CN117388491A

CN117388491A - SVA neutralizing antibody liquid phase blocking ELISA detection kit

Info

Publication number: CN117388491A
Application number: CN202311263433.1A
Authority: CN
Inventors: 郭慧琛; 穆素雨; 孙世琪; 潘颂佳; 张韵; 尹双辉; 白满元; 吴金恩; 滕志东; 周静静; 丁耀忠; 董虎
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2023-09-27
Filing date: 2023-09-27
Publication date: 2024-01-12

Abstract

The invention belongs to the technical field of biotechnology, and particularly discloses a liquid-phase blocking ELISA kit for a neutralizing antibody of a porcine Seika virus, which comprises a porcine Seika virus single domain antibody and a matched detection reagent, wherein the porcine Seika virus 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 of 52 groups, a heavy chain variable region of each group of single domain antibodies or antigen binding fragments comprises CDR-1, CDR-2 and CDR-3, the amino acid sequence of the heavy chain variable region CDR-1 (VH-CDR-1) of the 52 groups of single domain antibodies or antigen binding fragments is respectively shown as SEQ ID NO. 1-SEQ ID NO.52, the amino acid sequence of the heavy chain variable region CDR-2 (VH-CDR-2) is respectively shown as SEQ ID NO. 53-SEQ ID NO.104, and the amino acid sequence of the heavy chain variable region CDR-3 (VH-CDR-3) is respectively shown as SEQ ID NO. 105-SEQ ID NO. 156. The kit can be used for rapidly detecting the SVA neutralizing antibody level in serum, has high sensitivity, good specificity and stable result, and can be better applied to SVA vaccine efficacy evaluation.

Description

SVA neutralizing antibody liquid phase blocking ELISA detection kit

Technical Field

The invention belongs to the technical field of biology, relates to the technical field of veterinary diagnosis, and in particular relates to a preparation method and application of an SVA neutralizing antibody liquid phase blocking ELISA detection kit.

Background

The sai virus disease is an infectious disease characterized by vesicular damage of the mouth and mouth of a pig caused by sai virus a. In 2014, SVA-induced diseases are exploded in the United states and Brazil, SVA is reported in Guangdong province in China for the first time in 2015, and then gradually escapes to a plurality of places, and 15 provinces and cities report the popularity of SVA at present. Genetic evolution and recombination analysis are carried out on SVA strains popular in the pig group in China, so that the strain popular in the pig group in China is found to be complex and diverse in genetic evolution, and new gene recombination strains appear, which suggest that the technology reserve for popular monitoring and prevention and control of SVA in China is required to be further enhanced.

SVA is a member of the genus Cork-wire virus of the family Picornaviridae, which is a single-stranded positive-strand RNA virus without a envelope, in an icosahedral structure, with a genome of full length 7300nt, comprising a 5 'non-coding region (5' -untranslated region,5 '-UTR) consisting of 666 nucleotides, a 3' non-coding region (3 '-untranslated region,3' -UTR) consisting of 71 nucleotides, and a unique open reading frame (Open Reading Frame, ORF) between the two non-coding regions. The ORF of SVA has a typical picornaviral genome L-4-3-4 structure, which encodes a polyprotein comprising 2181 amino acids, wherein the L region encodes the leader protein Lpro, the P1 coding region encodes the 4 structural proteins VP1, VP2, VP3 and VP4, the P2 coding region encodes the 3 nonstructural proteins 2A, 2B and 2C, and the P3 coding region encodes the 4 nonstructural proteins 3A, 3B, 3C and 3D. In recent studies, the immunogenicity of structural proteins such as VP2 proteins in type A Seneca viruses has been reported to be analyzed, and the immunogenicity of non-structural proteins has not been reported.

