Porcine Seneca Valley virus neutralizing liquid-phase blocking ELISA kit and its application

Abstract

The present invention discloses a porcine Seneca virus neutralization liquid phase blocking ELISA kit, which comprises an effective amount of porcine Seneca virus antigen, an effective amount of porcine Seneca virus single domain antibody 1, an effective amount of porcine Seneca virus single domain antibody 2, and a matching detection reagent. The porcine Seneca virus antigen is porcine Seneca virus particles purified by sucrose density gradient centrifugation, and the particle size of the purified porcine Seneca virus particles is 20 to 30 nm. The amino acid sequence of the heavy chain variable region of the porcine Seneca virus single domain antibody 1 is shown in SEQ ID NO.1. The amino acid sequence of the heavy chain variable region of the porcine Seneca virus single domain antibody 2 is shown in SEQ ID NO.2. The kit of the present invention has the advantages of high efficiency, simplicity, sensitivity, stability, etc., has a good correlation with the traditional diagnostic method virus neutralization experiment, and is suitable for a wide range of applications.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the field of veterinary diagnosis technology, in particular to a porcine Seneca virus neutralizing liquid-phase blocking ELISA kit and a preparation method and application thereof. Background Art

[0002] Porcine Senecavirus, also known as Senecavirus A (SVA) and Seneca Valley virus (SVV), is the sole member of the genus Senecavirus within the family MicroRNA. SVA is a single-stranded, positive-sense RNA virus with a genome length of approximately 7.3 kb. Its structure shares many similarities with other small RNA viruses. Chemical structure analysis revealed that the complete SVA virus particle is a naked, symmetrical icosahedral structure with a diameter of 25 to 30 nm and a spherical appearance. Research progress on swine Seneca virus disease The viral genome contains only one open reading frame (ORF), and there is a type IV internal ribosome entry site in the 5' non-coding region, which mediates the translation of the virus's only ORF into a polyprotein; the polyprotein is cleaved into the leader protein L, P1, P2, and P3 protein intermediates SVA under the action of proteases. They follow the standard L-4-3-4 layout of the picornavirus genome and are processed into mature viral proteins by virus-encoded proteases. P1 is cleaved into four structural proteins, VP1, VP2, VP3, and VP4. The three capsid proteins VP1, VP2, and VP3 have similar shapes and folding arrangements and are exposed on the surface of the virus, while VP4 is located inside.

[0003] At the end of 2014, an outbreak of SVA in pigs was first reported in Brazil, outside the United States and Canada. Subsequently, SVA began to spread widely around the world, including China, Thailand, Colombia, and Vietnam. While SVA strains worldwide share a high homology of 95.8% to 99.9%, the homology with the SVA-001 strain is lower (93.8% to 94.6%), indicating that SVA is constantly evolving during its epidemic. Retrospective SVA surveillance conducted by the China Animal Health and Epidemiology Center revealed that SVA has been detected in multiple provinces (autonomous regions and municipalities) in my country, including Guangdong, Guangxi, Yunnan, Guizhou, Hunan, Fujian, Jiangxi, Sichuan, Hubei, Henan, Shandong, Xinjiang, Shanghai, Liaoning, and Heilongjiang, indicating that SVA is now widespread in many regions of my country.

[0004] The hallmark clinical signs of SVA are nasal and coronary ligament cystic lesions, resulting in anorexia and lameness in 10%–90% of pigs. Affected skin displays corneal hyperkeratosis, epidermal hyperplasia, ulceration, neutrophil infiltration, and cellular debris. Experimentally infected pigs develop multifocal dermal detachments, inflammatory cell infiltration, necrotic keratinocytes, hemorrhage, and fibrin accumulation. During the first 3–7 days post-infection, lymphoid hyperplasia is seen in the tonsils, spleen, and lymph nodes, while the lungs present with mild palpitations and congestion. Within a week, piglets develop a wide range of symptoms, including weakness, salivation, skin congestion, neurologic signs, diarrhea, and sudden death. Necrosis manifests as renal petechiae, hemorrhagic ulcerative glossitis, and coronary ligament cystic lesions and ulcerative dermatitis.

[0005] Currently, several domestic institutions have obtained clinical trial approval for inactivated porcine Seneca virus vaccines, and some products have entered the registration stage. However, the immune efficacy of vaccines after immunization is directly related to the level of neutralizing antibodies produced. Neutralizing antibody detection is mainly based on the cell culture virus neutralization test (VNT), which is time-consuming, labor-intensive, and has complex experimental steps. Therefore, the development of simple, efficient, rapid, and accurate detection methods is crucial for the prevention and control of Seneca. Summary of the Invention

[0006] In view of the above existing problems and defects, the object of the present invention is to provide a porcine Seneca virus neutralizing liquid phase blocking ELISA kit and its preparation method and application.

