Monoclonal antibody 3B3 specifically combined with group A porcine rotavirus and application thereof

Abstract

The invention discloses a monoclonal antibody 3B3 capable of being specifically combined with a group A porcine rotavirus and application, the monoclonal antibody 3B3 is a murine monoclonal antibody, the heavy chain type of the monoclonal antibody 3B3 is IgG1, and the light chain type of the monoclonal antibody 3B3 is kappa; the monoclonal antibody 3B3 for resisting the group A porcine rotavirus is obtained by taking recombinant VP6 protein expressed by a CHO system as an immunogen to immunize a Balb / c mouse and screening through a hybridoma technology, has very excellent biological performance, not only has high affinity to rotavirus VP6 protein, but also has good reactivity to the group A porcine rotavirus. The sensitivity of the kit can reach 1: 256, and the kit has no cross reaction with positive serum of antibodies of six common diseases in pig farms. The method is relatively good in repeatability, and the coincidence rate with an indirect immunofluorescence detection result reaches 95.65%.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a monoclonal antibody 3B3 that specifically binds to group A porcine rotavirus and its applications. Background Technology

[0002] Porcine rotavirus-A (PoRVA) is a double-stranded RNA (dsRNA) virus belonging to the genus Rotavirus in the family Reoviridae. It is a predominantly circulating serotype worldwide and is generally transmitted via the fecal-oral route. Piglets without maternal antibodies have a high mortality rate, with nearly 100% mortality in piglets under one week old infected with PoRVA, posing an extremely serious threat. PoRVA is widely distributed globally, with an individual prevalence of 3.3%–67.3% and a positive rate of 1%–74% in large-scale pig farms. Furthermore, co-infection with pathogens such as PEDV, TGEV, and pathogenic Escherichia coli is very common, exacerbating diarrhea symptoms and increasing piglet mortality. Studies have also shown that PoRVA can cross the interspecies barrier, causing severe diarrhea in young children. Controlling PoRVA is crucial not only for the healthy development of the livestock industry but also for the prevention and control of zoonotic diseases.

[0003] Currently, vaccinating sows to produce maternal antibodies to protect piglets from viral infection is the main prevention and control measure. However, issues such as poor vaccine efficacy and insufficient immune protection in pig herds persist. Therefore, monitoring PoRVA antibody levels in pig herds is crucial for assessing immunization effectiveness, developing reasonable immunization programs, and thus achieving more comprehensive PoRVA control. Currently, clinical use relies on indirect ELISA detection methods, which are more prone to false positives compared to blocking ELISA, thus lacking accurate and effective tools for evaluating vaccine immunization.

[0004] Monoclonal antibodies are the core reagents for developing blocking ELISA methods; therefore, selecting appropriate target sites to prepare specific monoclonal antibodies is crucial. Hence, there is a need to develop a detection kit for PoRVA blocking ELISA antibodies. Summary of the Invention

[0005] The purpose of this invention is to address the problems of high false positive rates and unstable results in clinical PoRVA testing by providing a monoclonal antibody 3B3 that specifically binds to group A porcine rotavirus and its application. This invention has discovered that VP6 is a group-specific protein of PoRVA, the most abundant, containing 397 amino acids, with a molecular weight of approximately 45 kDa, accounting for about 51% of the total viral protein, and is highly conserved. VP6 has high immunogenicity; after PoRVA infection or oral immunization, the body first produces an antibody immune response against VP6. Therefore, VP6 is an ideal target for establishing PoRVA antibody detection technology. Based on this, this invention has developed a murine monoclonal antibody with blocking effect and established for the first time a highly sensitive and specific PoRVA blocking ELISA antibody detection kit, providing an efficient and useful new tool for monitoring PoRVA antibodies in pig farms, which is of great significance for the prevention and control of diarrheal diseases in pig farms.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a monoclonal antibody 3B3 that specifically binds to group A porcine rotavirus. The monoclonal antibody 3B3 is a murine monoclonal antibody, with the heavy chain type being IgG1 and the light chain type being kappa. The heavy chain variable region of monoclonal antibody 3B3 includes three complementarity-determining regions (CDR regions), namely 3B3-CDR-H1, 3B3-CDR-H2 and 3B3-CDR-H3, whose amino acid sequences are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively. The light chain variable region of monoclonal antibody 3B3 includes three complementarity-determining regions (CDR regions), namely 3B3-CDR-L1, 3B3-CDR-L2 and 3B3-CDR-L3: their amino acid sequences are QEISGY (as shown in SEQ ID NO.8), AAS and LQYANYPFT (as shown in SEQ ID NO.9), respectively.

