A liquid-phase blocking elisa assay kit for sva neutralizing antibodies

Abstract

The application belongs to the technical field of biotechnology and relates to the field of veterinary diagnosis, and particularly discloses a liquid-phase blocking ELISA kit for porcine Senecavirus neutralizing antibody, which contains porcine Senecavirus single-domain antibody and matched detection reagents, and the porcine Senecavirus single-domain antibody can also be an antigen-binding fragment of the single-domain antibody, the single-domain antibody is selected from one or more groups in 52 groups, the heavy chain variable region of each group of single-domain antibodies or antigen-binding fragments contains CDR-1, CDR-2 and CDR-3, the amino acid sequences of the heavy chain variable region CDR-1 (VH-CDR-1) of the 52 groups of single-domain antibodies or antigen-binding fragments are respectively shown in SEQ ID NO. 1-SEQ ID NO. 52, the amino acid sequences of the heavy chain variable region CDR-2 (VH-CDR-2) are respectively shown in SEQ ID NO. 53-SEQ ID NO. 104, and the amino acid sequences of the heavy chain variable region CDR-3 (VH-CDR-3) are respectively shown in SEQ ID NO. 105-SEQ ID NO. 156. The kit can quickly detect the SVA neutralizing antibody level in serum, has high sensitivity, good specificity and stable results, and can be better applied to SVA vaccine efficacy evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically the field of veterinary diagnostic technology, and particularly relates to the preparation method and application of an SVA neutralizing antibody liquid-phase blocking ELISA detection kit. Background Technology

[0002] Seneca virus disease (SVA) is an infectious disease in pigs caused by SVA, characterized by vesicular lesions on the mouth and hooves. In 2014, SVA outbreaks occurred in the United States and Brazil. In 2015, SVA was first reported in Guangdong Province, my country, and has since spread to many other regions, with current reports of SVA epidemics in 15 provinces and municipalities. Genetic evolution and recombination analysis of prevalent SVA strains in Chinese pig populations revealed complex and diverse genetic evolution, including the emergence of new recombinant strains. These findings suggest the need to further strengthen the monitoring and control technologies for SVA in my country.

[0003] Senecavirus (SVA) is a member of the genus Senecavirus in the family Picornaviridae. It is a non-enveloped, single-stranded, positive-sense RNA virus with an icosahedral structure. Its genome is 7300 nt in length, comprising a 5' untranslated region (5'-UTR) of 666 nucleotides, a 3' untranslated region (3'-UTR) of 71 nucleotides, and a unique open reading frame (ORF) between the two untranslated regions. The SVA ORF exhibits the typical L-4-3-4 structure of a pituitary genome, encoding a polyprotein of 2181 amino acids. The L region encodes the leader protein Lpro, the P1 coding region encodes four structural proteins VP1, VP2, VP3, and VP4, the P2 coding region encodes three non-structural proteins 2A, 2B, and 2C, and the P3 coding region encodes four non-structural proteins 3A, 3B, 3C, and 3D. Recent studies have reported on the immunogenicity of structural proteins such as VP2 in Seneca virus type A, but the immunogenicity of non-structural proteins has not been reported.

[0004] Currently, there are no practical applications of SVA vaccines. A recent study evaluated the immunogenicity of inactivated, attenuated, and virus-like particle (VLP) SVA candidate vaccines and demonstrated protective efficacy against homologous viral challenge. Similar to other piconera virus vaccines, such as foot-and-mouth disease virus, nucleic acids (NAs) are detected via neutralization assays, which are crucial data points. Standard techniques for detecting NAs include plaque reduction neutralization assays, microneutralization assays, and fluorescent antibody-virus neutralization assays (VNTs). However, these methods are laborious and time-consuming, requiring manual precision and the use of clean-grade intercellular materials. Furthermore, they are unsuitable for field monitoring of NAs after SVA vaccination. In contrast, ELISA is cost-effective, less time-consuming than standard neutralization assays, and overcomes the aforementioned drawbacks. However, current SVA-ELISA kits appear unsuitable for assessing the protective efficacy of vaccination against SVA infection or vaccination itself, as the overall antibody level does not directly reflect NA titers. Moreover, the production of NAs is crucial for a protective response in the body. Therefore, understanding the correlation between NAs and protective responses is beneficial for advancing vaccine development and reducing animal use.

