Porcine Seneca complete virus particle sandwich ELISA kit and its application

Abstract

The present invention discloses a porcine Seneca virus complete virion sandwich ELISA kit, which comprises an effective amount of porcine Seneca virus complete virions, 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 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 present invention uses porcine Seneca virus complete virions as diagnostic antigens to establish a double-antibody sandwich ELISA kit for quantitatively detecting Seneca virus complete virions. The kit has the advantages of high efficiency, simplicity, sensitivity, stability, etc., can be used for vaccine quality control evaluation, and has good application prospects.

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 complete virus particle sandwich 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] Porcine Senecavirus has only one serotype and is the only species in the genus Senecavirus. Porcine Senecavirus disease is caused by Senecavirus type A (SVA) of the genus Senecavirus in the family Picornaviridae. Pigs are considered the natural reservoir for SVA. Clinical signs of SVA infection in pigs include fluid-filled and ruptured vesicles or bullae, ulcerative lesions of the coronary ligament, lameness, anorexia, lethargy, skin congestion, and fever. Pigs of all ages are susceptible, and affected adult pigs often experience mobility difficulties. SVA infection is associated with neonatal morbidity and mortality, primarily in piglets less than seven days of age, with morbidity rates exceeding 70% and mortality rates ranging from 15% to 30%. SVA infection has affected more than half of China's provinces, autonomous regions, and municipalities. A few Chinese strains have undergone genetic recombination, and asymptomatic SVA infection has also been reported in some areas, causing significant economic losses to the Chinese livestock industry.

[0004] Currently, several domestic institutions have received clinical trial approval for inactivated porcine Seneca virus vaccines, and some products have entered the registration phase. Vaccine quality is closely related to the antigen content within the vaccine, making it particularly important to assess vaccine quality. Therefore, developing simple, efficient, rapid, and accurate detection methods is crucial for Seneca virus prevention and control. Summary of the Invention

[0005] In view of the above existing problems and defects, the object of the present invention is to provide a porcine Seneca intact virus particle sandwich ELISA kit and its preparation method and application.

[0006] Therefore, on one hand, the present invention provides a porcine Seneca virus complete particle sandwich ELISA kit, which comprises an effective amount of porcine Seneca virus complete virus particles, 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 complete virus particles are the standard of the kit, and the concentrations of the standard are: 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.06125 μg / ml, 0.03125 μg / ml, and 0 μg / ml respectively.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] Preferably, the detection kit of the present invention includes a sample diluent, a PBST buffer, a substrate solution, and a stop solution.

[0012] In another aspect, the present invention further provides a detection method of the kit, comprising the following steps:

[0013] (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;

[0014] (2) Wash the ELISA plate coated in step (1) 3-5 times with PBST, spin dry, add 200 μL / well of PBST buffer containing 2% BSA to the ELISA plate, and incubate at 37°C for 2 h;

[0015] (3) adding samples and standards of different concentrations, wherein the standard is intact Seneca suis virus particles, and the concentrations thereof are 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.06125 μg / ml, 0.03125 μg / ml, and 0 μg / ml, respectively: washing the blocked ELISA plate in step (2) with PBST 3-5 times, drying, adding antigen, 100 μL / well, and incubating at 37°C for 60 min;

[0016] (4) 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;

[0017] (5) Color development: Add 100 μL / well of TMB substrate solution to the ELISA plate and incubate at 37°C for 15 min.

[0018] (6) Termination: Add 50 μL / well of the stop solution to the ELISA plate, mix well, and read the OD450 nm value of the sample on a microplate reader;

[0019] (7) Calculation of results: A standard curve is established based on the concentration of the standard sample and the corresponding OD450nm value. Its R2 should be ≥0.99. The OD450nm value of the sample to be tested is substituted into the standard curve to calculate the concentration of the sample to be tested. If the concentration of the sample to be tested is high, it can be diluted before testing.

[0020] In another aspect, the present invention also provides a use of the kit in the quantitative detection of intact virions of Seneca porcine.

[0021] In another aspect, the present invention also provides a use of the porcine Seneca virus single-domain antibody 2 in preparing a porcine Seneca virus complete virus particle detection reagent.