[0007] Therefore, on one hand, the present invention provides a porcine Seneca virus neutralizing liquid phase blocking ELISA kit, which comprises an effective amount of porcine Seneca virus antigen, an effective amount of porcine Seneca virus single domain antibody 1, an effective amount of porcine Seneca virus single domain antibody 2 and a matching detection reagent.

[0008] Preferably, the porcine Seneca virus antigen of the present invention is porcine Seneca virus particles purified by sucrose density gradient centrifugation, and the particle size of the purified porcine Seneca virus particles is 20 to 30 nm.

[0009] Preferably, the working concentration of the porcine Seneca virus antigen of the present invention is 1 μg / mL.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the porcine Seneca virus single-domain antibody 1 of the present invention is as shown in SEQ ID NO.1.

[0011] Preferably, the porcine Seneca virus single-domain antibody 1 of the present invention is a capture antibody, and its optimal coating concentration is 1 μg / mL.

[0012] Preferably, the amino acid sequence of the heavy chain variable region of the porcine Seneca virus single-domain antibody 2 of the present invention is as shown in SEQ ID NO.2.

[0013] Preferably, the porcine Seneca virus single domain antibody 2 described in the present invention is a detection antibody, which is labeled with HRP before use, and the optimal dilution ratio of the labeled porcine Seneca virus single domain antibody 2 is 1:20,000.

[0014] Preferably, the detection kit of the present invention includes positive and negative controls, sample diluent, PBST buffer, substrate solution, and stop solution.

[0015] In one aspect, the present invention further provides a detection method of the kit, comprising the following steps:

[0016] (1) Coating the capture antibody, wherein the capture antibody is a single domain antibody 1: dilute the capture antibody to 1 μg / ml with 0.05 M carbonate buffer solution (pH 9.6), add 100 μL / well to the ELISA plate, shake for 30 seconds, and place at 4°C for coating overnight;

[0017] (2) Antigen-antibody binding: The serum to be tested and the positive and negative controls were serially diluted to set up 4 wells for antigen control, 100 μl / well, porcine Seneca virus antigen was diluted to 1 μg / mL with sample diluent, 100 μl / well was added to the serum well, incubated at 4°C overnight; the positive control was SVA-positive serum diluted 1:8 times, and the negative control was SVA-negative serum;

[0018] (3) Blocking: Wash the ELISA plate coated in step (1) 3 to 5 times with PBST, spin dry, add PBST buffer containing 2% BSA to the ELISA plate at 200 μL / well, and incubate at 37°C for 2 h.

[0019] (4) Wash the blocked ELISA plate in step (3) with PBST 3-5 times, spin dry, add the antigen-antibody mixture in step (2) at 100 μL / well, and incubate at 37°C for 60 min;

[0020] (5) Add detection antibody, which is HRP-labeled single domain antibody 2, and dilute it with sample diluent 1:20,000 before use: wash the plate 3-5 times with PBST, spin dry, add detection antibody, 100 μL / well, and incubate at 37°C for 30 min;

[0021] (6) Color development: Wash the plate 3–5 times with PBST, spin dry, add 100 μL / well of TMB substrate solution to the ELISA plate, and incubate at 37°C for 15 min.

[0022] (7) Termination: Add 50 μL / well of the stop solution to the ELISA plate, mix well, detect on a microplate reader, and read the OD450nm value of the sample.

[0023] (8) Test validity standard: Each plate has 4 wells for virus antigen control. The OD450nm value of the virus antigen control should be above 1.0. The titer of the positive control antibody should be within 1:1024±1 titer, and the titer of the negative control antibody should be <1:8.

[0024] (9) Determination of serum antibody titer: For the viral antigen control wells, discard the highest and lowest OD450nm values, calculate the average OD450nm value of the remaining two wells, and divide it by 2, which is the 50% control value. This value is the critical value, indicating the control OD450nm value that blocks 50% of the reaction. Wells with OD450nm values of the tested serum greater than the critical value are negative wells, and wells with OD450nm values less than or equal to the critical value are positive wells. If the critical value is the same as the OD450nm value of the highest dilution multiple positive well, the highest dilution multiple of the tested serum positive well is used as the antibody titer of the serum; if the critical value is between the OD450nm values of two dilution wells, the antibody titer is the middle value of the antilogarithm of the dilution multiples of the adjacent positive and negative wells. If it is between 1:64 and 1:128, the antibody titer of the serum is determined to be 1:90.

[0025] In another aspect, the present invention also provides a use of the kit in detecting porcine Seneca virus antibodies.

[0026] This study used intact Seneca porcine virus particles purified by sucrose density gradient centrifugation as antigens, single-domain antibody 1 expressed in a eukaryotic expression system as the capture antibody, and single-domain antibody 2 as the detection antibody. Matrix titration and ROC curve drawing were used to determine the optimal reaction conditions and critical values, thereby establishing a standard ELISA detection kit. The kit parameters were evaluated using specificity tests, intra-batch and inter-batch repeatability tests, and VNT correlation tests. Clinical serum samples were tested using the assembled kit, with a specificity of 100% and a sensitivity of 98.85%. The Pearson coefficient in the VNT correlation test was 0.86, and the coefficient of variation for both intra-batch and inter-batch tests was less than 10%. This indicates that the kit of the present invention has good specificity, sensitivity, and repeatability, and is suitable for large-scale applications.