[0005] Inactivated SVA or VLP vaccines have been shown to produce high titers of NA, thereby generating a protective response that may be attributed to their conformational epitopes. However, the process of developing NA quantification methods using ELISA requires stable antibody molecules with strong affinity, high neutralizing activity, and excellent specificity. Therefore, selecting intact viral particles or VLPs as immunogens is an ideal approach. Currently, two main types of antibodies are used. The first type includes conventional antibodies composed of heavy and light chains. Based on monoclonal antibody screening, ELISA methods for detecting serum NAs of FMDV (types A and O), rabies, Severe Acute Respiratory Syndrome Coronavirus 2, Classical Swine Fever, and SVA have been established. The other type is camel-derived heavy chain antibodies. In 1993, another antibody in camel blood, besides conventional IgG antibodies, was first reported. Its receptor-binding single-domain antibody fragment (VHH) is the smallest available intact antigen-binding fragment with a molecular weight of approximately 15 kDa. VHH exhibits better stability and solubility than conventional antibodies and is readily recombinant and expressed in E. coli. Therefore, VHH has been used to combat various viruses, such as FMDV types A and O, rabies, Severe Acute Respiratory Syndrome Coronavirus 2, PEDV, and other pathogens. VHH has also been used to detect antibodies, antigens, and for treatment. However, there are currently no reports on the use of VHH in SVA. Summary of the Invention:

[0006] This invention obtains single-domain antibodies against SVA-VHH through screening and develops a liquid-phase blocking (LPB)-ELISA technique using these single-domain antibodies to detect neutralizing antibodies (NAs) against Seceavirus A (SVA). This invention is based on this technique.

[0007] In a first aspect, the present invention provides a liquid-phase blocking ELISA kit for neutralizing porcine Seneca virus, the kit containing an effective amount of porcine Seneca virus single-domain antibody and matching detection reagents.

[0008] Furthermore, the porcine Seneca virus single-domain antibody may also be an antigen-binding fragment of a single-domain antibody.

[0009] Furthermore, the single-domain antibody is selected from one or more of the 52 groups, and the heavy chain variable region of each group of single-domain antibody or antigen-binding fragment includes CDR-1, CDR-2 and CDR-3. The amino acid sequences of the heavy chain variable region CDR-1 (VH-CDR-1) of the 52 groups of single-domain antibody or antigen-binding fragment are shown in SEQ ID NO.1 to SEQ ID NO.52, the amino acid sequences of the heavy chain variable region CDR-2 (VH-CDR-2) are shown in SEQ ID NO.53 to SEQ ID NO.104, and the amino acid sequences of the heavy chain variable region CDR-3 (VH-CDR-3) are shown in SEQ ID NO.105 to SEQ ID NO.156.

[0010] Furthermore, the heavy chain constant region of the single-domain antibody or its antigen-binding fragment is selected from porcine IgG, human IgG, and / or chicken IgY.

[0011] Furthermore, the heavy chain constant region IgG is selected from one or more of IgG1, IgG2a, IgG2b and IgG4.

[0012] Furthermore, the amino acid sequence of the heavy chain variable region shown in SEQ ID NO.1 to SEQ ID NO.52, SEQ ID NO.53 to SEQ ID NO.104, or SEQ ID NO.105 to SEQ ID NO.156 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or a variant of no more than 8 amino acids.

[0013] Furthermore, the porcine Seneca virus neutralizing antibody liquid phase blocking ELISA kit also includes porcine Seneca virus antigen.

[0014] Furthermore, the porcine Seneca virus antigen is a mixture of antigen and serum.

[0015] In one embodiment, the porcine Seneca virus single-domain antibody includes a detection antibody and a capture antibody, wherein the detection antibody is Fc-VHH-27-HRP and the capture antibody is Fc-VHH-27.

[0016] Furthermore, the porcine Seneca virus single-domain antibody heavy chain variable region includes CDR-1, CDR-2, and CDR-3. The amino acid composition of the heavy chain variable region CDR-1 is SEQ ID NO.27, the amino acid composition of the heavy chain variable region CDR-2 is SEQ ID NO.79, and the amino acid composition of the heavy chain variable region CDR-3 is SEQ ID NO.131.

[0017] Secondly, the present invention provides the application of a single-domain antibody against porcine Seneca virus in the preparation of an antibody liquid-phase blocking ELISA reagent or kit.

[0018] Furthermore, the detection reagent or kit also includes reagents for detecting the single-domain antibody.

[0019] Furthermore, the porcine Seneca virus single-domain antibody may also be an antigen-binding fragment of a single-domain antibody.

[0020] Furthermore, the single-domain antibody is selected from one or more of the 52 groups, and the heavy chain variable region of each group of single-domain antibody or antigen-binding fragment includes CDR-1, CDR-2 and CDR-3. The amino acid sequences of the heavy chain variable region CDR-1 (VH-CDR-1) of the 52 groups of single-domain antibodies are shown in SEQ ID NO.1 to SEQ ID NO.52, the amino acid sequences of the heavy chain variable region CDR-2 (VH-CDR-2) are shown in SEQ ID NO.53 to SEQ ID NO.104, and the amino acid sequences of the heavy chain variable region CDR-3 (VH-CDR-3) are shown in SEQ ID NO.105 to SEQ ID NO.156.

