Porcine rotavirus vp4 protein monoclonal antibody, kit and application

Abstract

The application discloses a porcine rotavirus VP4 protein monoclonal antibody, a kit and application, and belongs to the field of porcine rotavirus antibody detection.The porcine rotavirus VP4 protein monoclonal antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown as SEQ ID NO.1, and the amino acid sequence of the light chain is shown as SEQ ID NO.2.Innovatively taking the monoclonal antibody targeting a VP4 conservative functional epitope as the core, an ELISA detection kit and a detection method are constructed, the method can significantly reduce cross-reactions, and can truly reflect a protective immune level;without a species-specific secondary antibody, non-specific binding and serum source interference are avoided, the method has strong universality, good repeatability, is suitable for high-throughput application, and fills a technical blank, and provides a new tool with high specificity, high correlation and high practicability for precise immune monitoring and vaccine evaluation of PoRV.

Description

Technical Field

[0001] This invention relates to the field of porcine rotavirus antibody detection, and in particular to a porcine rotavirus VP4 protein monoclonal antibody, kit, and application. Background Technology

[0002] Porcine rotavirus (PoRV) is one of the main pathogens causing viral diarrhea in piglets and is widespread in global pig production systems. This virus primarily infects piglets during the suckling period and early weaning stage, leading to watery diarrhea, vomiting, dehydration, and growth retardation. In severe cases, secondary bacterial infections can occur, increasing treatment costs and affecting survival rates. PoRV spreads rapidly in pig herds, with morbidity rates exceeding 80%, although mortality is usually relatively low. Nevertheless, the cumulative economic losses it causes to feed conversion ratios, time to market, and farming efficiency are significant, making it one of the key pathogens requiring prevention and control in current pig herd health management.

[0003] VP4 is a key surface spike protein of PoRV, which is highly variable and determines the P serotype of the virus (such as P

[23] , P[7], P

[13] , etc.), and mediates the adsorption and invasion of the virus to host cells. It is one of the main antigens that induce neutralizing antibodies. VP4 is cleaved into VP8 and VP5 by trypsin. VP8 is located at the top of the viral surface spike and is mainly responsible for recognizing and binding receptors (such as glycolipids and glycoproteins) on host cells, mediating the initial adsorption of the virus. VP5 participates in the fusion process between the virus and the cell membrane. It undergoes conformational changes in the low pH environment of the endosome, promotes the fusion of the viral capsid with the cell membrane, and helps the viral genome enter the cytoplasm. Its sequence and structure are more conserved. Studies have shown that antibodies against VP4 protein are highly correlated with viral neutralizing antibodies. Anti-VP4 antibodies produced by the body after natural infection or vaccine immunization can effectively block the binding of the virus to host cells, significantly inhibit viral invasion and replication, and have clear neutralizing activity. Therefore, detecting the level of anti-VP4 antibodies in serum can truly reflect the actual immune protection status of animals against the virus. Using recombinant VP4 protein as the detection target not only demonstrates high specificity and low cross-reactivity, but also shows good consistency between the measured antibody levels and neutralizing antibody titers, exhibiting excellent immunorelevance. Furthermore, this antigen exhibits good batch stability, is easy to standardize production, and is suitable for high-throughput, automated detection platforms. It provides pig farms with accurate and reliable means for immune assessment and epidemiological monitoring, making it an ideal choice for optimizing existing antibody detection methods.