[0022] In this study, a single-domain antibody that specifically recognizes and binds to SVA complete virions was used to establish a SVA complete virion-specific quantitative ELISA to detect the antigen content in the vaccine.

[0023] To establish a quantitative ELISA assay for intact SVA virions, this study used matrix titration and sensitivity curve analysis to determine optimal reaction conditions and critical values. Specificity, sensitivity, and repeatability tests were also used to evaluate the kit parameters. The assembled kit was used to test samples with different disease antigens. The developed ELISA showed no cross-reactivity with foot-and-mouth disease virus types O and A, porcine circovirus type 2, porcine parvovirus, swine fever, encephalomyocarditis virus, and canine parvovirus. It was positive only for porcine Seneca, demonstrating the ELISA's excellent specificity. The sensitivity curve showed that the lowest protein dilution was 3.9 ng / ml when diluted 256-fold, and the coefficient of variation in repeatability tests was less than 10%.

[0024] In this study, a double-antibody sandwich ELISA kit for the quantitative detection of intact Seneca swine virus particles was established using intact Seneca swine virus particles as diagnostic antigens. This kit has the advantages of high efficiency, simplicity, sensitivity, and stability. It can be used for vaccine quality control evaluation and has good application prospects.

[0025] 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.

[0026] It should be noted that, based on the technical solution of this invention, those skilled in the art can use porcine Seneca virus particles prepared by other methods (such as porcine Seneca virus particles prepared according to the preparation method of porcine Seneca virus particles disclosed in Chinese invention patent CN 110279855 B). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Particle size distribution of porcine Seneca virus antigen. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] Example 1: Preparation of porcine Seneca virus antigen

[0031] 1. Preparation (Sucrose Density Gradient Centrifugation)

[0032] (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.

[0033] (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;

[0034] (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;

[0035] (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;

[0036] (5) Grind on ice for 2 h, centrifuge at 6000 rpm for 30 min, and discard the precipitate;

[0037] (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.

[0038] 2. Detection

[0039] 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.

[0040] 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.

[0041] Example 2: Preparation of porcine Seneca virus single domain antibodies

[0042] 1. Immunity

[0043] The porcine Seneca virus antigen prepared in the example was emulsified with an adjuvant (such as ISA 206, etc.) to prepare a vaccine.

[0044] 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.

[0045] 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.

[0046] 2. Isolation of Lymphocytes

[0047] 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.

[0048] 3. RNA Extraction and cDNA Preparation

[0049] 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.

[0050] 4. Establishment of Alpaca Antibody VHH Library, Construction and Screening of Phage Library, and Construction of Expression Vector

[0051] 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.

[0052] 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:

[0053] Single domain antibody 1 (SEQ ID NO. 1) is:

[0054] DVQLVESGGGSVQAGGSLRLSCAASGYTYSSNCMGWFRQAPGKEREGVAAIYTAGGNTYYADSVKGRFTISQDNAKNTFYLQMNSLKPEDTAMYYCAAEIDPIWSTFTCGDWEADFSYWGQGTQVTVSS.

[0055] Single domain antibody 2 (SEQ ID NO. 2) is:

[0056] DVQLVESGGAAAQAGGSLRLSCAASAYTFSSNCVGWFRQVPGKEREGVARYDGSGDPRYADSVKGRFTISTDNAKATLYLQMDSLKPEDTAMYYCAAGWALGWPATCDYSYWGQGTLVTVSS.

[0057] 5. Preparation of Single Domain Antibodies

[0058] (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.

[0059] (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.

[0060] (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;

[0061] (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.

[0062] (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.

[0063] (6) The single-domain antibodies collected by PBS dialyzation were quantitatively divided and stored at -80°C for future use.

[0064] 6. Detection of Single Domain Antibodies

[0065] (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%.

[0066] (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.

[0067] (3) Affinity detection of single domain antibodies and antigens (indirect ELISA)

[0068] 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;

[0069] 2) Washing: Wash 3-5 times with 300 μl PBST per well and pat dry;

[0070] 3) Blocking: Block with PBST buffer containing 2% skim milk powder for 2 h; 200 μl per well;

[0071] 4) Washing is the same as 2);

[0072] 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.