[0021] Furthermore, the application also includes measuring antibodies in the serum of test subjects for the diagnosis of viral diseases.

[0022] Furthermore, the application is to determine the antibody titer of immune serum or to evaluate the effectiveness of vaccination.

[0023] Furthermore, the reagent is a test kit.

[0024] Beneficial effects:

[0025] This invention utilizes SVA-specific neutralizing single-domain antibodies to establish a kit for detecting SVA neutralizing antibodies, enabling rapid detection of SVA neutralizing antibody levels in serum. This invention exhibits high sensitivity, good specificity, and stable results, making it better suited for evaluating the efficacy of SVA vaccines. Attached Figure Description

[0026] Figure 1 Flowchart of phage display technology for screening single-domain antibodies against SVA (A: immunization of camels with SVA antigen using Freund's adjuvant; B: screening of single-domain antibodies that are reactive to SVA antigen using phage ELISA).

[0027] Figure 2 ELISA is used to detect the affinity and specificity of expressed antibodies.

[0028] Figure 3 Neutralization assays are used to determine the neutralizing activity of antibodies.

[0029] Figure 4 The titers of negative and positive sera are dispersed.

[0030] Figure 5 Determination of the sensitivity and specificity values ​​of NA-ELISA.

[0031] Figure 6 Correlation between VNT titer and NA-ELISA titer.

[0032] Figure 7 Correlation between LBP-ELISA, VNT, and detection protection rate. Detailed Implementation

[0033] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0034] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0035] Example

[0036] 1. Cells, viruses, and animals

[0037] IBRS-2 cells were cultured at 37°C in 5% CO2 DMEM medium (Sigma-Aldrich, MA, US), supplemented with 10% fetal bovine serum (FBS; Gibco, CA, USA), 3 mM glutamine, penicillin (100 IU / mL), streptomycin (50 μg / mL), and gentamicin (25 μg / mL, Gibco). The SVA strain (GenBank: MN2922286) was isolated from Hubei Province. Two adult camels were from a livestock farm in Jinchang City, Gansu Province, China.

[0038] 2. Serum sample

[0039] A total of 87 serum samples from clinically healthy pigs were collected, and the SVA antibody was negative by VNT and ELISA tests.

[0040] A total of 105 serum samples were collected from experimentally infected pigs or vaccine-treated pigs. Serum samples from pigs infected with PRRSV, PCV2, CSFV, and FMDV were tested, and SVA antibodies were positive. Of these, 68 positive serum samples were used for Pearson coefficient testing to calculate the correlation between VNT and LBP-ELISA. Furthermore, among the 68 positive serum samples, 36 were from 0-7 weeks (8 weeks) post-vaccination (pig) and 1-4 weeks (pig from our laboratory). Of these, 12 were from SVA infection 1-4 weeks post-infection (pigs 1, 2, and 3); 12 were from pigs 4, 5, and 6; and 12 were from infected pigs (pigs 7, 8, and 9).

[0041] Example 1: Preparation and Screening of SVA Single-Domain Antibodies

[0042] 1. SVA preparation and camel immunization

[0043] 1.1 Select two healthy, age-appropriate camels and number them S-1# and S-2# respectively;

[0044] 1.2 Before immunization, 5 ml of peripheral blood was collected, serum was separated, and used as a negative control when detecting titer;

[0045] 1.3 At 400ug SVA antigen (see...) Figure 1 An equal volume of Freund's adjuvant was added to the mixture and thoroughly emulsified. Two camels were then immunized via subcutaneous injection at multiple points in the neck. The initial immunization used Freund's complete adjuvant, and subsequent immunizations used Freund's incomplete adjuvant. The immunization interval was 14 days.

[0046] 1.4 Two weeks after the fourth immunization, 5 ml of whole blood was collected, serum was separated, and serum titer was measured (see...). Figure 1 ).

[0047] 1.5 Take 200ml of camel peripheral blood, separate PBMCs using the Ficoll method, and use them to construct a camel VHH antibody library. The library is constructed by mixing two camels.

[0048] Purified SVA antigen was emulsified with complete and incomplete Freund's adjuvant and injected subcutaneously and at multiple sites (more than 3 sites) in the neck at weeks 0, 2, 4, and 6 (see [link to relevant documentation]). Figure 1 A).

[0049] 2. Construction of a camel nanobody phage library

[0050] 2.1 PBMC cells were lysed using TriZol lysis buffer to isolate total RNA. Then, reverse transcription PCR was performed using oligo-dT primers to prepare cDNA for subsequent amplification of antibody genes.