[0027] The present invention uses intact Seneca suis virus particles as diagnostic antigens to develop a neutralizing liquid-phase blocking ELISA kit for detecting neutralizing antibodies against Seneca suis. This kit offers advantages such as high efficiency, simplicity, sensitivity, and stability, and correlates well with traditional diagnostic methods such as virus neutralization tests. This kit can be used to assess Seneca neutralizing antibody levels in livestock herds and has promising application prospects.

[0028] It should be noted that, based on the technical solution of this invention, those skilled in the art can use other porcine Senecaviruses (such as the porcine Seneca Valley virus A / ZJ / 2015 strain deposited in Chinese invention patent CN 109679927 B, with a deposit number of CGMCC No. 15034, where porcine Seneca Valley virus is another translation of porcine Senecavirus) to prepare porcine Senecavirus antigens according to the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Particle size distribution of porcine Seneca virus antigen. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0031] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0032] Single-domain antibodies (SDAs) are a novel and unique class of antigen-binding fragments derived from naturally occurring heavy-chain antibodies in camel serum. Their advantageous properties, such as small size, high stability, strong antigen-binding capacity, and water solubility, have led to their widespread use in biotechnology research.

[0033] Example 1: Preparation of porcine Seneca virus antigen

[0034] 1. Preparation (Sucrose Density Gradient Centrifugation)

[0035] (1) IBRS-2 cells were inoculated into T175 cell flasks. When the cells grew to a full monolayer, the culture medium was discarded and 1 MOI of porcine Seneca virus solution (provided by Zhejiang Hongsheng Biotechnology Co., Ltd.) was inoculated. Maintenance medium was added and the cells were collected after 10-12 hours.

[0036] (2) Place the culture flask in a -70°C refrigerator, freeze and thaw repeatedly 2 to 3 times, and inactivate with 0.2% formaldehyde at 37°C for 24 h;

[0037] (3) The inactivated virus solution was centrifuged at 6000 rpm for 1 h to remove cell debris and concentrated to 80 mL using a membrane bag;

[0038] (4) The concentrated virus solution was divided into ultracentrifuge tubes, centrifuged at 35,000 rpm for 2.5 h, the supernatant was discarded, and the suspension was resuspended in 2 ml of PBS;

[0039] (5) Grind on ice for 2 h, centrifuge at 6000 rpm for 30 min, and discard the precipitate;

[0040] (6) Add trichloroethylene to the supernatant at a 1:1 ratio, pipette repeatedly to mix, and centrifuge at 6000 rpm for 30 min. The supernatant is subjected to a sucrose density gradient with a sucrose density of 15% to 45%, centrifuged at 36,000 rpm for 2.5 h, and aliquoted into 500 μl tubes. Store at -80°C until use.

[0041] 2. Detection

[0042] The prepared porcine Seneca virus antigen was subjected to TEM test. The results showed that ( Figure 1 ), the particle size of porcine Seneca virus antigen is about 20 to 30 nm, and has good uniformity.

[0043] The concentration of porcine Seneca virus antigen was detected using a Bradford protein concentration detection kit, and the results showed that the concentration of the antigen was 480 μg / mL.

[0044] Example 2: Preparation of porcine Seneca virus single domain antibodies

[0045] 1. Immunity

[0046] The porcine Seneca virus antigen prepared in the example was emulsified with an adjuvant (such as ISA 206, etc.) to prepare a vaccine.

[0047] After selecting the alpaca and ensuring its suitability, record the ear number and begin the immunization experiment. Each injection is given at two sites near the alpaca's neck lymph nodes, one on each side, with 0.4 mL of the mixed antigen injected at each site. Observe the alpaca for half an hour after immunization to ensure it is in good condition and has no symptoms. Vaccinations are performed every two weeks for a total of seven times.

[0048] Blood was collected for immune evaluation before the 4th, 5th and 6th immunizations. Blood was collected from the alpaca's neck vein, and 5 mL of blood was collected each time. The blood was centrifuged at 400 g for 30 minutes in a pre-cooled 25-degree centrifuge on the same day to separate and preserve the upper serum.

[0049] 2. Isolation of Lymphocytes

[0050] First add 3mL of cell separation solution to a 15mL centrifuge tube, and then slowly add 3mL of blood. Be careful and slow when adding blood to prevent the blood and separation solution from mixing. After that, precool the centrifuge to 25 degrees, centrifuge at 400g for 30 minutes, observe the blood separation in the centrifuge tube, and use a 200μl pipette to carefully draw out the cotton-like upper immune cells in the middle into a new 15ml centrifuge tube. The upper serum is stored in a new centrifuge tube at -80 degrees. Add 10mL of PBS buffer at room temperature to each tube, and centrifuge at 25 degrees and 400g for 20 minutes. Remove the supernatant, add 5mL of PBS buffer at room temperature to each tube, and centrifuge at 25 degrees and 400g for 20 minutes. Use a hemocytometer to count the number of cells. Remove the supernatant, and use RNAiso Plus to dissolve the separated lymphocytes according to the number of cells to obtain 10 7 / mL dissolution solution, stored at -80 degrees.