[0004] Currently, PoRV antibody detection primarily employs indirect ELISA based on whole virus or VP6 antigen. The former is susceptible to non-specific interference and has poor specificity; the latter, while highly sensitive, detects mostly non-neutralizing antibodies with low correlation to immune protection. Although VP4-based indirect ELISAs have been reported, they still rely on the secondary antibody system and are easily affected by serospecific differences in serum. Currently, there are no publicly reported reports of blocking ELISA based on anti-VP4 monoclonal antibodies in the field of PoRV antibody detection. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody against porcine rotavirus VP4 protein, a reagent kit, and its applications, to address the problems existing in the prior art. This invention innovatively uses a monoclonal antibody targeting a conserved functional epitope of VP4 as the core to construct a blocking ELISA detection kit and method. This method has significant advantages: the monoclonal antibody is highly specific, significantly reducing cross-reactivity; it detects antibodies that can block neutralizing sites through a competitive mechanism, and the results show a good correlation with serum neutralizing activity, accurately reflecting the level of protective immunity; it does not require species-specific secondary antibodies, avoiding non-specific binding and serum-derived interference; it is highly versatile and reproducible, suitable for high-throughput applications. This invention fills a technological gap and provides a novel tool with high specificity, high correlation, and high practicality for precise immune monitoring and vaccine evaluation of PoRV.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a monoclonal antibody against porcine rotavirus VP4 protein, characterized in that the monoclonal antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO.1 and the amino acid sequence of the light chain is shown in SEQ ID NO.2.

[0008] The present invention also provides a nucleic acid molecule encoding the monoclonal antibody described above.

[0009] This invention also provides the use of the monoclonal antibody or the nucleic acid molecule described herein in any of the following:

[0010] (1) Application in the preparation of a kit for detecting porcine rotavirus antibodies;

[0011] (2) Application in the preparation of a kit for detecting porcine rotavirus.

[0012] Furthermore, the kit includes a blocking ELISA kit.

[0013] The present invention also provides a blocking ELISA kit for detecting porcine rotavirus antibodies, comprising the aforementioned monoclonal antibody.

[0014] Preferably, the blocking ELISA kit further includes an ELISA plate coated with porcine rotavirus VP4 protein, porcine rotavirus positive control serum, porcine rotavirus negative control serum, serum diluent, washing buffer, TMB substrate solution, and stop solution.

[0015] Preferably, porcine rotavirus antibodies are detected using a blocking ELISA method based on the monoclonal antibody.

[0016] Preferably, the detection of porcine rotavirus antibodies by the blocking ELISA method includes the following steps:

[0017] (1) Dilute the serum to be tested with serum diluent at an equal volume ratio and add it to an ELISA plate coated with porcine rotavirus VP4 protein, and incubate;

[0018] (2) Add horseradish peroxidase-labeled porcine rotavirus VP4 protein monoclonal antibody and incubate;

[0019] (3) Spin-dry the ELISA plate, wash it, and pat it dry;

[0020] (4) Add TMB substrate solution and develop color at room temperature;

[0021] (5) Add the stop solution;

[0022] (6) Select the OD450nm reading, calculate the blocking rate, and determine whether porcine rotavirus antibodies are present.

[0023] Preferably, in step (1), the incubation conditions are: incubation at 37°C for 45 minutes;

[0024] In step (2), the horseradish peroxidase-labeled porcine rotavirus VP4 protein monoclonal antibody is diluted at a ratio of 1:2000; the incubation conditions are: incubation at 37°C for 1 hour.

[0025] Preferably, the volume ratio of the diluted test serum, the diluted horseradish peroxidase-labeled porcine rotavirus VP4 protein monoclonal antibody, the TMB substrate solution, and the stop solution is 1:1:2:1.

[0026] Preferably, the criteria for judgment are as follows: a blocking rate ≥ 26.92% is judged as positive, that is, the serum to be tested is positive for porcine rotavirus antibodies; a blocking rate < 26.92% is judged as negative, that is, the serum to be tested is negative for porcine rotavirus antibodies.

[0027] The present invention discloses the following technical effects:

[0028] (1) High specificity: The present invention uses VP4 protein monoclonal antibody, and the specificity of the antibody is determined by ROC curve. The specificity reaches 98.28%, and there is no cross-reaction with the positive serum of eight common porcine viral pathogens.

[0029] (2) High sensitivity: The sensitivity of the present invention reaches 96.00%, which is higher than that of existing detection methods, and can detect low-titer antibodies more accurately.