Currently, there is no practical use of SVA vaccines. A recent study assessed the immunogenicity of inactivated, attenuated and virus-like particle (VLP) SVA candidate vaccines and demonstrated protection against homologous virus challenge. Nucleic acids, like other picornavirus vaccines, are detected as key data by neutralization experiments. Standard techniques for detecting NA include plaque reduction neutralization assays, micro-neutralization assays, and fluorescent antibody-virus neutralization assays (VNT). However, these methods are laborious and time consuming, require manual precision, and require the use of clean grade intercellular material. Furthermore, they are not suitable for on-site monitoring of NA after immunization with SVA vaccine. In contrast, ELISA is cost effective, less time consuming than standard neutralization assays, and can overcome the above drawbacks. However, current SVA-ELISA kits do not appear to be suitable for assessing the protective efficacy of vaccination against SVA infection or vaccination, as the level of whole antibodies does not directly reflect the titer of NA. Furthermore, NAs production is critical to produce a protective response in the body. Thus, understanding the correlation between NAs and protective responses is beneficial to advance vaccine development and reduce animal use.

Inactivated SVA or VLP vaccines have been demonstrated to produce NA at high titers, leading to protective responses that may be attributed to their conformational epitopes. However, the process of developing NAs quantification methods by using ELISA requires stable antibody molecules with strong affinity, high neutralizing activity and excellent specificity. Therefore, the selection of intact virions or VLPs as immunogens is an ideal approach. Currently, two main types of antibodies are used. The first type includes conventional antibodies consisting of heavy and light chains. Based on monoclonal antibody screening, ELISA methods for detecting FMDV (type A, type O), rabies, severe acute respiratory syndrome coronavirus type 2, swine fever and SVA serum Nas are established. The other is a heavy chain antibody produced by camel. In 1993, another antibody in camelid blood was first reported in addition to the traditional IgG antibody. The receptor binding single domain antibody fragment (VHH) is the smallest intact antigen binding fragment available and has a molecular weight of about 15kDa. VHH has better stability and solubility than traditional antibodies and is easy to recombine and express in e. Thus, VHH has been used to combat a variety of viruses, such as FMDV type a and O, rabies, severe acute respiratory syndrome coronavirus type 2, PEDV and other pathogens. VHH are also used to detect antibodies, antigens and therapies. However, there is currently no report on the use of VHH in SVAs.

The invention comprises the following steps:

the invention provides a method for detecting Neutralizing Antibodies (NAs) of Seceavirus A (SVA) by screening to obtain single domain antibodies of SVA-VHH and developing a Liquid Phase Blocking (LPB) -ELISA technology by using the single domain antibodies. The present invention has been completed in this technology.

In a first aspect, the invention provides a liquid phase blocking ELISA kit for a porcine saikovirus neutralizing antibody, wherein the kit contains an effective amount of porcine saikovirus single domain antibody and a matched detection reagent.

Further, the porcine saikovirus single domain antibody may also be an antigen binding fragment of a single domain antibody.

Further, the single domain antibody is selected from one or more of 52 groups, the heavy chain variable region of each single domain antibody or antigen binding fragment comprises CDR-1, CDR-2 and CDR-3, the amino acid sequence of the heavy chain variable region CDR-1 (VH-CDR-1) of the 52 groups of single domain antibodies or antigen binding fragments is shown as SEQ ID NO. 1-SEQ ID NO.52, the amino acid sequence of the heavy chain variable region CDR-2 (VH-CDR-2) is shown as SEQ ID NO. 53-SEQ ID NO.104, and the amino acid sequence of the heavy chain variable region CDR-3 (VH-CDR-3) is shown as SEQ ID NO. 105-156.

Further, the heavy chain constant region of the single domain antibody or antigen binding fragment thereof is selected from the group consisting of porcine IgG, human IgG and/or chicken IgY.

Further, the heavy chain constant region IgG is selected from one or more of IgG1, igG2a, igG2b, and IgG 4.

Further, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1-SEQ ID NO.52, SEQ ID NO. 53-SEQ ID NO.104 or SEQ ID NO. 105-SEQ ID NO. 156; variants having 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%, at least 99% sequence identity or no more than 8 amino acids.

