adjuvant and BEV antigen, and injected subcutaneously at multiple sites. Booster immunizations were then administered every 2 weeks for a total of 3-4 booster immunizations. Two weeks after the three immunizations, serum was collected and separated. Serum titers were determined using ELISA. A final booster immunization was administered when the titer exceeded 4000-fold, and a fusion assay was performed 3 days later.

[0016] Example 2. Preparation and Identification of Bovine Epidemic Fever Virus Monoclonal Antibody I. Cell Fusion Three days after the final booster immunization, mice were sacrificed, and spleens were aseptically removed and placed in a culture dish containing pre-chilled RPMI 1640 medium. The spleen was gently ground with a syringe needle core to prepare a spleen cell suspension, which was then filtered through a 200-mesh cell sieve to remove tissue fragments, yielding a single-cell suspension. Mouse myeloma cells in the logarithmic growth phase (SP2 / 0) were taken, washed twice with RPMI 1640 medium, centrifuged at 1000×g for 5 minutes, and the supernatant was discarded. Spleen cells and myeloma cells were mixed at a 5:1 ratio in a 50 mL centrifuge tube, and preheated RPMI 1640 medium (37°C) was added. The mixture was gently pipetted and centrifuged at 1000×g for 5 minutes, and the supernatant was discarded. In a 37°C water bath, 1 mL of 50% PEG 1500 solution was slowly added while gently stirring for 90 seconds. Then, 10 mL of preheated RPMI 1640 medium (37°C) was rapidly added to terminate the fusion reaction. Centrifuge at 1000×g for 5 minutes and discard the supernatant. Resuspend the cell pellet in HAT selective medium and seed the cell suspension into 96-well cell culture plates, 100 μL per well, and incubate at 37°C in a 5% CO2 incubator.

[0017] II. Hybridoma Cell Screening and Subcloning On day 10 after cell fusion, the hybridoma cell culture supernatant was screened using an indirect ELISA method.

[0018] 1. Dilute bovine ephemeral fever virus antigen to 1 μg / mL with carbonate buffer (pH 9.6), coat ELISA plates with 100 μL per well, and incubate overnight at 4°C.

[0019] 2. The next day, discard the coating solution, wash three times with PBS containing 0.05% Tween-20 (PBST), add 200 μL of 5% skim milk to each well, and block at 37°C for 1 hour.

[0020] 3. Discard the blocking solution, add 100 μL of hybridoma cell culture supernatant to each well, and set up a negative control (normal mouse serum) and a positive control (known anti-bovine ephemeral fever virus serum). Incubate at 37°C for 1 hour.

[0021] 4. After washing 3 times, add 100 μL of HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution) to each well and incubate at 37°C for 1 hour.

[0022] 5. After washing 5 times, add substrate solution (TMB) 100 μL per well and react at room temperature in the dark for 15 minutes.

[0023] 6. Stop the reaction by adding 2 M sulfuric acid (100 μL per well). Measure the OD value at 450 nm using a microplate reader.450 Value ≥2.1× Negative control OD 450 The value is used as the positive criterion to screen out positive hybridoma cells.

[0024] Clonalization of positive hybridoma cells was performed using a limiting dilution method. Positive hybridoma cells were diluted to an appropriate concentration with HT medium and seeded into 96-well cell culture plates, with 0.5-1 cells per well. The plates were then incubated at 37°C in a 5% CO2 incubator. When the cell clones grew to 1 / 3-1 / 2 of the well bottom area, the culture supernatant was analyzed using an indirect ELISA method to screen for hybridoma cell lines that stably secreted specific antibodies. After three rounds of subclonal screening and identification, a stable hybridoma cell line, 33G2, secreting monoclonal antibodies was obtained. The results are as follows: Figure 2 As shown.