[0030] (3) Elimination of species differences: The blocking ELISA method of the present invention can effectively eliminate the differences in the species origin of serum samples, making the detection results more accurate and reliable.

[0031] (4) Simple and fast operation: The detection process takes only 2 hours, the operation steps are simplified, no special equipment is required, and it is suitable for large-scale detection.

[0032] (5) High-throughput detection: Suitable for high-throughput detection of a large number of field serum samples, and can detect 96 samples at the same time.

[0033] (6) Accurate quantification: It can accurately reflect antibody titer and is suitable for long-term monitoring of antibody growth and decline trends.

[0034] (7) Molecular-level innovation: The specific sequence of monoclonal antibodies was determined by antibody sequencing technology, which provided a molecular-level theoretical basis for the detection method and significantly improved the reliability and scientific nature of the detection. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is an SDS-PAGE image of purified monoclonal antibody; M represents the standard protein molecule, and 1 and 2 represent the samples.

[0037] Figure 2 Determination of the detection threshold for a porcine rotavirus antibody detection blocking ELISA kit;

[0038] Figure 3 Diagnostic specificity and sensitivity analysis of a porcine rotavirus antibody detection blocking ELISA kit;

[0039] Figure 4 Analysis of the detection specificity of the porcine rotavirus antibody detection blocking ELISA kit;

[0040] Figure 5Sensitivity analysis of a porcine rotavirus antibody detection blocking ELISA kit. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] Example 1: Preparation of monoclonal antibody against porcine rotavirus VP4 protein

[0047] This invention utilizes VP4 protein 26-476aa of porcine rotavirus strain AHFY2022 (GenBank accession number: OQ979283.1) as an antigen.

[0048] 1. Preparation of VP4 monoclonal antibody

[0049] 1.1 Animal Immunization

[0050] VP4 protein and ISA TM201 adjuvants were mixed in equal volumes and emulsified to prepare a water-in-oil-in-water formulation. Mice were immunized by subcutaneous injection at multiple sites on the back, 50 µg / mouse, for a total of 3 immunizations, with an interval of 2 weeks between immunizations. Seven days after the third immunization, serum was collected via orbital vein and its ELISA titer was determined.

[0051] 1.2 Screening and Identification of Monoclonal Antibodies

[0052] When the ELISA titer was greater than 80,000, the spleen was removed after sterilization, and a spleen cell suspension was prepared. This suspension was then fused with resuscitated myeloma cells using PEG. After centrifugation and washing, a single-cell suspension was prepared using HAT medium and seeded into 96-well plates. On day 7 of fusion, clonal formation was visible to the naked eye. Initial screening was performed using an indirect ELISA method, and cells from strongly positive wells were cultured in 24-well plates for expansion. Using a limiting dilution method, multiple cloning operations were performed until the positive rate reached 100%, at which point a hybridoma cell line was obtained. The cell line supernatant was used for blocking ELISA screening, and clone 16 (mAb16) was found to have good blocking activity. Following the instructions of the monoclonal antibody subclass identification kit, the monoclonal antibody subtype was identified, and the mAb16 genotype was determined to be IgG2a / kappa.

[0053] 1.3 Ascites preparation and purification

[0054] Female mice were sensitized by intraperitoneal injection of 0.5 mL of liquid paraffin, and 1-2 weeks later, 1-2 × 10⁻⁶ g of paraffin was injected. 6 Hybridoma cells were collected. Ascites fluid was collected and centrifuged after significant peritoneal distension in mice. The ascites fluid was crudely purified using ammonium sulfate precipitation: the supernatant was collected, and saturated ammonium sulfate was added dropwise to a semi-saturated state at 4°C, stirred for 30 min, and then centrifuged. The precipitate was resuspended, and the ammonium sulfate concentration was adjusted again to 33%. The precipitate was collected by centrifugation, dissolved, and dialyzed for 12 h. The product was stored at -20°C. Further purification was then performed using a Protein G affinity chromatography column, followed by neutralization and dialysis after elution. Antibody concentration was determined using the BCA method, and the antibody was aliquoted and stored at -80°C. Finally, antibody purity was identified by SDS-PAGE gel electrophoresis, and the purification effect was judged by observing the bands. Figure 1 ).