[0051] 3. RNA Extraction and cDNA Preparation

[0052] Total RNA was extracted from the isolated alpaca lymphocytes using an RNA extraction kit, and the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. The cDNA was quantitatively divided and stored at -80°C for future use.

[0053] 4. Establishment of Alpaca Antibody VHH Library, Construction and Screening of Phage Library, and Construction of Expression Vector

[0054] Using the cDNA prepared above as a template, a VHH library was established, which was then used as a basis for constructing a phage library. Suitable antibodies were screened from the library, and expression vectors were constructed based on the antibody sequences. Specific methods can be found in CN 112898421 A and CN 110256564 A.

[0055] Through the above screening, two good single-domain antibodies were screened, named single-domain antibody 1 and single-domain antibody 2, and their amino acid sequences are:

[0056] Single domain antibody 1 (SEQ ID NO. 1) is:

[0057] DVQLVESGGGSVQAGGSLRLSCAASGYTYSSNCMGWFRQAPGKEREGVAAIYTAGGNTYYADSVKGRFTISQDNAKNTFYLQMNSLKPEDTAMYYCAAEIDPIWSTFTCGDWEADFSYWGQGTQVTVSS.

[0058] Single domain antibody 2 (SEQ ID NO. 2) is:

[0059] DVQLVESGGAAAQAGGSLRLSCAASAYTFSSNCVGWFRQVPGKEREGVARYDGSGDPRYADSVKGRFTISTDNAKATLYLQMDSLKPEDTAMYYCAAGWALGWPATCDYSYWGQGTLVTVSS.

[0060] 5. Preparation of Single Domain Antibodies

[0061] (1) Sample preparation: Transfect the single domain antibody-expressing plasmid pcDNA3.1-SV1 (single domain antibody 1 expression vector) or pcDNA3.1-SV2 (single domain antibody expression vector 2) in a 125 mL shake flask. For 25 mL, the density is 1.0×10 6 25 μg pDNA was added to 1200 μL 293SFM medium at a concentration of 100 cells / mL. After mixing, 100 μg PEI was added and vortexed. After incubation for 5 minutes, the mixture was added dropwise to a shake flask. After culturing on a shaker for 5 days, the cell supernatant was harvested by centrifugation at 5000 rpm for 15 minutes. The supernatant was mixed with binding buffer in a 1:1 ratio.

[0062] (2) Equilibrate the Protein A affinity chromatography column: drain the 20% ethanol used to store the column, fill the column with deionized water, drain, and repeat washing three times to remove any remaining ethanol. Fill the column with elution buffer, drain, and wash once to remove any residual protein from the previous purification. Add 5 mL of binding buffer, drain, and repeat washing three times. Place at 4°C until ready for use.

[0063] (3) Binding of protein to Protein A affinity chromatography column: Add the sample to the column and allow it to bind at room temperature for 10 min;

[0064] (4) Elution of impurities: Place the column on ice and let it stand for 2 minutes. When stratification occurs, place the column on an iron rack, open the outlet, and rinse the column with binding buffer. The specific operation is as follows: cover the outlet, add 5 mL of binding buffer, let it stand on ice for 3-4 minutes, open the outlet, let it flow out, and repeat the washing three times until there is no protein in the binding solution.

[0065] (5) Elution of target protein: Elute the target antibody with 10 mL of 0.1 mol / L, pH 3.0 citrate buffer. Collect 1 mL of sample per tube and add 50 μL of 1 mol / L, pH 9.0 Tris-HCl buffer to each tube. Determine the protein content in each collection tube by SDS-PAGE and combine the protein tubes.

[0066] (6) The single-domain antibodies collected by PBS dialyzation were quantitatively divided and stored at -80°C for future use.

[0067] 6. Detection of Single Domain Antibodies

[0068] (1) SDS-PAGE assay: 10 μL of each of the purified single-domain antibodies 1 and 2 was mixed with 2 μL of 5×SDS loading buffer and subjected to SDS-PAGE assay. The results showed that the purity of both single-domain antibodies was above 90%.

[0069] (2) Protein concentration determination: The concentration of concentrated protein was detected using a Bradford protein quantification kit. The results showed that the concentrations of the two single-domain antibodies were 540 μg / mL and 480 μg / mL, respectively.