[0073] 6) Washing is the same as 2);

[0074] 7) Color development: Add 50 μl / well of TMB substrate solution to the ELISA plate and incubate at 37°C for 15 min.

[0075] 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.

[0076] 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.

[0077] (4) Neutralizing activity of antibodies

[0078] 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.

[0079] 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;

[0080] 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.

[0081] The results showed that both single-domain antibodies had neutralizing activity, and the neutralizing antibody titers were both 1:1024.

[0082] Example 3: Preparation of porcine Seneca virus complete particles (SVA VLPs)

[0083] Preparation and identification of 1SVA VLPs

[0084] 1.1 Preparation of target protein

[0085] The structural proteins (VP0, VP1, and VP3) of porcine Senecavirus were expressed using a prokaryotic expression system. For specific methods, please refer to "Zhang Yongning, Zhang Zhou, Zhu Mingxin, et al. Prokaryotic expression of Senecavirus VP2 protein type A and preparation of its polyclonal antibody [J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2019.", "Mo Yaxia, Song Pin, Mu Suyu, et al. Prokaryotic expression of Senecavirus capsid protein type A and assembly of its pentamer [J]. Chinese Journal of Veterinary Science, 2019, 49(8):8.". A brief description is as follows:

[0086] Prepare the required amount of LB according to the test requirements, sterilize under high pressure and cool to room temperature. In the clean bench, add the positive expression bacteria E. coli BL21 containing the recombinant plasmid of SVA structural protein (VP0, VP1, VP3) to LB at a volume ratio of 1:100, and then add ampicillin (50 mg / mL) and kanamycin (10 mg / mL) at a volume ratio of 1:1000. Incubate on a shaker at 37°C and 220 r / min for 3.5 to 4 hours. Until the bacteria reach the logarithmic growth phase, that is, the bacterial solution OD 600 The cell culture medium was incubated at a pH of 0.6-0.8, and 0.5 mM / mL IPTG was added for induction. The cells were cultured on a shaker at 16°C and 200 rpm for 16-18 hours to induce expression of the target protein. The cells were harvested by centrifugation at 4°C and 4500 rpm for 30 minutes. The pellet was resuspended in Buffer A on ice water and sonicated for 20-25 minutes using an ultrasonic cell disruptor. The supernatant was collected by centrifugation at 4°C and 11000 rpm for 30 minutes. The proteins were purified using a nickel column.

[0087] 1.2 VLP Assembly

[0088] Add SUMO protease to the sample solution obtained above at a volume ratio of 1:50, mix thoroughly, transfer to a dialysis bag, and place in a beaker containing the assembly solution. Place the beaker on a magnetic stirrer and stir slowly at 4°C overnight. The next day, pour the liquid from the dialysis bag into a pre-equilibrated Ni2+ affinity chromatography column to separate the cleaved His-SUMO tag from the assembled VLPs. Add glycerol to the VLPs, mix thoroughly, aliquot in fixed quantities, and store at -80°C.

[0089] Identification of 2SVA VLPs

[0090] The expression of the three SUMO-tagged capsid proteins was confirmed by SDS-PAGE and TEM experiments. The results showed that the band sizes of the three SUMO-tagged capsid proteins were consistent with the theoretical values. SVA VLPs highly bound to SVA immune serum, indicating that SVA VLPs have good reactivity and can be used as diagnostic antigens in ELISA methods for detecting SVA antibodies.

[0091] Example 4: Establishment of a porcine Seneca complete virus particle sandwich ELISA kit

[0092] 1. Determination of capture and detection antibodies

[0093] 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.

[0094] 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.

[0095] 2. Determination of the Optimal Dilution of Detection Antibodies

[0096] Coat the capture antibody at the optimal concentration and dilute the reference serum at the optimal dilution. Dilute the detection antibody (HRP-labeled single-domain antibody) at 1:10,000, 1:20,000, and 1:30,000. Determine the optimal dilution based on the OD450nm value, assuming all other conditions remain the same.