[0051] 2.2 Using cDNA as a template, the antibody gene was amplified separately. The first round of PCR amplification was performed using primers with characteristic sequences upstream of the antibody heavy chain variable region (GTC.CTG.GCT.GCT.CTT.CTA.CAA.GG) and downstream of CH2 (GGT.ACG.TGC.TGT.TGA.ACT.GTT.CC). A band of approximately 600 bp was obtained by electrophoresis and used as a template for the second round of PCR. The second round of PCR amplification used primers with characteristic sequences of VHH.

[0052] 2.3 Enzyme restriction sites were added to both ends of the primers used in the second round of PCR amplification to facilitate subsequent ligation into the vector (F: AACATGCCATGACTCGCGGCTCAACCGG CCATGGCTGA K GT B CAG CTGCAG GC GTCTGGRGGAGG; R: GTTATTATTATTCAGATTATTAGT GCGGCCGCTGGAGACGGTGACC W GGGTCC). After obtaining the VHH gene by PCR, it was digested with restriction endonucleases, and the digested fragments were ligated into pre-digested phage display vectors using T4 DNA ligase. After ethanol precipitation and desalting, the ligation products were transformed into TG1 bacterial competent cells by electroporation to construct a library with a volume of 1.2 × 10⁻⁶. 9 The antibody library is used for subsequent screening.

[0053] 3. Phage ELISA screening for virus-specific VHH antibodies followed by sequencing.

[0054] 3.1 Coating: Dilute the target protein in ELISA plates with pH 9.6 coating buffer (50 μL / well) and incubate at 37°C for 1 hour or 4°C overnight in a humidified chamber. Blocking: Discard excess coating buffer, invert the plate and tap it on a clean paper towel to remove residual liquid, then wash the plate three times with PBS.

[0055] 3.2 Add blocking solution, 300 μL / well, and incubate at 37°C in a humidified chamber for 1 hour. Primary antibody: Discard excess phage solution, invert the plate and tap it on a clean paper towel to remove residual liquid, wash the plate 3 times with PBS. Dilute HRP rabbit anti-M13 1:1000 with 1% Milk-PBS, add 50 μL / well, and incubate at 37°C in a humidified chamber for 1 hour.

[0056] 3.3 Colorimetric Development: Discard excess solution and invert the plate onto a clean paper towel to remove residual liquid. Wash the plate 4 times with PBS. Add 50 μL of TMB colorimetric solution to each well, stop with 2 M H₂SO₄, and detect at 450 nm.

[0057] 3.4 Sequencing of reactive monoclonal samples.

[0058] The results showed that the VHH phase library was constructed from camel peripheral blood lymphocyte RNA, with SVA particles as the target antigen, FMDV particles as the negative antigen, and no antigen as a blank control. After three rounds of screening, an output phase of 1.74 × 10³ was obtained. Phage ELISA was performed on 372 phage monoclonal antibodies and the target antigen SVA particles, yielding 220 positive clones. Sanger sequencing yielded 52 unique VHH antibody sequences (see Tables 1-3). These 52 unique VHH antibodies were fused with human Fc cells and expressed in 293T cells (see Table 3). Figure 1 B).

[0059] Three to four weeks after immunization, camels exhibited a plateau in specific antibodies against SVA (1:2,560,000) and NA levels (>16,384). Then, separable peripheral blood lymphocytes were constructed, containing 1.3 × 10⁻⁶ cells. 9 CFU / ml phage library Figure 1 A and B). Subsequently, blank control (smear solution), negative control (FMDV antigen), and target cell plate (SVA) were designed.

[0060] Table 1. Amino acid sequence of CDR-1 in the VHH heavy chain variable region.

[0061]

[0062]

[0063]

[0064] Table 2. Amino acid sequence of CDR-2 in the VHH heavy chain variable region.

[0065]

[0066]

[0067]

[0068] Table 3. Amino acid sequence of CDR-3 in the VHH heavy chain variable region.

[0069]

[0070]

[0071]

[0072] 4. After phage display, VHH was screened for SVA.

[0073] Intact SVA antigen was encapsulated overnight in 96-well plates, and phage selection was performed as follows (see...). Figure 1 B):

[0074] 4.1 Add 300 μL of blocking solution (2% milk dissolved in PBS) to each well to block the SVA antigen and incubate at 37°C for 1 h.

[0075] 4.2 Wash the plate three times with PBS to infer the background. Add 10 μL of the phage library (approximately 1.0 μL). 13 Mix PFU with 1% milk PBS, then add 100 μL / well to each blank well. Let stand at room temperature for 1 hour.

[0076] 4.3 Wash three times with PBS to ensure proper binding of the sealed SVA antigen. Add 100 μL of the stock solution from step 2 to the well from step 1. Incubate at room temperature for 1 hour.

[0077] 4.4 Wash the plate with 0.1% PBST.