[0070] (3) Affinity detection of single domain antibodies and antigens (indirect ELISA)

[0071] 1) Coating antigen: The porcine Seneca virus antigen prepared in Example 1 was diluted to 1 μg / ml with carbonate buffer solution (0.05 M, pH = 9.6), added to the coating plate, 100 μl per well, and incubated at 4°C overnight;

[0072] 2) Washing: Wash 3-5 times with 300 μl PBST per well and pat dry;

[0073] 3) Blocking: Block with PBST buffer containing 2% skim milk powder for 2 h; 200 μl per well;

[0074] 4) Washing is the same as 2);

[0075] 5) Adding antibodies: The purified antibodies were first labeled with HRP (using a commercial kit), then diluted 5000-fold, 10000-fold, 20000-fold, and 30000-fold with PBST, and 100 μl was added to each well of the coated plate and incubated at 37°C for 1 h.

[0076] 6) Washing is the same as 2);

[0077] 7) Color development: Add 50 μl / well of TMB substrate solution to the ELISA plate and incubate at 37°C for 15 min.

[0078] 8) Termination: Add 50 μl / well of the stop solution (2 M sulfuric acid) to the ELISA plate, mix well, detect on a microplate reader, and read the OD450 nm value of the sample.

[0079] The results showed that when the dilution of the two single-domain antibodies was 1:20000, the OD450nm was greater than 1.1, and were 1.325 and 1.256 respectively, indicating that both single-domain antibodies had good affinity.

[0080] (4) Neutralizing activity of antibodies

[0081] 1) The two single-domain antibodies were serially diluted 2-fold in DMEM medium (containing 1% double antibody) in a 96-well microplate, starting from 1:4 and diluted in 8 steps. Each dilution was diluted to 4 wells, with 50 μL per well.

[0082] 2) Dilute porcine Seneca virus to 200 TCID using DMEM medium (containing 1% double antibody) 50 50 μL was added to each well of the dilution plate, and the virus titer was set as a control. The virus solution was diluted from 200 TCID 50 Serially dilute 6 times in 10-fold gradients and incubate in a 37°C 5% CO2 incubator for 1 h;

[0083] 3) After trypsinization, the cultured IBRS-2 cells were diluted with 2% serum-containing DMEM medium (containing 1% double-antibody) to prepare a cell suspension. 50 μL / well was added to a 96-well plate containing the antigen-antibody mixture described above. The plate was cultured in a 37°C 5% CO2 incubator for 48 h. The results were calculated according to the Reed-Muench method.

[0084] The results showed that both single-domain antibodies had neutralizing activity, and the neutralizing antibody titers were both 1:1024.

[0085] Example 3: Establishment of a neutralizing liquid-phase blocking ELISA method for porcine Seneca virus

[0086] 1. Determination of capture and detection antibodies

[0087] Single domain antibody 1 and single domain antibody 2 were respectively diluted to 1 μg / mL with coating solution, and 100 μL / well was added to the coated plate and incubated at 4°C overnight; after washing, the porcine Seneca virus antigen prepared in Example 1 was diluted to 5 μg / mL with PBST, 100 μL / well was added to the plate, and incubated at 37°C for 1 hour; after washing, the two HRP-labeled single domain antibodies were diluted 20,000 times and cross-added to the plate, 100 μL / well, and incubated at 37°C for 30 minutes; after washing, TMB was added at 100 μL / well and incubated at 37°C for 15 minutes; finally, the stop solution was added, and the absorbance at OD450 was measured using a microplate reader.

[0088] The results showed that when single-domain antibody 1 was coated, HRP-labeled single-domain antibody 2 had the highest OD450nm value of 2.65 when detected. However, when single-domain antibody 2 was coated, HRP-labeled single-domain antibody 1 had an OD450nm value of 0.56. Therefore, single-domain antibody 1 was selected as the coating antibody and HRP-labeled single-domain antibody 2 as the detection antibody.

[0089] 2. Determination of the optimal dilution of porcine Seneca virus antigen and serum

[0090] The cross-checkerboard titration method was used. The single-domain antibody 1 was diluted to 1μɡ / mL with coating buffer and added to the enzyme-labeled plate; the porcine Seneca virus antigen of known concentration was diluted to 1, 3, 5, and 7μɡ / mL with PBST, and 100μL was added to each well of the dilution plate. At the same time, the negative and positive reference sera were diluted in multiples from 1:2 to 1:1024, and added to each well from top to bottom. A 4-well antigen control was set up, with 100μL per well at 4°C overnight; the next day, the liquid in the enzyme-labeled plate was poured out, washed 3-4 times with washing solution, and patted dry. Then 200μL of PBST buffer containing 2% BSA was added to each well, blocked at 37°C for 120 minutes, and then washed. The antigen-serum mixed solution was added to the enzyme-labeled plate, 100μL per well, and incubated at 37°C for 60 minutes; the liquid in the enzyme-labeled plate was poured out, washed 3-4 times with washing solution, and patted dry. The HRP-labeled single-domain antibody 2 was washed with PBST Dilute 1:20,000, add 100 μL per well, incubate at 37°C for 30 minutes, then wash. After washing, add 100 μL of TMB to each well and incubate at 37°C for 15 minutes. After color development, add 50 μL of stop solution (2 M H2SO4) to each well and measure at 450 nm on a microplate reader. The optimal antigen and serum dilution is determined by the same antigen concentration and serum dilution as the known serum titer.