[0025] III. Monoclonal Antibody Preparation and Purification Female Balb / c mice aged 6-8 weeks were selected, and each mouse was injected intraperitoneally with 0.5 mL of liquid paraffin. 7-10 days later, the selected hybridoma cell line 33G2 was adjusted to a concentration of 2 × 10⁻⁶ cells using RPMI 1640 medium. ５ 0.1 mL was administered intraperitoneally to each mouse. After 7-10 days, when the mice's abdomens were significantly distended, ascites fluid was collected. The ascites fluid was centrifuged at 3000×g for 15 minutes at 4°C, and the supernatant was further purified using a Protein G affinity chromatography column. The column was equilibrated with PBS buffer, the antibody solution was loaded, and unbound proteins were eluted with PBS buffer. Finally, the target antibody was eluted with 0.1 M citrate buffer (pH 3.0), and the elution peak was collected. The elution buffer was immediately neutralized with 1 M Tris-HCl buffer (pH 9.0), and the purified antibody was dialyzed against PBS buffer to remove small molecule impurities, yielding high-purity monoclonal antibody 33G2.

[0026] Sequencing results of bovine ephemeral fever virus monoclonal antibody 33G2: Heavy chain VH gene: (SEQ ID NO.1) CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGATTTTCACTGAGCACTTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCACCAACCAGVTCTTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCGAGGGAATTACGCAGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAG; Heavy chain VH amino acids: (SEQ ID NO.2) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWVAHIYWDDDKRYNPSLKSRVTISKDTSTNQVFLKITSVDTADTATYYCARGNYADVWGAGTTVTVSS; Heavy chain CDR-H1: GFSLSTSGMG; (SEQ ID NO.3) Heavy chain CDR-H2: IYWDDDKR; (SEQ ID NO.4) Heavy chain CDR-H3: GNYADV; (SEQ ID NO.5) Light chain VL gene: (SEQ ID NO.6) CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTTCCAACAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATACCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCAAAGGAGTCGTTACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAATACG; Light chain VL amino acids: (SEQ ID NO.7) QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWIYTTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCHQRSRYPFTFGSGTKLEIIR; Light chain CDR-L1: SASSSVSYMH; (SEQ ID NO.8) Light chain CDR-L2: TT SNLA; (SEQ ID NO.9) Light chain CDR-L3: HQRSRYPFT (SEQ ID NO.10).

[0027] IV. Monoclonal antibody specificity identification Bovine eperythritis virus antigen, akaban virus, bovine coronavirus antigen, bovine infectious rhinotracheitis virus antigen, and bovine viral diarrhea virus antigen were diluted to 1 μg / mL with carbonate buffer (pH 9.6) and coated onto ELISA plates, 100 μL per well, and incubated overnight at 4°C. The next day, the coating solution was discarded, and the plates were washed three times with PBST. 200 μL of 5% skim milk powder was added to each well, and the plates were blocked at 37°C for 2 hours. The blocking solution was discarded, and 100 μL of purified monoclonal antibody 33G2 (1:1000 dilution) was added to each well. A negative control (PBS) and a positive control (known anti-bovine eperythritis virus serum) were also included. The plates were incubated at 37°C for 1 hour. After three washes, 100 μL of HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution) was added to each well, and the plates were incubated at 37°C for 1 hour. After three washes, 100 μL of substrate solution (TMB) was added to each well, and the plates were incubated at room temperature in the dark for 15 minutes. The reaction was terminated by adding 2 M sulfuric acid. 100 μL was added to each well, and the OD was measured using a microplate reader. 450nm The results showed that monoclonal antibody 33G2 only reacted positively with bovine ephemeral fever virus antigen (OD). 450 Value ≥2.1× Negative control OD 450 nm value) Figure 3 The antibody tested negative for both the virus antigen and other viral antigens, indicating that it has good specificity.

[0028] V. Identification of target proteins of monoclonal antibodies The target proteins of the monoclonal antibody were identified using an indirect immunofluorescence method. BHK21 cells were transfected with the eukaryotic expression vector pCAGGS, containing BEFV G, N, and P proteins, respectively, as samples. The following steps were taken: using a monoclonal antibody as the primary antibody (monoclonal antibody 33G2) and a fluorescently labeled secondary antibody (HRP-labeled goat anti-mouse IgG antibody) as the detection antibody; preparation of reagents such as monoclonal antibody (0.8 μg / mL) and fluorescent secondary antibody (1:100~1:1000); After fixation, washing, and blocking, the sample was incubated with monoclonal antibody at 37°C for 1 hour, washed, and then incubated with secondary antibody at 37°C for 1 hour in the dark, followed by washing.