[0078] 4.5 Elute with glycine, adding 100 μL / well and gently shaking for 8 minutes at room temperature. Stop elution by aspirating 15 μL / well Tris-HCl (pH 9.1) to obtain the eluent.

[0079] 4.6 Amplification of the elution product using the TG1 monoclonal strain. The TG1 monoclonal strain was inoculated into 20 mL of 2×YT medium and shaken at 37°C and 250 rpm until the logarithmic phase was reached (OD = 0.5). After infecting TG1 with the elution product, M13KO7 helper phage was added, and the mixture was incubated at 37°C for 20 min. The bacteria were collected and resuspended (5000 rpm, 10 min), and the medium was amplified with ×YT + Amp (final concentration 50 μg / mL) + Kan (final concentration 10 μg / mL).

[0080] 4.7 Obtain the amplified elution product by PEG precipitation. Centrifuge at 10000×g for 10 minutes at 4°C, discard the precipitate, add 1 / 4 volume of PEG6000 (Sigma) to the supernatant, incubate at 4°C for 4 hours, centrifuge at 10000μg for 20 minutes at 4°C, resuspend the precipitate in 0.5mL PBS, centrifuge at 10000μg for 10 minutes at 4°C, collect the supernatant to obtain the amplification product.

[0081] 4.8 Enrichment of high-affinity phages.

[0082] TG1 cells with an infective index were infected using eluted phage. Binding of randomly selected clones to SVA was assessed using a 96-well phage ELISA assay. The specific procedure was as follows: First, ELISA plates were diluted with 50 μL / well of 1 μg / mL SVA in carbonate solution at pH 9.6 and incubated overnight at 4°C. After discarding the coating buffer, the ELISA plates were washed three times with PBS and blocked with 5% milk (300 μL / well) at 37°C for 1 h. Subsequently, the plates were washed three times as described above and incubated with HRP-conjugated rabbit anti-M13 antibody diluted 1:1000 in PBS containing 1% milk (50 μL / well) at 37°C for 1 h. Finally, the ELISA plates were washed and the reaction was initiated by adding soluble TMB substrate solution, followed by termination with 2M H2SO4. The optical density of each well was measured at 450 nm using a microplate reader.

[0083] 5. Sequence Analysis

[0084] The VHH sequences encoding CDR1, CDR2, and CDR3 were analyzed using primer analysis software.

[0085] 6. Antibody expression and purification

[0086] The VHH sequence was cloned into the pTT5 eukaryotic vector using NcoI and NotI restriction sites. The vector contained the secretion signal peptide IL2 (amino acid sequence: EVQLVESGGGSVQAGGSLRLSCAVSG) and human IgG1-Fc. The cell density of 293F cells was adjusted to 1x10⁻⁶. 7 The recombinant plasmid was transferred to 293F cells at a concentration of / mL. After 3 days of expression, the cells were centrifuged at 4000×g for 30 minutes to obtain the supernatant. The antibody was purified using protein A (General Electric Company) according to the manufacturer's instructions.

[0087] 7. VHH ELISA titration

[0088] The antigen SVA was diluted to 1 μg / mL with carbonate coating solution at pH 9.6 and incubated in a 96-well ELISA plate at 4°C. 100 μL of the coating solution was added to each well and incubated overnight. The liquid was discarded in the wells, washed three times with PBST, sealed with 4% skim milk PBS, 200 μL / well, incubated at 37°C for 1 h. After washing with PBST, 100 μL / well of SVA-VHH was added, and FMDV or culture medium was added as a control. The wells were washed at 37°C for 1 h. After washing with PBST, HRP goat anti-human IgG (Fc) (Abcam, USA) was added at a 1:5000 dilution, 100 μL / well, diluted at 37°C for 1 h. After washing with PBST, TMB chromogenic solution was added, 100 μL / min per well, and chromogenic was developed in the dark. Finally, the reaction was terminated with 50 μL of 2M sulfuric acid. The OD450 nm value was read by enzyme-linked immunosorbent assay (ELISA). Data points were plotted using GraphPadPrism 9, and the 50% inhibition concentration (IC50) was calculated using a three-parameter nonlinear model. 50 )value.

[0089] 8. Virus neutralization test

[0090] Virus neutralization assay (VNT) was performed using antibody at an initial concentration of 1 μg / mL, serially diluted 1:2, with each dilution repeated in four wells. The diluted antibody was incubated with 100 TCID50 CH-HuB-2017 viruses at 37°C for 1 hour. 50 μl of IBRS-2 cells were added to 96-well plates and incubated at 37°C in a 5% CO2 incubator for 72 hours. Pathological changes in the cells were then observed using an inverted microscope. Finally, the NAs value was calculated using the Reed-Munch method.