[0091] The results showed (Table 1) that, based on the serum titer, the porcine Seneca virus antigen concentration was 1 μg / mL and the serum titer was 1:45. Therefore, the optimal dilution concentration of porcine Seneca virus antigen was determined to be 1 μg / mL.

[0092] Table 1 Determination of the optimal concentration of porcine Seneca virus antigen and the optimal dilution of serum

[0093] Antigen concentration 1 μg / mL 3 μg / mL 5 μg / mL 7 μg / mL Antibody titer 1:45 1:25 1:25 1:16

[0094] 3. Capture Antibody and Detection Antibody Dilution

[0095] Dilute the capture antibody (single domain antibody 1) of known concentrations to 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL with coating buffer and add it to the ELISA plate. Coat 100 μL per well at 4°C overnight. Dilute the positive and negative reference serum to the optimal dilution and add 100 μL per well to the dilution plate. Dilute the antigen to the optimal concentration and add 100 μL per well to the serum. Mix by shaking at room temperature for 30 seconds and incubate at 4°C overnight. The next day, discard the liquid from the ELISA plate, wash 3-4 times with detergent, and pat dry. Add 200 μL of PBST buffer containing 2% BSA to each well and block at 37°C for 120 minutes, followed by washing. Add 100 μL of the antigen-serum mixture and antigen solution to each well of the ELISA plate, incubate at 37°C for 60 minutes, and then wash. Then, dilute the detection antibody (HRP-labeled single-domain antibody 2) serially from 1:5000 to 1:40,000 and add 100 μL to each well from top to bottom. Incubate at 37°C for 30 minutes, then wash. After washing, add 100 μL of TMB to each well. After developing at 37°C for 15 minutes, add 50 μL of stop solution (2 M H2SO4) to each well and measure at 450 nm on a microplate reader. The value with the largest positive / negative ratio (N / P) is used as the optimal coating antibody concentration and the optimal working concentration of the detection antibody.

[0096] The results showed that the N / P value (2.74) was maximized when the capture antibody concentration was 1 μg / mL and the detection antibody was diluted 1:20,000. Therefore, the coating concentration of the capture antibody (single domain antibody 1) was determined to be 1 μg / mL, and the dilution of the detection antibody (HRP-labeled single domain antibody 2) was determined to be 1:20,000.

[0097] 4. Determination of the optimal incubation time for antigen and serum, blocking solution, blocking time, enzyme-labeled antibody reaction time, and substrate TMB reaction time

[0098] Using the optimal method, we selected one condition at a time for testing and exploration, and determined the optimal detection method by comparing the N / P ratio. Testing determined that the optimal incubation time for antigen and serum was 60 minutes at 37°C; the blocking solution was PBST buffer containing 2% BSA, and the blocking time was 2 hours at 37°C; the reaction time for the enzyme-labeled antibody (HPR-labeled single-domain antibody 2) was 30 minutes at 37°C; and the reaction time for the substrate TMB was 15 minutes at 37°C.

[0099] 5. Establishment of ELISA method judgment criteria

[0100] The method established in this study was used to detect 120 non-immune negative sera and 85 vaccine-immune positive sera, and the specificity and sensitivity were calculated. The cutoff value was determined at the maximum specificity and sensitivity.

[0101] Sensitivity and specificity analysis: 205 serum samples with different antibody titers were tested using the established ELISA method, and their serum titers were calculated. The sensitivity of the method was calculated according to the following formula: Sensitivity = number of true positives / (number of true positives + number of false negatives)*100%

[0102] The established ELISA method was used to test 205 serum samples with different antibody titers, and their serum titers were calculated. The specificity of the method was calculated according to the following formula: Specificity = number of true negatives / (number of true negatives + number of false positives)*100%

[0103] The results showed that when the cutoff value was set at 1:45, the sensitivity and specificity values were optimal, reaching 100% specificity and 98.85% sensitivity. When the cutoff value was set at 1:45, the sensitivity and specificity values were maximized, and the consistency rate with the identified serum samples was the highest. Therefore, the judgment criteria were determined as an antibody titer of <1:45 for negative and an antibody titer of ≥1:45 for positive.

[0104] Example 4: Kit Performance Testing

[0105] 1. Specificity test

[0106] The established detection method was used to detect positive sera for various swine diseases. As shown in Table 2, the established ELISA method had no cross-reaction with porcine circovirus type 2, classical swine fever, blue ear disease, and foot-and-mouth disease, and only judged the positive serum of porcine Seneca as positive, indicating that the ELISA method has good specificity.