[0007] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3B3 is shown in SEQ ID NO.2; the amino acid sequence of the light chain variable region of 3B3 is shown in SEQ ID NO.4.

[0008] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3B3 is shown in SEQ ID NO.1; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3B3 is shown in SEQ ID NO.3.

[0009] This invention provides the application of the above-mentioned monoclonal antibody 3B3 in the preparation of a group A porcine rotavirus blocking ELISA antibody detection kit.

[0010] The present invention also provides a group A porcine rotavirus blocking ELISA antibody detection kit, the detection kit comprising the above-mentioned monoclonal antibody 3B3 labeled with HRP (abbreviated as HRP-3B3).

[0011] Furthermore, the test kit also includes an antigen-coated plate, 1×PBST, blocking buffer, serum diluent, antibody diluent, stop solution, single-component chromogenic solution (brand: IPANDA), positive control, and negative control; wherein, Preparation of each component of the above Group A porcine rotavirus blocking ELISA antibody detection kit 1. Preparation of HRP-labeled monoclonal antibody HRP-3B3 a. The ultrafiltration purified monoclonal antibody 3B3 was dissolved in 0.05M carbonate buffer; b. Weigh 6 mg HRP and dissolve it in 1.2 mL of double-distilled water. Pre-cool at 4 °C in the dark for 30 min. Add 0.72 L of freshly prepared 0.05 M NaIO4 solution and mix well. Stir at 4 °C in the dark for 30 min. At room temperature, slowly add 0.6 mL of 0.16 M ethylene glycol aqueous solution to the stirred HRP. React at room temperature in the dark for 30 min to obtain a crude HRP solution after oxidation. The oxidized crude HRP solution was transferred into an ultrafiltration tube (molecular weight 10 kDa, to remove unreacted reagents and reaction byproducts by ultrafiltration) and ultrafiltered at 3000 g / min for 15-20 min; then diluted to a suitable volume with 0.05 M carbonate buffer to obtain an oxidized HRP solution, wherein the HRP concentration in the solution was 4 mg / mL. c. According to the calculated feed amount, add the purified monoclonal antibody 3B3 to the oxidized HRP solution under low-speed stirring. Stir overnight at 4°C in the dark; add 4 mg / mL NaBH4 solution to make the final concentration 20 mM, mix by inversion, and let stand at room temperature in the dark for 2 h; add the above solution to a dialysis bag and dialyze overnight with 0.02 M PBS, changing the medium 3-5 times throughout the process; add an equal volume of glycerol and 2% BSA and store at -20°C; obtain the HRP-labeled monoclonal antibody HRP-3B3.

[0012] 2. Preparation of other components and articles 1×PBST: 1×PBS 500mL is added to 250μL of Tween-20 and mixed.

[0013] The blocking solution was PBST containing 2% BSA and 5% skim milk by mass.

[0014] The serum diluent was PBST containing 6% NaCl and 2% BSA by mass.

[0015] The antibody diluent was PBST containing 2% BSA by mass.

[0016] Stop solution (2M H2SO4): Take 150mL of ultrapure water, add 22.2mL of concentrated sulfuric acid, and make up to 200mL.

[0017] Positive control: PoRVA VP6 protein-immunized porcine serum.

[0018] Negative control: SPF porcine serum.

[0019] Furthermore, the antigen coated on the antigen-coated plate is the VP6 protein, wherein the preparation of the antigen-coated plate is as follows: a. Antigen coating solution (carbonate buffer, pH=9.6): 1.59g Na2CO3 and 2.97g NaHCO3 are added to ultrapure water and brought to a final volume of 1000mL; b. Dilute VP6 protein to 100 ng / mL using antigen coating buffer, coat overnight at 4°C, add 100 μL / well of 1×PBST to the wells of an ELISA plate, tap off the absorbent material, and repeat three times; block with 120 μL / well of blocking buffer at 37°C for 2 h, add 100 μL / well of 1×PBST to the wells, tap off the absorbent material, and repeat three times; obtain the antigen-coated plate.

[0020] The beneficial effects of this invention are: 1. The monoclonal antibody 3B3 against group A porcine rotavirus of the present invention was obtained by immunizing Balb / c mice with recombinant VP6 protein expressed in the CHO system as an immunogen and screened by hybridoma technology. Its biological performance is excellent. It not only has high affinity for rotavirus VP6 protein, but also has good reactivity to group A porcine rotavirus.

[0021] 2. The amino acid and nucleotide sequences of the variable regions of the heavy and light chains of the monoclonal antibody 3B3 against group A porcine rotavirus of the present invention are newly discovered sequences with no repetition.