[0091] 9. Experimental Results

[0092] (1) SVA single-domain antibody screening results

[0093] Three to four weeks after immunization, camels exhibited a plateau in specific antibodies against SVA (1:2,560,000) and NA levels (>16,384). Then, separable peripheral blood lymphocytes were constructed, containing 1.3 × 10⁻⁶ cells. 9 CFU / ml phage library Figure 1 A). Subsequently, blank control (spread solution), negative control (FMDV antigen), and target cell plate (SVA) were designed. After phage display, VHH underwent three rounds of screening against SVA. Phage binding to SVA showed significant growth, with an enrichment factor (input / output) of 1.74 × 10⁻⁶. 3 In addition, a total of 372 phage single clones were used for phage ELISA, of which 220 were positive for both phage and SVA. These 220 positive clones were then sequenced and aligned with the CDR1, CDR2, and CDR3 regions, thereby identifying 52 VHH clones with unique sequences. Figure 1 B).

[0094] (2) Detection of SVA single-domain antibody specificity and binding activity

[0095] Expressed in 293F cells, of the 52 VHHs, only VHH-11 and VHH-51 did not bind to SVA. Furthermore, the remaining 50 VHHs exhibited SVA binding activity, and none of them reacted with FMDV, demonstrating good specificity (see [link to relevant documentation]). Figure 2 (and Table 4).

[0096] Table 4 Results of SVA single-domain antibody specificity and binding activity assay

[0097]

[0098]

[0099] (3) Detection of neutralizing activity of SVA single-domain antibody

[0100] Neutralization assays were performed using an initial antibody concentration of 1 μg / mL, serially diluted 1:2, with each dilution replicated in four wells. The diluted antibody was incubated with 100 TCID50 CH-HuB-2017 virus cells at 37°C for 1 hour. 50 μL of LIBRS-2 cells were added to 96-well plates and incubated at 37°C with 5% CO2 for 72 hours. Cellular lesions were then observed using an inverted microscope. Finally, the neutralizing antibody levels were calculated using the Reed-Muench method.

[0101] The results showed that neutralizing antibodies with high neutralizing activity were obtained by neutralizing live SVA with an initial concentration of 1 μg / ml and then serially diluted 2-fold (see [link to results]). Figure 3 (and Table 5).

[0102] Table 5. Results of SVA Single-Domain Antibody Neutralization Activity Detection

[0103]

[0104]

[0105] Example 2: Preparation of SVA Neutralizing Liquid Phase Blocking ELISA Antibody Detection Kit

[0106] 1. Preparation of SVA antigen

[0107] (1) IBRS-2 cells were cultured in a 37°C incubator for 24 hours, inoculated with 1 MOI of SVA virus solution, and the virus was collected after 10 hours. The collected virus solution was repeatedly frozen and thawed three times and then inactivated with 1.2% to 1.4% BEI at 30°C for 28 hours. Then, 4% blocking agent (50% sodium thiosulfate) was added.

[0108] (2) Centrifuge the inactivated virus solution at 6000 rpm for 1 hour to remove cell debris, and concentrate it to 80 ml using a membrane.

[0109] The concentrated virus solution was aliquoted into ultrafiltration tubes, centrifuged at 35,000 rpm for 2.5 h, the supernatant was discarded, the virus was resuspended in 2 ml of PBS, ground on ice for 2 h, centrifuged at 6,000 rpm for 30 min, and the precipitate was discarded.

[0110] (3) Add the supernatant to trichloroethylene at a ratio of 1:1, mix by repeatedly blowing and stirring, centrifuge at 6000 rpm for 30 min, and retain the supernatant.

[0111] (4) Centrifuge the supernatant using a sucrose density gradient (15%–45% sucrose density) at 36,000 rpm for 2.5 h, dispense 500 μl / tube, and measure OD. 260 .

[0112] (5) Use transmission electron microscopy (TEM) to analyze and identify virus particles.

[0113] After centrifuging the SVA virus solution with sucrose density gradient, it was adsorbed onto a carbon-coated copper grid at room temperature for 2.5 min. Excess liquid on the copper grid was removed with filter paper. The sample was then negatively stained with 2% tungsten phosphate for 5 min, and the excess liquid was removed with filter paper. Finally, the sample was observed under a transmission electron microscope.

[0114] 2. HRP (horseradish peroxidase) labeling of VHH

[0115] According to the manufacturer's instructions, Fc-VHH-27 was labeled using the HRP Coupling Kit (Abcam).