[0055] 2. Antibody sequencing

[0056] The heavy and light chain variable region sequences of the screened monoclonal antibodies were determined. The results showed that the heavy chain sequence of the monoclonal antibody was SEQ ID NO.1, and the light chain sequence was SEQ ID NO.2.

[0057] Example 2 Preparation of porcine rotavirus VP4 protein

[0058] The VP4 gene fragment (gene sequence SEQ ID NO:3), synthesized after codon optimization, was cloned into the XhoI and SalI multiple cloning sites of the pCold-sumo vector to construct the prokaryotic expression plasmid pCold-sumo-VP4. Enzyme digestion confirmed the correct plasmid construction, and sequencing confirmed the absence of base mutations. The correctly identified recombinant plasmid pCold-sumo-VP4 was transformed into E. coli BL21(DE3), and positive clones were selected for expansion culture. Expression was induced by IPTG, and the plasmid was cultured at 16℃ in a shaker for 24 h. After sonication, SDS-PAGE electrophoresis was performed, and a specific protein band appeared at the expected location, indicating stable expression mainly in a soluble form. The target protein VP4 was purified with high purity using a soluble protein purification method.

[0059] Example 3: Composition of a porcine rotavirus antibody detection blocking ELISA kit

[0060] 1. Preparation of porcine rotavirus VP4 protein

[0061] VP4 protein was prepared according to the method in Example 2, and the protein concentration was determined by the BCA method. The purified VP4 protein was stored at -80°C for later use.

[0062] 2. Serum preparation

[0063] Porcine rotavirus positive serum: Serum was collected 21 days after inoculation of PoRV antigen and antibody double-negative pigs with strain AHFY2022 (GenBank accession number: OQ979280.1~OQ979290.1).

[0064] Porcine rotavirus negative serum: derived from antigen-antibody double-negative porcine serum from a pig farm in Jiangsu Province.

[0065] 3. Monoclonal antibody preparation

[0066] The porcine rotavirus VP4 protein monoclonal antibody was obtained according to the method in Example 1.

[0067] 4. Assembly of the blocking ELISA kit

[0068] The blocking ELISA kit includes a coated plate, positive control serum, negative control serum, HRP-labeled monoclonal antibody, serum diluent, washing buffer, substrate solution, and stop solution. More specifically:

[0069] (1) ELISA plates coated with porcine rotavirus VP4 protein (0.1~5 ng / μL, 100 μL / well, incubate at 37℃ for 2 h or at 4℃ overnight).

[0070] (2) Porcine rotavirus positive control serum (serum collected 21 days after AHFY2022 strain was inoculated into PoRV antigen and antibody double negative pigs);

[0071] (3) Porcine rotavirus negative control serum (PoRV antigen and antibody double negative porcine serum);

[0072] (4) Horseradish peroxidase-labeled monoclonal antibody against porcine rotavirus VP4 protein (diluted at 1:1000~3000).

[0073] (5) Serum diluent (PBS solution containing 0.05% Tween (V / V), pH 7.4);

[0074] (6) Washing solution (PBST solution);

[0075] (7) TMB substrate solution;

[0076] (8) Termination solution (2M H2SO4).

[0077] Example 4: Method for detecting porcine rotavirus antibodies, comprising the following steps

[0078] (1) Dilute the serum to be tested with serum diluent at a ratio of 1:1 (50 μL / well) and add it to an ELISA plate coated with porcine rotavirus VP4 protein;

[0079] (2) Set up negative and positive control wells and add negative control serum and positive control serum diluted 1:1 (50 μL / well).