[0097] The results showed (Table 1) that the OD450 was closest to 1.5 at a dilution of 20,000. Therefore, the optimal dilution of the detection antibody was determined to be 1:20,000.

[0098] Table 1 Determination of the optimal dilution of detection antibodies

[0099] Optimal dilution 1:10000 1:20000 1:30000 OD450 value 2.56 1.45 0.87

[0100] 3. Determination of the optimal incubation time for antigens

[0101] The antigen was incubated with the optimal coating amount at 37°C for 30 min, 60 min, and 90 min, respectively. Under the same conditions, the optimal incubation time of the antigen was determined according to the P / N value.

[0102] The results showed (Table 2) that the P / N value was the highest when incubated at 37°C for 60 min. Therefore, the optimal incubation time for the antigen was 37°C for 60 min.

[0103] Table 2 Determination of optimal incubation time for antigens

[0104] 37℃ incubation time 30min 60min 90 minutes P / N value 28.6 30.6 29.5

[0105] 4. Determination of blocking solution

[0106] The coated ELISA plates were blocked with 1% BSA, 2% BSA, 5% BSA and unblocked plates, and the control was unblocked. Under the same conditions, the best blocking agent was determined according to the P / N value.

[0107] The results (Table 3) showed that the P / N value was the highest when the blocking solution was 2% BSA. Therefore, the optimal blocking solution was determined to be 2% BSA.

[0108] Table 3 Determination of the optimal blocking solution

[0109]

[0110]

[0111] 5. Determination of the blocking effect time

[0112] The coated ELISA plate was blocked with the optimal blocking agent at 37°C for 60 min, 90 min, and 120 min, respectively. Under the same conditions, the optimal blocking time was determined based on the P / N value.

[0113] The results (Table 4) show that the P / N value is the highest when the blocking solution is applied for 120 minutes. Therefore, the optimal blocking solution application time is 120 minutes.

[0114] Table 4 Determination of the optimal time for blocking solution

[0115] Different action time 60min 90 minutes 120 minutes P / N value 29.2 28.9 30.5

[0116] 6. Determination of Antibody Action Time

[0117] Dilute the detection antibody at the optimal dilution ratio, add the same volume to the ELISA plate, mix thoroughly, and incubate at 37°C for 30, 60, and 90 minutes, respectively. While other conditions remain unchanged, test various samples and determine the enzyme-labeled antibody incubation time based on the P / N value.

[0118] The results (Table 5) show that the P / N value is greater when the exposure time is 30 minutes. Therefore, the optimal exposure time is 30 minutes.

[0119] Table 5 Determination of the optimal time for antibody action

[0120] Different action time 30min 60min 90 minutes P / N value 27.6 21.5 18.6

[0121] 7. Determination of the TMB substrate action time

[0122] The substrate solution was added to the reaction system under the same other conditions, and the color was developed at 37°C in the dark for 10 min, 15 min, and 20 min, respectively. The optimal color development was determined according to the P / N value.

[0123] The results (Table 6) show that, according to the P / N value, when the substrate action time is 15 minutes, the P / N value is larger and the error is smaller. Therefore, the optimal substrate action time is 15 minutes.

[0124] Table 6 Determination of the optimal time for substrate action

[0125] Different action time 10min 15min 20min P / N value 25.6 28.2 23.8

[0126] Example 5: Performance evaluation of the porcine Seneca complete virus particle sandwich ELISA kit

[0127] 1. Specificity Analysis

[0128] The established detection method was used to detect positive sera for various swine diseases. As shown in Table 7, the established ELISA method had no cross-reaction with foot-and-mouth disease type O and A, porcine circovirus type 2, porcine parvovirus, classical swine fever, encephalomyocarditis virus, and canine parvovirus, and was only positive for porcine Seneca, indicating that the ELISA method has good specificity.