[0116] 3. LPB-ELISA Design

[0117] The amino acid sequences of the CDR-1, CDR-2, and CDR-3 regions of the SVA-VHH-27 heavy chain variable region are as follows:

[0118] CDR-1:YIERHYCMG; (SEQ ID NO.27)

[0119] CDR-2:TVAYEGSTTYAESVKG; (SEQ ID NO.79)

[0120] CDR-3:RTTYFCTPRANDFTY. (SEQ ID NO.131)

[0121] The amino acid sequences of FR-1, FR-2, and FR-3 from the camel antibody heavy chain constant region are as follows:

[0122] FR-1: WFRQAPGKEREGVA; (SEQ ID NO.157)

[0123] FR-2: RFTISRDNAKNTLYLQINSLKPEDTGIYYCAA; (SEQ ID NO.158)

[0124] FR-3: WGQGTRVTVSS. (SEQ ID NO.159)

[0125] The amino acid sequence of the antibody used as LPB-ELISA is as follows:

[0126] EVQLVESGGGSVQAGGSLRLSCAVSGYIERHYCMGWFRQAPGKEREGVATVAYEGS TTYAESVKGRFTISRDNAKNTLYLQINSLKPEDTGIYYCAARTTYFCTPRANDFTYWGQG TRVTVS. (SEQ ID NO.160)

[0127] The optimal concentrations of the capture antibody (Fc-VHH-27) and the detection antibody (Fc-VHH-27-HRP) were determined using a checkerboard titration method. The incubation time and blocking buffer were optimized based on the ratio of negative (N) to positive (P) serum readings.

[0128] The optimal conditions for LPB-ELISA are: a mixture of SVA antigen 1 μg / mL and serum at 37°C for 1 hour, followed by coating of capture antibody onto a 96-well microplate. In NY, USA, the antibody was incubated overnight at 4°C at 0.16 μg / mL, and the detection antibody was incubated at 37°C at 0.1 μg / mL for 1 hour. The blocking solution was 5% milk, incubated at 37°C for 1 hour, and the color development time was 15 minutes (all dilution buffers were PBST).

[0129] Step 1: Plate Coating. Spread 96-well microplates overnight at 4°C with diluted Fc-VHH-150 in carbonate / bicarbonate buffer (pH 9.6) to provide capture antibodies. Subsequently, wash three times with PBST, add 200 μL of 5% milk to each well, block at 37°C for 1 h, and wash three times with PBST.

[0130] Step 2: Antigen and serum mixing. Mix 50 μL of diluted test serum with 50 μL of SVA 1 μg / mL antigen in a 96-well microplate and incubate at 37°C for 1 hour. Then, transfer 100 μL of this mixture to a 96-well microplate coated with capture antibody.

[0131] Step 3, antibody detection. Add 50 μL of diluted Fc-VHH-150-HRP (0.1 μg / mL) to each well, incubate at 37°C for 1 hour, and wash 3 times with PBST.

[0132] Step 4, substrate. Add 50 μL / well of tetramethylbenzyl substrate (TMB), incubate at 37°C for 15 minutes, and finally add 2M sulfuric acid to terminate the reaction.

[0133] Step 5, Result Confirmation. OD is measured using an automated microplate reader (BioTek, USA). 450 A wavelength of nm.

[0134] Step 6, Control Settings. Simultaneously measure positive control serum samples (reference serum samples with high titers) and negative control serum samples (from unvaccinated pigs) with known titers on the ELISA plate as references. Positive sera should be diluted in the range of 1:4 to 1:512, while negative sera should be diluted in the range of 1:4 to 1:32 for detection. Use four wells for antigen controls, which should exhibit 100% reactivity. The cutoff value for these control wells is half the optical density (OD) of the four antigen control wells. Express the antibody titer as the reciprocal of the serum dilution (log10).

[0135] Step 7: Result Confirmation. An antigen control is considered effective when the average OD value is approximately 1.65. The titer (log10) of a strong positive serum control should be 2.25 ± 0.3, and the titer (log10) of a negative serum control should be less than 0.9.

[0136] 3. Preparation of other solutions in the kit:

[0137] ① Sample dilution solution: 0.01 mol / L phosphate-buffered saline (PBS) containing 0.1% Tween-20 at a pH of 7.2-7.4;

[0138] ② Washing solution: Add 1 mL of Tween-20 to 1000 mL of 0.01 M PBS solution;

[0139] ③ Termination solution: Take 54.34 mL of 98% concentrated sulfuric acid and add distilled water to make 1000 mL.

[0140] 4. Data and Statistical Analysis

[0141] Receiver operating characteristic (ROC) curve analysis was used to determine the cutoff value, sensitivity, Pearson coefficient test, and specificity of the assay by using serum from piglets and SVA-positive pig serum from experimentally infected or immunized animals. P < 0.05 was considered statistically significant.

[0142] 5. Results

[0143] (1) Production and characterization of SVA antigen

[0144] The SVA supernatant was concentrated and purified in a linear sucrose gradient of 10%–50% (wt / vol).