Further, the liquid phase blocking ELISA kit for the neutralizing antibody of the porcine Session initiation virus also comprises the antigen of the porcine Session initiation virus.

Furthermore, the porcine Session inner card virus antigen is a mixed solution of antigen and serum.

In one embodiment, the porcine Sesinkavirus single domain antibody comprises a detection antibody and a capture antibody, wherein the detection antibody is Fc-VHH-27-HRP and the capture antibody is Fc-VHH-27.

Further, the heavy chain variable region of the porcine Sesinkavirus single domain antibody comprises a CDR-1, a CDR-2 and a CDR-3CDR-1, wherein the amino acid of the heavy chain variable region CDR-1 is shown as SEQ ID NO.27, the amino acid of the heavy chain variable region CDR-2 is shown as SEQ ID NO.79 and the amino acid of the heavy chain variable region CDR-3 is shown as SEQ ID NO. 131.

In a second aspect, the invention provides an application of a single domain antibody of porcine saikovirus in preparing a neutralizing antibody liquid phase blocking ELISA reagent or kit.

Further, the detection reagent or kit further comprises a reagent for detecting the single domain antibody.

Further, the single domain antibody is selected from one or more of 52 groups, the heavy chain variable region of each group of single domain antibody or antigen binding fragment comprises 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 are respectively shown as 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 as 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 as SEQ ID NO. 105-SEQ ID NO. 156.

Further, the use further comprises assaying antibodies in the serum of the subject for diagnosis of viral diseases.

Further, the application is to determine the antibody titer of immune serum or the evaluation of the effect after vaccination.

Further, the reagent is a detection kit.

The beneficial effects are that:

the invention establishes a kit for detecting the SVA neutralizing antibody by utilizing the SVA specific neutralizing single domain antibody, and can rapidly detect the SVA neutralizing antibody level in serum; the invention has high sensitivity, good specificity and stable result, and can be better applied to SVA vaccine efficacy evaluation.

Drawings

FIG. 1 shows a flowchart of a phage display technique for screening single domain antibodies against SVA (A is an immune camel against SVA antigen by Freund's adjuvant; B is a phage ELISA screening single domain antibodies reactive against SVA antigen).

FIG. 2ELISA detects affinity and specificity of expressed antibodies.

FIG. 3 neutralization assay determines the detection of neutralizing activity of antibodies.

FIG. 4 titer dispersion of negative and positive sera.

FIG. 5 determination of sensitivity and specificity values for NA-ELISA.

FIG. 6 correlation between VNT titres and NA-ELISA assay titres.

FIG. 7 correlation of LBP-ELISA, VNT and detection protection rate.

Detailed Description

The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments described below may be combined with each other as long as they do not collide with each other.

The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.

Examples

1. Cells, viruses and animals

IBRS-2 cells at 37℃and 5% CO ₂ Is cultured in DMEM medium (Sigma-Aldrich, MA, US) supplemented with 10% fetal bovine serum (FBS; gibco, CA, USA), 3mM glutamine, penicillin (100 IU/mL), streptomycin (50. Mu.g/mL) and gentamicin (25. Mu.g/mL, gibco). The SVA strain (GenBank: MN 2922286) is a strain isolated from Hubei province. Two adult camels are from some animal farm of Jin Changshi Gansu province in China.

2. Serum sample

Serum samples of 87 clinically healthy pigs were collected in total and negative for SVA antibodies were detected by VNT, ELISA.

A total of 105 serum samples were collected from experimentally infected pigs or from vaccine pigs obtained, and tested by serum from PRRSV, PCV2, CSFV and FMDV infected pigs for positive SVA antibodies. Of these, 68 positive sera were used for pearson coefficient test, and correlation between VNT and LBP-ELISA was calculated. Furthermore, of the 68 positive serum samples, 36 from 0-7 weeks (8 weeks) after vaccination (pic) and 1-4 weeks (pic in this laboratory), of which 12 were serum from 1-4 weeks after SVA infection, from pic 1,2,3;12 parts from pic 4,5,6;12 parts were from infected pigs, pig 7,8,9.