[0022] 3. This invention establishes a blocking ELISA antibody detection method using the monoclonal antibody 3B3 against group A porcine rotavirus. ROC analysis shows that at a cutoff value of 23.23%, it possesses a diagnostic sensitivity and specificity of 98.04%, effectively distinguishing between positive and negative sera. The sensitivity of this invention reaches 256, and it shows no cross-reactivity with positive sera from six common swine disease antibodies (CSFV, PEDV, PRV, PRRSV, PCV2, and ASFV). This invention exhibits good reproducibility, with a concordance rate of 95.65% with indirect immunofluorescence detection results.

[0023] 4. This invention is a useful tool for field monitoring and epidemiological research of pig herds, filling the gap in highly specific PoRVA blocking ELISA antibody detection methods, and is of great significance for the prevention and control of swine diarrhea. Attached Figure Description

[0024] Figure 1 The image shows the purification results of monoclonal antibody 3B3 for group A porcine rotavirus. Figure 2 The image shows the results of indirect immunofluorescence assay for the identification of monoclonal antibody 3B3 against group A porcine rotavirus. Figure 3 The image shows the subtype identification results of monoclonal antibody 3B3 for group A porcine rotavirus. Figure 4 The figure shows the titer of HRP-labeled monoclonal antibody 3B3 for group A porcine rotavirus. Figure 5 The results of determining the optimal conditions for VP6 coating concentration and 3B3-HRP dilution ratio in the blocking ELISA antibody detection method are shown in the figure. Figure 6 The results of determining the optimal incubation time for samples in the blocking ELISA antibody detection method are shown in the figure. Figure 7 The graph shows the results of ROC analysis on the specificity and sensitivity of the blocking ELISA antibody detection method. Figure 8 A graph showing the results of determining the cut-off value for the blocking ELISA antibody detection method; Figure 9 The graph shows the sensitivity analysis results of the blocking ELISA antibody detection method. Figure 10 The image shows the results of a specific analysis of the blocking ELISA antibody detection method. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0026] Example 1: Preparation, purification, and identification of monoclonal antibodies against porcine group A rotavirus I. Hybridoma cells 3B3 a. VP6 protein (The amino acid sequence of VP6 protein is shown in SEQ ID NO. 10: EVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIVTMNGNEFQTGGIGNLPIRNWTFDFGLLGTTLLNLDANYVENARTTIEYFIDFIDNVCMDEIARESQRNGIAPQSEALRKLSGIKFKRINFDNSSDYIENWNLQNRRQRTGFVFHKPNILPYSASFTLNRSQPAHDNLMGTMWINAGSEIQVAGFDYSCAFNAPANIQQFEHVVPLRRA) LTTATITLLPDAERFSFPRVINSADGTTTWYFNPVILRPSNVEVEFLLNGQIINTYQARFGTIIARNFDTIRLSFQLVRPPNMTPAVANLFPQAPPFIFHATVGLTLRIESAVCESVLADASETLLANVTSVRQEYAIPVGPVFPPGMNWTELITNYSPSREDNLQRVFTVASIRSMLIK) was emulsified with Freund's adjuvant and immunized with BALB / c mice every 2 weeks at a final concentration of 50 μg / mouse. When the titer of the mice reached 10... 4 Afterwards, a shock immunization was performed every four days. After three shock immunizations, the mice were euthanized by cervical dislocation and the spleens were harvested. SP2 / 0 cells were fused with immune spleen cells using polyethylene glycol solution (Sigma, USA) and cultured for 7 days at 37°C with 5% carbon dioxide in RPMI-1640 medium (Cytiva, USA) containing 20% ​​FBS and 2% hypoxanthine methotrexate thymidine (HAT) (Sigma, USA).

[0027] b. Coat VP6 protein with 50 μg / mL solution at 4°C overnight, and set up a prokaryotic expression control for other proteins. Wash 3 times with PBST, and block each well with 120 μL of PBST solution containing 2% BSA and 5% skim milk at 37°C for 2 h, then wash 3 times with PBST solution.

[0028] c. Subsequently, the cells were incubated with 100 μL of cell supernatant at 37°C for 30 min, washed three times with PBST solution, and then incubated with 1:10000 diluted goat anti-mouse IgG (Southern Biotech, USA) at 37°C for 30 min, followed by three washes with PBST solution. d. After developing the color with 100 μL of single-component TMB per well for 10 min, terminate the reaction with 50 μL of 2M H2SO4 and read the OD. 450nmThe numerical values ​​are S for sample wells and N for control wells. Wells with S / N > 2 are considered to be positive hybridoma cells.