[0145] like Figure 4 As shown in Figure A, 10 mL of sample was evenly distributed in 20 test tubes, and the OD was measured. 260 / 280 The value was used to generate a peak. The 12th tube containing the peak was removed, and spherical particles with a diameter of 30 nm were observed using a transmission electron microscope. Dynamic light scattering technology further confirmed the particle size of the sample tube to be 28 nm. Figure 4 B). Finally, SDS-PAGE was used to identify the VP2, VP3, and VP1 bands of the SVA (B). Figure 4 C).

[0146] (2) Sensitivity and specificity analysis:

[0147] The established ELISA method was used to detect 192 serum samples with different antibody titers, and the specificity reached 100% and the sensitivity was 98.85%.

[0148] (3) Sensitivity and specificity of LPB-ELISA

[0149] A total of 87 SVA-negative sera and 105 sera with previously confirmed SVA infection or immunity were selected to determine the cutoff value for LPB-ELISA. The cutoff value for LPB-ELISA was 1.65 log [missing value]. 10 At that time, the optimal sensitivity and specificity values ​​were obtained. The sensitivity and specificity were 100% and 99.04%, respectively. Figure 5 A and 5C). The area under the ROC curve (AUC) of the LPB-ELISA was calculated to be 0.9996 (standard error [SE] = 0.0005261). The 95% confidence interval is 0.9986–1.000 ( Figure 5 B).

[0150] (4) Correlation between LPB-ELISA and VNT

[0151] Antibodies were detected in 68 serum samples obtained from SVA-infected or immunized pigs using the established LPB-ELISA and VNT assays to determine whether the established method could detect NA. Figure 6 As shown in A and 6B, all 68 serum samples were positive, and the results of LPB-ELISA and VNT assays were consistent (R 2 =0.83, P<0.0001), and the Pearson correlation confirms this.

[0152] The dynamics of NA changes in SVA-infected or immunized pigs were analyzed to further validate the effectiveness of the established method. NA level changes were monitored in two groups of SVA-VLP-immunized pigs (1–4 weeks and 0–7 weeks) and one group of SVA-infected pigs (1–4 weeks), and similar trends were observed in all three groups. Figure 7 C-7H). Both batches of vaccines provided 100% protection against porcine aggressive SVA; therefore, when log2 > 6.49 (1.65log2 mentioned in this paper), 10 =6.49log2), which can achieve full protection (Table 6).

[0153] Table 6. Correlation between LBP-ELISA, VNT, and detection protection rate

[0154]

Claims

1. A liquid-phase blocking ELISA kit for neutralizing porcine Seneca virus (PSV) antibodies, the kit containing a single-domain PSV antibody and matching detection reagents, wherein the heavy chain variable region of the PSV antibody comprises CDR1, CDR2, and CDR3, the amino acid of the heavy chain variable region CDR1 is SEQ ID NO. 27, the amino acid of the heavy chain variable region CDR2 is SEQ ID NO. 79, and the amino acid of the heavy chain variable region CDR3 is SEQ ID NO. 131; the detection reagents include a sample diluent, a washing buffer, and a stop solution; the sample diluent contains 0.01 mol / L of 0.1% Tween 20 and phosphate buffer with pH = 7.2-7.4; the washing buffer is prepared by adding 1 mL of Tween 20 to 1000 mL of 0.01 M phosphate buffer; the stop solution is prepared by adding 54.34 mL of 98% concentrated sulfuric acid to distilled water to a final volume of 1000 mL.

2. The porcine Seneca virus neutralizing antibody liquid-phase blocking ELISA kit as described in claim 1, wherein the porcine Seneca virus single-domain antibody is an antigen-binding fragment of a single-domain antibody.

3. The kit according to claim 1, wherein the heavy chain constant region of the single-domain antibody or its antigen-binding fragment is derived from the camel antibody heavy chain constant region FR1, FR2, and FR3; the amino acid sequence of FR1 is WFRQAPGKEREGVA (SEQ ID NO. 157), the amino acid sequence of FR2 is RFTISRDNAKNTLYLQINSLKPEDTGIYYCAA (SEQ ID NO. 158), and the amino acid sequence of FR3 is WGQGTRVTVSS (SEQ ID NO. 159).

4. The kit according to claim 1, characterized in that, The porcine Seneca virus neutralizing antibody liquid phase blocking ELISA kit also includes porcine Seneca virus antigen, which is a mixture of antigen and serum.

5. The application of a single-domain antibody against porcine Seneca virus in preparation and in an antibody liquid-phase blocking ELISA reagent, wherein the heavy chain variable region of the single-domain antibody against porcine Seneca virus comprises CDR1, CDR2 and CDR3, wherein the amino acid of the heavy chain variable region CDR1 is SEQ ID NO. 27, the amino acid of the heavy chain variable region CDR2 is SEQ ID NO. 79 and the amino acid of the heavy chain variable region CDR3 is SEQ ID NO. 131.

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