EXAMPLE 1 preparation and screening of SVA Single-domain antibodies

SVA preparation and camel immunization

1.1 selecting healthy camels with two heads and proper ages, wherein the numbers of the camels are S-1# and S-2#, respectively;

1.2 taking 5ml of peripheral blood before immunization, separating serum, and taking the serum as a negative control when the titer is detected;

1.3 adding equal volume Freund's adjuvant into 400ug SVA antigen (see figure 1), mixing, emulsifying, and immunizing two heads of camel respectively by neck subcutaneous multipoint injection method. The first immunization used Freund's complete adjuvant, and the subsequent immunization used Freund's incomplete adjuvant. The immunization interval was 14 days.

Two weeks after 1.4 th immunization, 5ml of whole blood was taken, serum was isolated, and serum titers were measured (see fig. 1).

1.5 separate from 200ml camel peripheral blood, ficoll method was used to construct camel VHH antibody libraries after separation of PBMC, and two-head camel mix to construct libraries.

Purified SVA antigen was emulsified with complete and incomplete Freund's adjuvant and injected subcutaneously and at multiple points (more than 3 sites) in the neck over 0, 2, 4 and 6 weeks, respectively (see FIG. 1A).

2. Construction of camel nanobody immune phage library

2.1 lysis of PBMC cells with TriZol lysate and isolation of total RNA. Then, reverse transcription PCR reaction is carried out by using oligo-dT as a primer to prepare cDNA for subsequent amplification of antibody genes.

2.2 amplification of antibody genes with cDNA as template. The first PCR amplification is carried out by adopting the upstream (GTC.CTG.GCT.GCT.CTT.CTA.CAA.GG) of the heavy chain variable region of the antibody and the downstream characteristic sequence primer (GGT.ACG.TGC.TGT.TGA.ACT.GTT.CC) of CH2, and the band DNA with about 600bp is obtained by electrophoresis separation and used as a template for the second PCR. The second round of PCR amplification was performed using the characteristic sequence primers of VHH.

2.3 addition of cleavage sites at both ends of the primer amplified in the second round of PCR facilitates subsequent ligation to the vector (F: AACATGCCATGACTCGCGGCTCAACCGG CCATGGCTGA K GT B CAG CTGCAG GC GTCTGGRGGAGG; R: GTTATTATTATTCAGATTATTAGT GCGGCCGCTGGAGACGGTGACC W GGGTCC). After obtaining the VHH gene by PCR, the VHH gene is digested by restriction enzyme, and the digested fragments are respectively connected into a pre-cut phage display vector by using T4 DNA ligase. The connection product is desalted by ethanol precipitation, and then adopts an electrotransformation method,transferring into TG1 bacteria to construct a stock capacity of 1.2X10 ⁹ For subsequent screening.

3. Phage ELISA screening for virus-specific VHH antibodies and sequencing

3.1 coating: the ELISA plate was diluted with pH9.6 coating solution, 50 ul/well, and allowed to stand in a wet box at 37℃for 1h or at 4℃overnight. Closing: and (3) throwing out the redundant coating liquid, inverting the flat plate, beating and throwing out the residual liquid on clean paper towels, and washing the PBS for 3 times.

3.2 adding blocking solution, 300 ul/hole, standing in wet box at 37deg.C for 1h. An antibody: and (3) throwing out the redundant phage solution, inverting the flat plate, beating and throwing out the residual liquid on a clean paper towel, and washing the plate for 3 times by using PBS. HRP rabbit anti-M13 was diluted with 1% Mill-PBS 1:1000, added to 50 ul/well and allowed to stand in a wet box at 37℃for 1h.

3.3 developing: and (5) throwing out redundant solution, inverting the flat plate, beating and throwing out residual liquid on clean paper towels, and washing the PBS for 4 times. TMB color development liquid, 50 ul/well, 2M H ₂ SO ₄ And (5) stopping detection at 450 nm.