[0080] (3) Incubate at 37℃ for 45 minutes without washing;

[0081] (4) Add horseradish peroxidase-labeled porcine rotavirus VP4 protein monoclonal antibody (100 μL / well) diluted at 1:2000 and incubate at 37°C for 1 h;

[0082] (5) Shake the ELISA plate dry, wash it 3 times with PBST, and pat it dry with absorbent paper;

[0083] (6) Add TMB substrate solution (100 μL / well) and develop color at room temperature for 10 min;

[0084] (7) Add stop solution (50 μL / well);

[0085] (8) Select OD 450nm Read the value and calculate the blocking rate:

[0086] Blocking rate = (OD value of negative control - OD value of serum to be tested) / OD value of negative control × 100%;

[0087] Judgment criteria: A blocking rate ≥ 26.92% is considered positive, and a blocking rate < 26.92% is considered negative.

[0088] Example 5: Effect Verification

[0089] 1. Validation of diagnostic specificity and sensitivity

[0090] Fifty positive sera from porcine rotavirus and 58 negative sera from porcine rotavirus, identified as positive by the virus-serum neutralization test, were selected and then detected using VP4-cELISA. The results showed that the sensitivity was 96.00%, the specificity was 98.28%, and the detection cutoff value was 26.92%. Figure 2 and Figure 3 ).

[0091] 2. Detection specificity verification

[0092] Positive sera for porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), foot-and-mouth disease virus type O (FMDV), classical swine fever virus (CSFV), and African swine fever virus (ASFV), as well as negative sera from pigs not infected with porcine rotavirus, were tested. The results showed that only the blocking rate of positive sera for porcine rotavirus was greater than 26.92%, proving that this invention does not have cross-reactivity with positive sera from common porcine viral pathogens. (See [link to relevant documentation]). Figure 4 .

[0093] 3. Sensitivity verification of the test

[0094] Three positive porcine rotavirus sera were serially diluted fourfold (1:1 to 1:1024). The results showed that all positive sera remained positive at a dilution of 1:256 (inhibition rate PI > 26.92%), while the signal disappeared at the 1:1024 dilution, and the inhibition rate was below the judgment threshold in all cases. Figure 5 ).

[0095] 4. Repeatability verification

[0096] Four rotavirus antibody-negative sera and four rotavirus antibody-positive sera were subjected to repeatability tests, with each serum sample tested three times. The results are shown in Table 1. The results showed that the intra-assay repeatability CV value was <5% and the inter-assay repeatability CV value was <6%.

[0097] Table 1 Repeatability test results

[0098]

[0099] 5. Comparative experiment with existing methods

[0100] This invention was compared with the neutralizing antibody method in the following tests: 50 known positive serum samples of porcine rotavirus antibodies and 58 known negative serum samples were tested. The results are as follows:

[0101] Table 2 Comparison of detection results between the method of the present invention and the neutralizing antibody method

[0102]

[0103] As shown in Table 2, the results indicate that the blocking ELISA method of the present invention has a good concordance rate with the neutralizing antibody detection method.

[0104] As can be seen from the above embodiments, this invention prepares a monoclonal antibody against porcine rotavirus VP4 protein. By using this monoclonal antibody and combining it with antibody sequencing technology, a blocking ELISA detection method was established, effectively solving the problems of low specificity, complex operation, and the influence of serum species on detection results in existing methods. The blocking ELISA kit of this invention has advantages such as high specificity (98.28%), high sensitivity (96.00%), simple and rapid operation (2 hours), elimination of serum species differences, and suitability for high-throughput detection. It can more accurately detect porcine rotavirus antibodies, providing strong technical support for the prevention and control of porcine rotavirus, and has significant innovation and application value.

[0105] The sequence involved in this invention:

[0106] Heavy chain of monoclonal antibody (SEQ ID NO.1):

[0107] 5'-EVQLQESGPSLVRPSQTLSLTCSVT RDSIPSGH WNWIRKFPDNKLEYMGY ISFSGQS DYNPSLKSRISISRDTSNNQYYLQLISVTSEDTATYYC TRGRWDGKDF WGQGTLVTVSA AKTTAPSVYPLAPVCGDTTGSSVTL GCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRG PTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRE DYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVT DFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTP GK -3'.