[0129] Table 7 Specificity analysis results

[0130]

[0131] 2. Sensitivity Analysis

[0132] The porcine Seneca virus antigen prepared in Example 1 was diluted to 1 μg / ml, and the dilution ratio was 2-fold starting from 1:2. The logarithm of the protein concentration was used as the abscissa and the absorbance was used as the ordinate to obtain the linear regression equation y=1.02x+2.11, R2 =0.993. The curve shows that the protein can still be detected when diluted to 256 times. Therefore, the minimum detectable amount of the kit is 3.9 ng / mL.

[0133] 3. Repeatability Analysis

[0134] 3.1 Intra-batch repeatability test Three kits assembled with antigen coating from the same batch were selected, and five identical SVA VLPs were tested under the same conditions. The concentration of each sample was calculated. Table 8 shows that the intra-batch coefficient of variation was less than 10%.

[0135] Table 8 Intra-batch repeated tests

[0136] sample Repeat 1 Repeat 2 Repeat 3 average value Standard deviation (SD) CV #1 57.12 59.62 61.25 59.33 2.08 3.51％ #2 118.56 121.21 119.43 119.73 1.35 1.13％ #3 256.29 249.95 261.56 255.93 5.81 2.27％ #4 303.11 306.16 296.92 302.06 4.71 1.56％ #5 315.25 309.65 311.65 312.18 2.84 0.91％

[0137] 3.2 Inter-batch repeatability test Three kits assembled with different batches of antigens were selected, and five identical SVA VLPs were tested under the same conditions. The concentration of each sample was calculated. Table 9 shows that the inter-batch coefficient of variation was less than 10%.

[0138] Table 9 Inter-batch repeat test

[0139] sample 01 batch 02 batch 03 batch average value Standard deviation (SD) CV #1 57.12 62.65 63.98 61.25 3.64 5.94％ #2 118.56 122.54 128.65 123.25 5.08 4.12％ #3 256.29 249.98 265.54 257.27 7.83 3.04％ #4 303.11 300.99 299.14 301.08 1.99 0.66％ #5 315.25 320.15 327.65 321.02 6.25 1.95％

[0140] 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.

[0141] Example 5: Porcine Seneca Virus Sandwich ELISA Kit Kit Operation Steps

[0142] (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;

[0143] (2) Wash the ELISA plate coated in step (1) 3-5 times with PBST, spin dry, add 200 μL / well of PBST buffer containing 2% BSA to the ELISA plate, and incubate at 37°C for 2 h;

[0144] (3) Adding samples and standards of different concentrations (SVA VLPs prepared in Example 3, with concentrations of 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.06125 μg / ml, 0.03125 μg / ml, and 0 μg / ml, respectively): Wash the blocked ELISA plate in step (2) 3-5 times with PBST, spin dry, add antigen, 100 μL / well, and incubate at 37°C for 60 min;

[0145] (4) 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;

[0146] (5) Color development: Add 100 μL / well of TMB substrate solution to the ELISA plate and incubate at 37°C for 15 min.

[0147] (6) Stopping: Add 50 μL / well of stop solution (2 M sulfuric acid) to the ELISA plate, mix well, and read the sample OD450nm value on a microplate reader;

[0148] (7) Calculation of results: A standard curve was established based on the concentration of the standard and the corresponding OD450nm value. 2 It should be ≥0.99. Substitute the OD450nm value of the sample to be tested into the standard curve to calculate the concentration of the sample to be tested. If the concentration of the sample to be tested is high, it can be diluted before testing.

[0149] It should be noted that when diluting samples with higher concentrations, a sample diluent is required, which is PBST buffer containing 1% BSA.

[0150] 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 complete particle sandwich ELISA kit, characterized in that: The kit includes an effective amount of porcine Seneca virus complete virus particles, 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 complete virus particles are the standard of the kit, and the concentrations of the standard are: 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.06125 μg / ml, 0.03125 μg / ml, and 0 μg / ml respectively; 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.

2. The kit according to claim 1, wherein The porcine Seneca virus single-domain antibody 1 is a capture antibody, and its optimal coating concentration is 1 μg / mL.

3. The kit according to claim 1, wherein 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.

4. The kit according to claim 1, wherein The matching detection kit includes sample diluent, PBST buffer, substrate solution and stop solution.

Images