3.4 sequencing of reactive monoclonal samples.

The results show that: the VHH phase library is constructed by camel peripheral blood lymphocyte RNA, SVA particles are respectively used as target antigens, FMDV particles are used as negative antigens, and no antigen is used as a blank control. The output phase is 1.74×103 after three rounds of screening. 372 phage monoclonal and target antigen SVA particles were selected for phage ELISA to obtain 220 positive clones, and 52 unique VHH antibody sequences were obtained by Sanger sequencing (see tables 1-3). 52 unique VHH antibodies were fused to human Fc and expressed in 293T cells (see fig. 1B).

After 3-4 weeks of immunization, camels showed specific antibodies (1:2560000) and NAs levels to SVA>16384 A) plateau. Then constructing separable peripheral blood lymphocytes comprising 1.3X10 ⁹ Phage libraries of CFU/ml (FIGS. 1A and B). Subsequently, a blank (coating solution), a negative control (FMDV antigen) and a target cell plate (SVA) were designed.

TABLE 1 CDR-1 amino acid sequence listing of VHH heavy chain variable region

TABLE 2 CDR-2 amino acid sequence listing of VHH heavy chain variable region

TABLE 3 CDR-3 amino acid sequence listing of VHH heavy chain variable region

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4. Screening VHH for SVA after phage display

Complete SVA antigen was packaged overnight on 96-well plates and phage selection was performed as follows (see fig. 1B):

4.1 to each well 300. Mu.L of blocking solution (2% milk in PBS) was added to block SVA antigen, which was left to stand at 37℃for 1h.

4.2 background was inferred by washing the plate three times with PBS. Phage library (number approximately 1.0) 10 ¹³ pfu was mixed with 1% milk PBS and 100. Mu.L/well was added to the blank wells. Standing at room temperature for 1h.

4.3 washing with PBS three times, sealed SVA antigen binding was good. 100. Mu.L of stock solution from step 2 was added to the wells from step 1. The mixture was left at room temperature for 1h.

4.4 plates were washed with 0.1% PBST.

4.5 elution with glycine, 100. Mu.L/well was added and the mixture was slowly shaken at room temperature for 8 minutes. The elution was stopped by inhalation of 15. Mu.L/well Tris-HCl (pH 9.1) to give an eluted product.

4.6 amplification of eluted product with TG1 monoclonal strain. TG1 monoclonal strain was inoculated in 20ml of 2 xyt medium and shaken at 37 ℃ and 250rpm until a logarithmic phase was reached (od=0.5). After TG1 was infected with the eluted product, M13KO7 helper phage was supplemented, and left to stand at 37℃for 20min. Bacteria (5000 rpm,10 minutes) were collected and resuspended ×YT+Amp (final concentration 50. Mu.g/mL) +Kan (final concentration 10. Mu.).

4.7 amplified eluted product was obtained by PEG precipitation. Centrifugation at 10000 Xg for 10 min at 4℃and discarding the pellet, adding 1/4 volume of PEG6000 (Sigma) to the supernatant, adsorbing for 4 hr at 4℃and centrifuging at 10000. Mu.g for 20min at 4℃and resuspending the pellet in 0.5mL PBS and centrifuging at 10000. Mu.g for 10 min at 4℃and collecting the supernatant to obtain amplified product.

4.8 enrichment of high affinity phages.

Exponentially growing TG1 cells were infected with eluted phage. Randomly selected clones were assessed for binding to SVA using a 96-well phage ELISA assay. The specific method comprises the following steps: first, ELISA plates were diluted with 50. Mu.L/well, 1. Mu.g/mL of SVA in carbonate solution at pH=9.6 and incubated overnight at 4 ℃. After discarding the coating buffer, the ELISA plates were washed three times with PBS and blocked with 5% milk (300. Mu.L/well) for 1h at 37 ℃. Subsequently, the plate was washed three times as described aboveAnd HRP conjugated rabbit anti-M13 antibody was diluted 1:1000 in PBS containing 1% milk (50 μl/well) and incubated for 1 hour at 37 ℃. Finally, ELISA plates were washed and developed by addition of soluble TMB substrate solution, followed by 2M H ₂ SO ₄ The solution was terminated. The optical density of each well was measured at 450nm using a microplate reader.