[0029] The results are as follows Figure 4 As shown, under a fluorescence microscope, the results are valid if specific fluorescence is observed and the negative control shows no signal. A: Eukaryotic expression of N protein; B: Eukaryotic expression of P protein; C: Eukaryotic expression of G protein; D: Positive control; E: Negative control.

[0030] VI. Monoclonal antibody competition identification The purified monoclonal antibody was identified by SDS-PAGE electrophoresis and Western blot to determine its purity and specificity. The isotype of the monoclonal antibody was identified as IgG1, and the light chain was identified as the κ chain, using an isotype identification kit.

[0031] This study used a competitive ELISA method to competitively identify the purified monoclonal antibody 33G2. The specific steps were as follows: First, the purified bovine ephemeral fever virus antigen was diluted to 1 μg / mL with carbonate buffer (pH 9.6), and 100 μL was used to coat the ELISA plate at 4°C overnight. The next day, the plate was blocked with PBST buffer containing 5% skim milk and incubated at 37°C for 1 h. After washing the plate, equal volumes of bovine ephemeral fever virus positive serum (BEFV inactivated vaccine immune serum), bovine ephemeral fever negative serum, and fetal bovine serum were added, followed by the monoclonal antibody (working concentration 1 μg / mL), and incubated at 37°C for 1 h. The mixture was discarded and the plate was washed. HRP-labeled anti-mouse secondary antibody was added, and the plate was incubated at 37°C for 60 min. After washing the plate again, TMB substrate solution was added for color development. Finally, the reaction was terminated with 2 mol / L H2SO4, and the OD value of each well was detected at 450 nm.

[0032] The results showed that the monoclonal antibody competitively bound to bovine ephemeral fever virus antigen and OD in positive serum. 450 The lowest nm value (0.112) Figure 5 ), exhibiting good competitiveness, while negative serum and fetal bovine serum do not compete with the monoclonal antibody, OD 450 The high nm value (1.5~1.6) indicates that monoclonal antibody 33G2 can be used to establish a competitive ELISA method.

[0033] Example 3. Bovine Epidemic Heat Competition ELISA Method I. Establishing a Competitive ELISA Method Monoclonal antibodies were selected as the competitive antibodies to establish a competitive ELISA method. The reaction conditions of the competitive ELISA were systematically optimized using a checkerboard titration method to determine the optimal experimental parameters. First, the antigen coating concentration and the working concentration of the monoclonal antibody were optimized: bovine ephemeral fever virus was used as the antigen to coat the ELISA plate at concentrations of 0.25, 0.5, 1, 2, and 10 μg / mL, respectively. Simultaneously, monoclonal antibody 33G2 was serially diluted at concentrations of 0.1, 0.25, 0.5, 1, and 2 μg / mL. The OD values ​​of each combination were detected by competitive ELISA. It was ultimately determined that when the antigen coating concentration was 2 μg / mL and the working concentration of monoclonal antibody 33G2 was 1 μg / mL, the difference in OD values ​​between the negative and positive controls was the largest, and the background value was the lowest. Subsequently, the incubation conditions were optimized, comparing the reaction effects of incubation at 37℃ for 30 min, 1 h, 2 h, and overnight at 4℃. The results showed that incubation at 37℃ for 1 h resulted in the highest reaction efficiency and the most stable results. Different dilutions of HRP-labeled goat anti-mouse IgG antibody (1:2500, 1:5000, 1:10000) were compared, and incubation at room temperature for 1 hour was performed. Furthermore, the types of blocking solutions (5% skim milk, 1% BSA, 1% gelatin, 10% rabbit serum) and substrate color development times (10 min, 15 min, 20 min) were screened, and 5% skim milk as the blocking solution and a color development time of 15 min at room temperature were determined to be the optimal conditions. Inhibition rate was defined as: (Negative control OD value - Sample OD value) / Negative control OD value × 100%. An inhibition rate ≥ 50% was considered positive, and < 50% was considered negative.