[0029] e. Using the limiting dilution method, the mixed hybridoma cells in the positive wells were subcloned three times to obtain single hybridoma cells 3B3.

[0030] II. Purification of Monoclonal Antibody 3B3 10 6 3B3 positive monoclonal hybridoma cells were intraperitoneally injected into BALB / c mice pretreated with ascites adjuvant one week prior. Ascites fluid was collected 10 days later. An appropriate amount of quartz sand was added to the ascites fluid, and the mixture was incubated overnight at 4°C on a rotary mixer to remove fat. Subsequently, the mixture was centrifuged at 12000 rpm for 5 min at 4°C, and the supernatant was collected. Three volumes of 0.06 M NaAc buffer (pH=4.5) were added to the collected ascites fluid. Caprylic acid was added dropwise at room temperature with stirring (33 μL of caprylic acid per mL of ascites fluid), ensuring each drop was completely dissolved before adding the next. After adding caprylic acid, the mixture was stirred for 30 min and incubated at 4°C for at least 2 h to allow for complete precipitation. The mixture was then centrifuged at 12000 rpm for 30 min at 4°C, and the supernatant was collected. The supernatant was filtered through ordinary filter paper, and 0.1 volumes of 10×PBS (0.1 mol pH 7.4) were added. The purified results (see...) Figure 1 ).

[0031] III. Identification of Monoclonal Antibody 3B3 a. Dilute PoRVA 05041-27 virus solution to MOI=1 and inoculate it into MA104 cells. Once the cytopathic effect reaches 50%, fix with 4% paraformaldehyde at room temperature for 1 hour. Then wash three times with pre-cooled PBS solution and permeabilize with 0.02% Triton-X solution at room temperature for 1 hour. Next, block with 10% goat serum at 37°C for 2 hours. After washing three times with pre-cooled PBS solution, incubate the purified monoclonal antibody 3B3 at 37°C for 1 hour. Positive mouse serum, negative mouse serum, and SP2 / 0 supernatant are used as controls.

[0032] Wash three times with pre-cooled PBS solution, then incubate with 1:1000 diluted goat anti-mouse IgG H&L (FITC, Beyotime, CHN) at 37°C in the dark for 1 hour. Wash three times with pre-cooled PBS solution, and finally incubate with DAPI solution at 37°C in the dark for 10 minutes. After washing three times with pre-cooled PBS solution, observe using an inverted fluorescence microscope.

[0033] The results are as follows Figure 2 As shown: Monoclonal antibody 3B3 reacts specifically with the virus.

[0034] Antibody subtype identification was performed using monoclonal antibody 3B3, following the instructions for use of the SBA Clonotyping System-HRP kit (Southern Biotech, USA).

[0035] The results are as follows Figure 3 As shown: the heavy chain type of monoclonal antibody 3B3 is IgG1, and the light chain type is Kappa.

[0036] IV. Reverse transcription of cDNA a. Hybridoma monoclonal cells 3B3 were cultured in 1640 complete medium at 37°C and 5% CO2. When the cell number reached 1×10⁻⁶... 7 Total RNA was extracted from cells using a total RNA extraction kit (purchased from Tiangen).

[0037] b. Perform reverse transcription according to the following reaction system and conditions: 5 μg RNA, 82 μL Oligo (dT), add DEPC H2O to 14 μL, incubate at 65℃ for 5 min, then immediately place on ice. Remove the sample and add 2 μL 10×RT buffer, 1 μL RNase Inhibitor, 2 μL dNTP (10 mM), and 1 μL MLV. Set the program to amplify at 42℃ for 60 min, then at 75℃ for 10 min; the reaction product is cDNA.

[0038] V. Sequence sequencing of monoclonal antibody 3B3 Universal upstream and downstream primers specific to the mouse heavy chain and light chain antibody genes were designed. PCR amplification was performed using cDNA as a template. The PCR system was 50 μL: 3 μL cDNA, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 25 μL 2×Phanta Max Buffer, 1 μL dNTP Mix (10 mM each), 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), and 16 μL ddH2O. PCR conditions were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 56℃ annealing for 15 s, and 72℃ extension for 30 s, for a total of 35 cycles; a final extension at 72℃ for 5 min. PCR products were subjected to 1% gel electrophoresis. Bands matching the expected sequence were excised and sent to a sequencing company to obtain the gene sequences of the 3B3 heavy and light chain variable regions of the monoclonal antibody. The nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 3B3 is shown in SEQ ID NO.1: GAGGTGCAGCTGGTGGAGTCAGGACCTGGCCTGGTTGACGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAAACAGTTATGGTATACACTGGATTCGCCAGCCTCCAGGAAAGAATCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAACACAAATTATAAT TCGGCTCTAATGTCCAGACTGAGCGTCAGCAAAGACAACTCCAAGGGCCAAGTTTTCTTAAAGATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAGACTATGATGGCGACCACGCCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCGCCGTCTCCTCA; The amino acid sequence encoding the variable region of the heavy chain of monoclonal antibody 3B3 is shown in SEQ ID NO.2: EVQLVESGPGLVTPSQSLSITCTVSGFSLNSYGIHWIRQPPGKNLEWLGVIWAGGNTNYNSALMSRLSVSKDNSKGQVFLKMNSLQTDDTAMYYCARDYDGDHAYAMDYWGQGTSVTVSS.