[0107] Table 2 Specificity test results

[0108]

[0109] 2. Correlation with virus neutralization experiments

[0110] The same 85 SVA-positive sera were tested using the ELISA method established in this study and the virus neutralization assay. Neutralization titers were calculated, and the Pearson coefficient was used to determine the correlation between ELISA titers and VNT titers (neutralizing antibody titers). The results showed a strong correlation between the ELISA method established in this study and VNT, as indicated by the Pearson coefficient of r = 0.85.

[0111] 3. Intra-batch and inter-batch replicate test results

[0112] 3.1 Intra-batch replicate testing

[0113] Three kits assembled with antigen coating from the same batch were selected, and 10 identical clinical serum samples were tested under the same conditions. The titer of each sample was calculated. As shown in Table 3, the intra-batch coefficient of variation was less than 10%.

[0114] Table 3 Intra-batch replicate test (OD450nm value)

[0115]

[0116]

[0117] 3.2 Inter-batch replication

[0118] Three kits assembled with antigen coating from different batches were selected, and 10 identical clinical serum samples were tested under the same conditions. The titer of each sample was calculated. As shown in Table 4, the intra-batch coefficient of variation was less than 10%.

[0119] Table 4 Inter-batch repetition test (OD450nm value)

[0120] Sample number 01 batch 02 batch 03 batch average value Standard deviation (SD) CV% #1 1.56 1.55 1.42 1.51 0.08 5.17％ #2 1.89 1.82 1.71 1.81 0.09 5.02％ #3 2.12 2.45 2.51 2.36 0.21 8.90％ #4 2.32 2.10 2.52 2.31 0.21 9.08％ #5 2.53 2.36 2.65 2.51 0.15 5.80％ #6 0.58 0.51 0.48 0.52 0.05 9.81％ #7 0.56 0.50 0.49 0.52 0.04 7.33％ #8 0.28 0.25 0.30 0.28 0.03 9.10％ #9 0.56 0.48 0.50 0.51 0.04 8.11％ #10 0.10 0.09 0.09 0.09 0.01 6.19％

[0121] The intra-assay and inter-assay coefficients of variation were both less than 10%, indicating that the kit assembled in this study had good reproducibility.

[0122] Example 3: Porcine Seneca virus neutralization liquid phase blocking ELISA kit operating steps

[0123] (1) Coating the capture antibody, wherein the capture antibody is a single domain antibody 1: dilute the capture antibody to 1 μg / ml with 0.05 M carbonate buffer solution (pH 9.6), add 100 μL / well to the ELISA plate, shake for 30 seconds, and place at 4°C for coating overnight;

[0124] (2) Antigen-antibody binding: Serum to be tested and positive and negative controls (positive control: SVA-positive serum diluted 1:8, negative control: SVA-negative serum) were serially diluted to set up 4 wells for antigen control, 100 μl / well, porcine Seneca virus antigen was diluted to 1 μg / mL with sample diluent (PBST buffer containing 1% BSA), 100 μl / well was added to the serum wells, and the cells were incubated at 4°C overnight;

[0125] (3) Blocking: Wash the ELISA plate coated in step (1) 3 to 5 times with PBST, spin dry, add PBST buffer containing 2% BSA to the ELISA plate at 200 μL / well, and incubate at 37°C for 2 h.

[0126] (4) Wash the blocked ELISA plate in step (3) with PBST 3-5 times, spin dry, add the antigen-antibody mixture in step (2) at 100 μL / well, and incubate at 37°C for 60 min;

[0127] (5) Add detection antibody, which is HRP-labeled single domain antibody 2, and dilute it with sample diluent 1:20,000 before use: wash the plate 3-5 times with PBST, spin dry, add detection antibody, 100 μL / well, and incubate at 37°C for 30 min;

[0128] (6) Color development: Wash the plate 3–5 times with PBST, spin dry, and add 100 μL / well of TMB substrate solution (commercial product, such as Beijing Solebow Technology Co., Ltd.) to the ELISA plate and incubate at 37°C for 15 min.

[0129] (7) Termination: Add 50 μL / well of the stop solution (2 M H2SO4) to the ELISA plate, mix well, detect on a microplate reader, and read the OD450nm value of the sample.

[0130] (8) Test validity standard: Each plate has 4 wells for virus antigen control. The OD450nm value of the virus antigen control should be above 1.0. The titer of the positive control antibody should be within 1:1024±1 titer, and the titer of the negative control antibody should be <1:8.

[0131] (9) Determination of serum antibody titer: For the viral antigen control wells, discard the highest and lowest OD450nm values, calculate the average OD450nm value of the remaining two wells, and divide it by 2, which is the 50% control value. This value is the critical value, indicating the control OD450nm value that blocks 50% of the reaction. Wells with OD450nm values of the tested serum greater than the critical value are negative wells, and wells with OD450nm values less than or equal to the critical value are positive wells. If the critical value is the same as the OD450nm value of the highest dilution multiple positive well, the highest dilution multiple of the tested serum positive well is used as the antibody titer of the serum; if the critical value is between the OD450nm values of two dilution wells, the antibody titer is the middle value of the antilogarithm of the dilution multiples of the adjacent positive and negative wells. If it is between 1:64 and 1:128, the antibody titer of the serum is determined to be 1:90.