[0034] II. Sensitivity of Competitive ELISA Methods To verify the sensitivity of the established bovine ephemeral fever virus (BOF) competitive ELISA method, a concordance test was conducted using the classic virus neutralization assay (VN assay) as a reference. Fifty bovine serum samples clinically suspected of being infected with BDF were selected (including 20 known positive, 15 known negative, and 15 unknown samples). All samples were free from hemolysis, turbidity, or other interference and were stored at -20°C. The competitive ELISA assay was performed strictly according to the previously optimized conditions.

[0035] The virus neutralization assay employed the fixed-virus dilution serum method, where samples were serially diluted twofold (1:4 to 1:512) in 96-well cell culture plates, with 100 TCID50 added to each well. 50 Bovine ephemeral fever virus solution was incubated at 37°C for 1 hour, and then sensitive cell suspension was added. The cells were cultured for another 5-7 days, and cytopathic effects (CPE) were observed. The highest serum dilution that could completely inhibit CPE was used as the neutralizing titer. A titer ≥1:4 was considered positive, and <1:4 was considered negative. The experimental results showed that out of 50 samples, 27 samples were both correctly identified as positive by the competitive ELISA and the neutralization test (true positives), and 22 samples were both correctly identified as negative (true negatives). The concordance rate was calculated as follows: Overall concordance rate = (true positives + true negatives) / total number of samples × 100% = (27 + 22) / 50 × 100% = 98% (Table 1). This indicates that the competitive ELISA and the neutralization test, which is considered the "gold standard," showed good consistency. This result suggests that the competitive ELISA can be used as a rapid detection method to replace the neutralization test for the batch screening of clinical samples of bovine ephemeral fever virus. Table 1. Comparison of Competitive ELISA and Neutralization Test

[0036] III. Specificity of Competitive ELISA Methods To evaluate the specificity of the competitive ELISA method for bovine ephemeral fever virus, five bovine disease-positive sera (bovine coronavirus, bovine respiratory syncytial virus, bovine infectious rhinotracheitis virus, bovine viral diarrhea virus, and akaban virus) were selected, along with a negative control (PBS). Each sample was tested in triplicate using the competitive ELISA method.

[0037] The results showed that the inhibition rate of bovine ephemeral fever virus positive serum was 95%, while the inhibition rates of negative control and other pathogen-positive serum were all below 20%. Figure 6 This indicates that monoclonal antibody 33G2 can specifically compete with bovine ephemeral fever virus (BOF) positive sera without interference from irrelevant pathogen antibodies. This competitive ELISA method has extremely high specificity and can accurately distinguish BOF positive sera from sera of other pathogens, making it suitable for the differential diagnosis of BOF.

Claims

1. A competitive monoclonal antibody to bovine epidemic fever virus, characterized in that, The amino acid sequence of CDR-H1 of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 3; the amino acid sequence of CDR-H2 is shown as SEQ ID NO. 4; and the amino acid sequence of CDR-H3 is shown as SEQ ID NO. 5; The amino acid sequence of CDR-L1 of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 8; the amino acid sequence of CDR-H2 is shown as SEQ ID NO. 9; and the amino acid sequence of CDR-H3 is shown as SEQ ID NO.

10.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 2; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.

7.

3. A nucleic acid molecule, characterized in that, The gene sequence encoding the heavy chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO. 1; and the gene sequence encoding the light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.

6.

4. The nucleic acid molecule of claim 3, wherein, 5. Use of the competitive monoclonal antibody of Bovine Epidemic Fever of claim 1 or 2 or the nucleic acid molecule of claim 3 or 4 in the preparation of a kit for detecting Bovine Epidemic Fever. The kit comprises the competitive monoclonal antibody of Bovine Epidemic Fever of claim 1 or 2, HRP-labeled goat anti-mouse IgG antibody and antigen; the antigen is Bovine Epidemic Fever.

6. A kit for detecting bovine ephemeral fever virus, characterized by, ​

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