[0039] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3B3 is shown in SEQ ID NO.3: GACATTGAGCTCACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGACAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGAAATTAGTGGTTACTTAAACTGGCTTCAGCAGAAACCAGATGGAACTATTAAACGCCTGATCTACGCCGCATCTAGTTTA GATTCTGGTGTCCCAAAAAGGTTCAGTGGCAGTAGATCTGGGTCAGAATATTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTTTGCAGACTATTATTGTCTACAATATGCTAATTATCCATTCACGTTCGGCTCGGGGACCAAGCTGGAAATAAAACGG; The amino acid sequence encoding the light chain variable region of monoclonal antibody 3B3 is shown in SEQ ID NO.4: DIELTQSPSSLSASLGQRVSLTCRASQEISGYLNWLQQKPDGTIKRLIYAASSLDSGVPKRFSGSRSGSEYSLTISSLESEDFADYYCLQYANYPFTFGSGTKLEIKR.

[0040] VI. Variable Region Amino Acid Sequence and Homology Analysis The heavy and light chain gene sequences of monoclonal antibody 3B3 were compared and analyzed in the NCBI database.

[0041] The analysis showed that the nucleotide sequence of the monoclonal antibody 3B3 heavy chain variable region gene had the highest homology with the mouse immunoglobulin heavy chain variable region (Sequence ID: M36217.1), with a homology of 333 / 359, or 93%. The amino acid sequence of the monoclonal antibody heavy chain variable region had the highest homology with the mouse immunoglobulin heavy chain variable region amino acid sequence (Sequence ID: ACY74426.1), with a homology of 101 / 120, or 84%.

[0042] The nucleotide sequence of the 3B3 light chain variable region gene of the monoclonal antibody showed the highest homology with the variable region of the mouse immunoglobulin light chain (Sequence ID: PP322080.1), with a homology of 307 / 321, representing a homology percentage of 96%. The amino acid sequence of the 3B3 light chain variable region showed the highest homology with the amino acid sequence of the variable region of the mouse immunoglobulin light chain (Sequence ID: WVW32498.1), with a homology of 100 / 107, representing a homology percentage of 97%.

[0043] Homology analysis of the gene and amino acid sequences encoding the heavy and light chain variable regions of monoclonal antibody 3B3 showed that no identical sequences were found.

[0044] By analyzing the sequences of the heavy chain variable region and the light chain variable region, their complementarity-determining regions (CDR regions) are obtained.

[0045] The amino acid sequences of the three CDR regions of the heavy chain variable region are shown in Table 1.

[0046] Table 1. Amino acid sequences of the three CDR regions in the heavy chain variable region. The amino acid sequences of the three CDR regions of the light chain variable region are shown in Table 2.

[0047] Table 2. Amino acid sequences of the three CDR regions in the light chain variable region. Example 2: Group A porcine rotavirus blocking ELISA antibody detection kit and its establishment The Group A porcine rotavirus blocking ELISA antibody detection kit includes HRP-labeled monoclonal antibody HRP-3B3, antigen-coated plate, 1×PBST, blocking buffer, serum diluent, antibody diluent, stop solution, single-component chromogenic solution (brand: IPANDA), positive control, and negative control; among which, Preparation of each component of the above Group A porcine rotavirus blocking ELISA antibody detection kit 1. Preparation of HRP-labeled monoclonal antibody HRP-3B3 a. The ultrafiltration purified monoclonal antibody 3B3 was dissolved in 0.05M carbonate buffer; b. Weigh 6 mg HRP and dissolve it in 1.2 mL of double-distilled water. Pre-cool at 4 °C in the dark for 30 min. Add 0.72 L of freshly prepared 0.05 M NaIO4 solution and mix well. Stir at 4 °C in the dark for 30 min. At room temperature, slowly add 0.6 mL of 0.16 M ethylene glycol aqueous solution to the stirred HRP. React at room temperature in the dark for 30 min to obtain a crude HRP solution after oxidation. The oxidized crude HRP solution was transferred into an ultrafiltration tube (molecular weight 10 kDa, to remove unreacted reagents and reaction byproducts by ultrafiltration) and ultrafiltered at 3000 g / min for 15-20 min; then diluted to a suitable volume with 0.05 M carbonate buffer to obtain an oxidized HRP solution, wherein the HRP concentration in the solution was 4 mg / mL. c. According to the calculated feed amount, add the purified monoclonal antibody 3B3 to the oxidized HRP solution under low-speed stirring. Stir overnight at 4°C in the dark; add 4 mg / mL NaBH4 solution to make the final concentration 20 mM, mix by inversion, and let stand at room temperature in the dark for 2 h; add the above solution to a dialysis bag and dialyze overnight with 0.02 M PBS, changing the medium 3-5 times throughout the process; add an equal volume of glycerol and 2% BSA and store at -20°C; obtain the HRP-labeled monoclonal antibody HRP-3B3.