[0132] (10) Result determination: an antibody titer of <1:45 indicates a negative antibody to porcine Seneca virus; an antibody titer of ≥1:45 indicates a positive antibody to porcine Seneca virus.

[0133] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A porcine Seneca virus neutralization liquid phase blocking ELISA kit, characterized in that: The kit comprises an effective amount of porcine Seneca virus antigen, an effective amount of porcine Seneca virus single domain antibody 1, an effective amount of porcine Seneca virus single domain antibody 2 and a matching detection reagent; The porcine Seneca virus antigen is prepared by purifying porcine Seneca virus particles using a sucrose density gradient centrifugation method. The particle size of the purified porcine Seneca virus particles is 20 to 30 nm. The working concentration of the porcine Seneca virus antigen is 1 μg / mL. The amino acid sequence of the heavy chain variable region of the porcine Seneca virus single domain antibody 1 is shown in SEQ ID NO. 1; the porcine Seneca virus single domain antibody 1 is a capture antibody, and its optimal coating concentration is 1 μg / mL; The amino acid sequence of the heavy chain variable region of the porcine Seneca virus single domain antibody 2 is shown in SEQ ID NO.2; the porcine Seneca virus single domain antibody 2 is a detection antibody, which is labeled with HRP before use. The optimal dilution ratio of the labeled porcine Seneca virus single domain antibody 2 is 1:20,000.

2. The kit according to claim 1, wherein The matching detection reagents include positive and negative controls, sample diluent, PBST buffer, substrate solution and stop solution.

3. A detection method of the kit according to claim 1, wherein the detection method is a detection method for non-disease diagnosis purposes, characterized in that: The method comprises the following steps: (1) Coating the capture antibody, wherein the capture antibody is a single domain antibody 1: dilute the capture antibody to 1 μg / ml with 0.05 M carbonate buffer solution (pH 9.6), add 100 μL / well to the ELISA plate, shake for 30 seconds, and place at 4°C for coating overnight; (2) Antigen-antibody binding: The serum to be tested and the positive and negative controls were serially diluted to set up 4 wells for antigen control, 100 μl / well, porcine Seneca virus antigen was diluted to 1 μg / mL with sample diluent, 100 μl / well was added to the serum well, incubated at 4°C overnight; the positive control was SVA-positive serum diluted 1:8 times, and the negative control was SVA-negative serum; (3) Blocking: Wash the ELISA plate coated in step (1) 3-5 times with PBST, spin dry, add 2% BSA-containing PBST buffer to the ELISA plate at 200 μL / well, and incubate at 37°C for 2 h; (4) Wash the blocked ELISA plate in step (3) with PBST 3-5 times, spin dry, add the antigen-antibody mixture in step (2) at 100 μL / well, and incubate at 37°C for 60 min; (5) Add detection antibody, which is HRP-labeled single domain antibody 2, and dilute it with sample diluent 1:20,000 before use: wash the plate 3-5 times with PBST, spin dry, add detection antibody, 100 μL / well, and incubate at 37°C for 30 min; (6) Color development: Wash the plate 3–5 times with PBST, spin dry, add 100 μL / well of TMB substrate solution to the ELISA plate, and incubate at 37°C for 15 min. (7) Stopping: Add 50 μL / well of the stop solution to the ELISA plate, mix well, and read the sample OD450nm value on a microplate reader; (8) Test validity standards: Each plate has 4 wells of virus antigen control, and the OD450nm value of the virus antigen control should be above 1.0; the titer of the positive control antibody should be within 1:1024±1 titer, and the titer of the negative control antibody should be <1:8; (9) Determination of serum antibody titer: For the viral antigen control wells, discard the highest and lowest OD450nm values, calculate the average OD450nm value of the remaining two wells, and divide it by 2 to obtain the 50% control value. This value is the critical value, which represents the control OD450nm value that blocks 50% of the reaction. Wells with OD450nm values of the tested serum greater than the critical value are negative wells, and wells with OD450nm values less than or equal to the critical value are positive wells. If the critical value is the same as the OD450nm value of the highest dilution multiple positive well, the highest dilution multiple of the tested serum positive well is used as the antibody titer of the serum. If the critical value is between the OD450nm values of two dilution multiples, the antibody titer is the midpoint of the antilogarithm of the dilution multiples of the adjacent positive and negative wells. (10) Result determination: an antibody titer of <1:45 indicates a negative antibody to porcine Seneca virus; an antibody titer of ≥1:45 indicates a positive antibody to porcine Seneca virus.

4. Use of the kit according to claim 1 in detecting antibodies against porcine Seneca virus, wherein the use is for non-disease diagnosis purposes.

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