[0108] Note: In the above heavy chain sequence, amino acids 26-33 form the CDR1 region, amino acids 51-57 form the CDR2 region, amino acids 96-105 form the CDR3 region, amino acids 117-446 form the constant region, and other amino acids located on both sides of the CDR region form the framework region.

[0109] The light chain of the monoclonal antibody (SEQ ID NO.2):

[0110] 5'-QIVLTQSPAIMSASLGEEITLTCSVS STMSY MRWYQQKSGTSPKLLIY RTSNLASGVPSRFSGSGSGTFFSLTISSVEAEDAADYYC HPVTSSSGT FGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPK DINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC -3'.

[0111] Note: In the light chain sequence above, amino acids 27-31 form the CDR1 region, amino acids 49-51 form the CDR2 region, amino acids 88-96 form the CDR3 region, amino acids 107-213 form the constant region, and other amino acids located on both sides of the CDR region form the framework region.

[0112] The nucleotide sequence of VP4 (26-476aa) (SEQ ID NO.3):

[0113]

[0114] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A porcine rotavirus VP4 protein monoclonal antibody, characterized in that, The monoclonal antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain is shown as SEQ ID NO.

2.

2. A nucleic acid molecule encoding the monoclonal antibody of claim 1.

3. Use of the monoclonal antibody of claim 1 or the nucleic acid molecule of claim 2 in any of the following: (1) use in preparing a kit for detecting porcine rotavirus antibody; (2) use in preparing a kit for detecting porcine rotavirus.

4. A blocking ELISA kit for detecting antibodies to porcine rotavirus, characterized by, The kit comprises the monoclonal antibody of claim 1.

5. The blocking ELISA kit of claim 4, wherein, The blocking ELISA kit further comprises an ELISA plate coated with porcine rotavirus VP4 protein, a positive control serum of porcine rotavirus, a negative control serum of porcine rotavirus, serum diluent, washing solution, TMB substrate solution and stop solution.

6. The blocking ELISA kit of claim 4, wherein Based on the monoclonal antibody, a blocking ELISA method is used to detect porcine rotavirus antibody.

7. The blocking ELISA kit of claim 6, wherein, The blocking ELISA method for detecting porcine rotavirus antibody comprises the following steps: (1) dilute the serum to be detected with serum diluent at equal volume ratio, and then add it to the ELISA plate coated with porcine rotavirus VP4 protein, and incubate; (2) add horseradish peroxidase-labeled porcine rotavirus VP4 protein monoclonal antibody, and incubate; (3) spin dry the ELISA plate, wash and pat dry; (4) add TMB substrate solution, and develop color at room temperature; (5) add stop solution; (6) Select OD 450nm Read values, calculate blocking rates, and determine whether the sample contains antibodies to porcine rotavirus.

8. The blocking ELISA kit of claim 7, wherein, In step (1), the incubation condition is 37℃ for 45 min; In step (2), the dilution ratio of the horseradish peroxidase-labeled porcine rotavirus VP4 protein monoclonal antibody is 1:2000; and the incubation condition is 37℃ for 1 h.

9. The blocking ELISA kit of claim 7, wherein, The volume ratio of the diluted serum to be detected, the diluted horseradish peroxidase-labeled porcine rotavirus VP4 protein monoclonal antibody, the TMB substrate solution and the stop solution is 1:1:2:

1.

10. The blocking ELISA kit of claim 7, wherein The judgment standard is that a blocking rate ≥ 26.92% is judged as positive, i.e. the serum to be detected is positive for porcine rotavirus antibody; and a blocking rate < 26.92% is judged as negative, i.e. the serum to be detected is negative for porcine rotavirus antibody.

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