[0048] 2. Preparation of other components and articles 1×PBST: 1×PBS 500mL is added to 250μL of Tween-20 and mixed.

[0049] The blocking solution was PBST containing 2% BSA and 5% skim milk by mass.

[0050] The serum diluent was PBST containing 6% NaCl and 2% BSA by mass.

[0051] The antibody diluent was PBST containing 2% BSA by mass.

[0052] Stop solution (2M H2SO4): Take 150mL of ultrapure water, add 22.2mL of concentrated sulfuric acid, and make up to 200mL.

[0053] Negative control: Porcine PoRVA antibody-negative serum.

[0054] Negative control: SPF porcine serum.

[0055] Preparation of antigen-coated plates: a. Antigen coating solution (carbonate buffer, pH=9.6): 1.59g Na2CO3 and 2.97g NaHCO3 are added to ultrapure water and brought to a final volume of 1000mL; b. Dilute VP6 protein to 100 ng / mL using antigen coating buffer, coat overnight at 4°C, add 100 μL / well of 1×PBST to the wells of an ELISA plate, tap off the absorbent material, and repeat three times; block with 120 μL / well of blocking buffer at 37°C for 2 h, add 100 μL / well of 1×PBST to the wells, tap off the absorbent material, and repeat three times; obtain the antigen-coated plate.

[0056] I. Screening of the above-mentioned Group A porcine rotavirus blocking ELISA antibody detection kit 1. Screening for the titer of HRP-labeled monoclonal antibody HRP-3B3 VP6 protein was diluted to 50 μg / mL using antigen coating buffer and coated overnight at 4°C. 100 μL / well of 1×PBST was added to each well of an ELISA plate, and the solution was tapped off onto absorbent material. This process was repeated three times. Blocking buffer was applied to each well at 37°C for 2 h, and 100 μL / well of 1×PBST was added to each well. This process was repeated three times. HRP-labeled monoclonal antibody HRP-3B3 was serially diluted 3-fold from 1:90 to 1:21870. 100 μL / well was added to each well of an ELISA plate. Simultaneously, three negative controls of serum dilutions were included for each dilution, with 100 μL / well added to each well. The plates were incubated at 37°C for 30 min. Wash three times with PBST solution, incubate 1:10000 diluted goat anti-mouse IgG (Southern Biotech, USA) at 37°C for 30 min, wash three times with PBST solution, add 100 μL of single-component TMB to each well for 10 min, then terminate with 50 μL of 2M H2SO4, and read the OD. 450nm The numerical values ​​are S for the sample wells and N for the negative control wells. A positive result is defined as S / N > 2.

[0057] The results showed that the titer of the HRP-labeled monoclonal antibody HRP-3B3 was 1:21870. Figure 4).

[0058] 2. Screening of HRP-3B3 dilution and serum sample incubation time Serum samples and negative controls were diluted 1:1, and 100 μL was added to each well of the antigen-coated plate. The plates were incubated at 37°C for 30 min, followed by three washes with PBST. Four dilutions of 3B3-HRP were set up: 1:1000, 1:2000, 1:4000, and 1:8000. 100 μL of each dilution was added to each well and incubated at 37°C for 30 min, followed by three washes with PBST. 100 μL of single-component TMB was added to each well and incubated at 37°C for 10 min. Finally, the reaction was terminated with 50 μL of 2M H2SO4 per well. 450nm The absorbance value is read. The PI value is calculated as follows: PI = 1 - (OD) 450nm Serum sample - OD 450nm (Negative control) × 100%.

[0059] The results showed that when the VP6 protein coating concentration was 100 ng / mL and the HRP-3B3 dilution was 1:2000, the PI value was 60.36%, which was the highest in the matrix. Figure 5 ).

[0060] In addition, incubation times for serum samples and negative controls were set at 15 min, 30 min, 45 min, and 60 min, respectively. The results showed that the PI values ​​were higher at serum incubation times of 30 min, 45 min, and 60 min than at 15 min. To save time, 30 min was chosen as the optimal serum incubation time. Figure 6 ).

[0061] 3. Measure the PI values ​​of 50 negative serum samples and 50 positive serum samples. Positive and negative serum samples and negative controls were diluted 1:1 with serum diluent, 100 μL per well, and incubated at 37°C for 30 min. 100 μL / well of 1×PBST was added to each well, and the solution was tapped off onto absorbent material. This process was repeated three times. 3B3-HRP was diluted to 1:2000 with antibody diluent, 100 μL per well, and incubated at 37°C for 30 min. 100 μL / well of 1×PBST was added to each well, and the solution was tapped off onto absorbent material. This process was repeated three times. Single-component TMB was added 100 μL per well and incubated at 37°C for 10 min. Finally, 50 μL of 2M H2SO4 was added to each well to stop the reaction. The reaction was then stopped at OD. 450nm The absorbance value is read. The PI value is calculated as follows: PI = 1 - (OD) 450nm Positive and negative serum samples - OD 450nm (Negative control) × 100%.

[0062] ROC analysis of the results showed an area under the curve (AUC) of 0.9973 (p < 0.0001). The optimal PI value (23.23%) was achieved when both specificity and sensitivity were 98.04%. Therefore, a PI < 23.23% was considered negative, and a PI ≥ 23.23% was considered positive. Figure 7 ).

[0063] The distribution of positive and negative results shows that positive and negative sera can be clearly distinguished, therefore this method is relatively accurate for detection. Figure 8 ).

[0064] II. The blocking ELISA antibody detection method of the above-mentioned Group A porcine rotavirus blocking ELISA antibody detection kit includes the following steps: 1) Dilute the serum sample to be tested and the negative control with serum diluent at a ratio of 1:1 to obtain diluted serum sample. Add 100 μL of diluted serum sample and diluted negative control to the antigen-coated plate at each well and incubate at 37°C for 30 min. Add 100 μL / well of 1×PBST to the wells of the antigen-coated plate and tap it off on absorbent material. Repeat three times. 2) Dilute 3B3-HRP to 1:2000 with antibody dilution buffer, add 100 μL to each well and incubate at 37°C for 30 min. Add 100 μL / well of 1×PBST to each well, tap off the excess material, and repeat three times. Add 100 μL of single-component chromogenic solution to each well and incubate at 37°C for 10 min. Finally, add 50 μL of stop solution to each well to terminate the reaction. [The last sentence appears to be incomplete and possibly refers to an OD value.] 450nm The absorbance reading and the PI value calculation method are as follows: PI = 1 - (OD) 450nm Serum sample - OD 450nm (Negative control) × 100%.

[0065] 3) Analyze and judge based on PI value: When PI < 23.23%, the result is considered negative, meaning that the serum sample to be tested does not have antibodies against group A porcine rotavirus VP6. A result is considered positive when the PI is ≥ 23.23%, meaning that the serum sample to be tested contains antibodies against group A porcine rotavirus VP6.

[0066] III. Sensitivity, specificity, and repeatability analysis of the above-mentioned Group A porcine rotavirus blocking ELISA antibody detection kit: 1. Sensitivity analysis of the blocking ELISA antibody detection method was performed using three positive serum samples. Based on the kit and method, a blocking ELISA was performed using three PoRVA VP6-immunized porcine serum samples. The results showed that when E18 serum was diluted to 1:512, the PI value was below 23.23%, resulting in a negative result. Therefore, the highest sensitivity of this blocking ELISA method was 1:256 (e.g., Figure 9 ).

[0067] 2. PI values ​​of serum samples positive for six common swine viral pathogens (CSFV, PEDV, PRV, PRRSV, PCV2, and ASFV).

[0068] Based on the kit and method, blocking ELISA detection was performed on positive sera of six viral pathogens.

[0069] The results showed that all values ​​were less than 23.23%, and the PI value of PoRVA-positive serum was 88.67%, indicating that the developed blocking ELISA method has good specificity (e.g., Figure 10 ).

[0070] 3. OD measured using 3 positive sera and 3 negative sera. 450nm The CV of intra-batch and inter-batch results in numerical computation.

[0071] The coefficient of variation for intra- and inter-assay detection of three PoRVA VP6-immunized porcine serum samples and three SPF porcine serum samples based on the kits and methods is calculated as follows: CV = (Standard deviation SD / Mean) × 100%.

[0072] The results showed that the CV values ​​for intra-batch experiments ranged from 0.72% to 10.17%, and the CV values ​​for inter-batch experiments ranged from 1.59% to 9.09%, indicating that the method has good repeatability and the results are stable and reliable (as shown in Table 3).

[0073] Table 3. Repeatability analysis of the blocking ELISA antibody detection method Example 3: Concordance test between the blocking ELISA method and the indirect immunofluorescence assay of the Group A porcine rotavirus blocking ELISA antibody detection kit: 116 PoRVA VP6-immunized porcine serum samples and 68 SPF porcine serum samples were tested using indirect immunofluorescence and antibody detection kits, respectively.

[0074] The specific steps of the indirect immunofluorescence method are as follows: PoRVA was seeded into MA104 cells at an MOI of 1. After lesions appeared, the cells were fixed with 4% paraformaldehyde at room temperature for 1 h. Then, the cells were washed three times with pre-chilled PBS and permeabilized with 0.02% Triton-X solution at room temperature for 1 h. Subsequently, the cells were blocked with 10% goat serum at 37°C for 2 h. After washing three times with pre-chilled PBS, the monoclonal antibody supernatant was incubated at 37°C for 1 h, with positive and negative sera as controls. The cells were then washed three times with pre-chilled PBS and incubated with 1:1000 diluted goat anti-mouse IgG H&L (FITC) (Beyotime, CHN) at 37°C in the dark for 1 h. After washing three times with pre-chilled PBS, the cells were incubated with DAPI solution at 37°C in the dark for 10 min. After washing three times with pre-chilled PBS, the results were observed using an inverted fluorescence microscope. Under 488 nm excitation light, 116 wells with obvious fluorescence were observed as positive wells, and 68 wells were observed as negative wells.

[0075] 184 serum samples were tested using a blocking ELISA antibody detection kit. The results showed that 114 out of 116 IFA-positive serum samples tested positive and 2 tested negative; 62 out of 68 IFA-negative serum samples tested negative and 6 tested positive.

[0076] Therefore, the results show that the positive serum concordance rate of this kit is 98.28%, the negative serum concordance rate is 91.18%, and the total concordance rate is 95.65%, indicating that the accuracy of this kit is very high and can be applied to clinical testing (Table 4).

[0077] Table 4. Conformity analysis of the blocking ELISA antibody detection method of the kit. All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody 3B3 that specifically binds to group A porcine rotavirus, characterized in that: The monoclonal antibody 3B3 consists of a heavy chain and a light chain; wherein... The heavy chain variable region of monoclonal antibody 3B3 includes three complementarity-determining regions, namely 3B3-CDR-H1, 3B3-CDR-H2 and 3B3-CDR-H3, whose amino acid sequences are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively. The light chain variable region of monoclonal antibody 3B3 includes three complementarity-determining regions, namely 3B3-CDR-L1, 3B3-CDR-L2 and 3B3-CDR-L3, whose amino acid sequences are QEISGY, AAS and LQYANYPFT, respectively.

2. The monoclonal antibody 3B3 according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3B3 is shown in SEQ ID NO.2; the amino acid sequence of the light chain variable region of 3B3 is shown in SEQ ID NO.

4.

3. The monoclonal antibody 3B3 according to claim 2, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3B3 is shown in SEQ ID NO.1; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3B3 is shown in SEQ ID NO.

3.

4. The use of the monoclonal antibody 3B3 according to claim 1 in the preparation of a group A porcine rotavirus blocking ELISA antibody detection kit.

5. A group A porcine rotavirus blocking ELISA antibody detection kit, characterized in that: The test kit includes HRP-labeled monoclonal antibody 3B3 as described in claim 1.

6. The detection kit according to claim 5, characterized in that: The test kit also includes an antigen-coated plate, 1×PBST, blocking solution, serum diluent, antibody diluent, stop solution, single-component chromogenic solution, positive control, and negative control; wherein... 1×PBST: Mix 500 mL of 1×PBS with 250 μL of Tween-20 to obtain PBS. The blocking solution was PBST containing 2% BSA and 5% skim milk by mass. The serum diluent was PBST containing 6% NaCl and 2% BSA by mass. The antibody diluent was PBST containing 2% BSA by mass. Termination solution: Take 150 mL of ultrapure water, add 22.2 mL of concentrated sulfuric acid, and make up to 200 mL; Positive control: PoRVA VP6 protein-immunized porcine serum; Negative control: SPF porcine serum.

7. The detection kit according to claim 5, characterized in that: The antigen coated on the antigen-coated plate is the VP6 protein.

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