5. Sequence analysis

The sequences of the VHH encoding CDR1, CDR2 and CDR3 were analyzed using analytical primer software.

6. Expression and purification of antibodies

The VHH sequence was cloned into a pTT5 eukaryotic vector containing the secretion signal peptide IL2 (amino acid sequence: EVQLVESGGGSVQAGGSLRLSCAVSG), human IgG1-Fc, using NcoI and NotI restriction sites. Cell density of 293F was adjusted to 1X10 ⁷ Per mL, the constructed recombinant plasmid was transferred into 293F cells, and after 3 days of expression, 4000×g was centrifuged for 30 minutes to obtain a supernatant. Protein a (universal electric company in the united states) was used to purify antibodies according to the instructions.

ELISA titration of VHH

Antigen SVA was diluted to 1. Mu.g/mL with carbonate coating solution pH9.6 and plated in 96-well ELISA plates at 4 ℃100 μl/well was coated overnight. The liquid was discarded in the microplate wells, washed three times with PBST, sealed with 4% nonfat dry milk PBS, 200. Mu.L/well, 37℃for 1h. PBST wash, 100. Mu.L/well SVA-VHH was added, FMDV or medium was added as control, and washing was performed at 37℃for 1h. PBST wash, HRP goat anti-human IgG (Fc) (Abcam, USA) was added, diluted 1:5000, 100. Mu.L/well, diluted 1h at 37 ℃, PBST wash, TMB chromogenic solution was added, 100. Mu.L/min well, and developed in the dark. Finally, the reaction was quenched with 50. Mu.L of 2M sulfuric acid. OD450nm values were read by enzyme-linked immunosorbent assay. Data points were plotted by GraphPad Prism 9 and 50% Inhibition Concentration (IC) was calculated using a three-parameter nonlinear model ₅₀ ) Values.

8. Virus neutralization assay

Virus neutralization assay (VNT) was performed using an initial concentration of 1ug/mL of antibody, serial dilutions starting at 1:2, and four sample wells were repeated for each dilution. The diluted antibodies were incubated with 100 TCID50 CH-HuB-2017 viruses for 1 hour at 37 ℃. Mu.l of IBRS-2 cells were added to 96-well plates at 37℃and 5% CO ₂ Culturing in an incubator for 72 hours. The cells were then observed for pathological changes using an inverted microscope. Finally, NAs values were calculated using the Reed-Munch method.

9. Experimental results

(1) SVA single domain antibody screening results

After 3-4 weeks of immunization, camels showed specific antibodies (1:2560000) and NAs levels to SVA>16384 A) plateau. Then constructing separable peripheral blood lymphocytes comprising 1.3X10 ⁹ Phage library of CFU/ml (FIG. 1A). Subsequently, a blank (coating solution), a negative control (FMDV antigen) and a target cell plate (SVA) were designed. Following phage display, VHH were subjected to three rounds of screening for SVAs. Phage binding to SVA showed significant growth with an enrichment factor (input/output) of 1.7X10 ³ . In addition, a total of 372 phage monoclonal were used in phage ELISA, 220 of which reacted positively with both phage and SVA. These 220 positive clones were then sequenced and aligned with CDR1, CDR2 and CDR3 regions, thereby identifying 52 VHHs with unique sequences (fig. 1B).

(2) SVA single domain antibody specificity and binding Activity detection

Expressed in 293F cells, only VHH-11 and VHH-51 did not bind SVA out of 52 VHH. Furthermore, the remaining 50 VHHs showed binding activity to SVA, and none of them reacted with FMDV, with better specificity (see fig. 2 and table 4).

TABLE 4 detection of SVA Single-domain antibody specificity and binding Activity

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(3) SVA single domain antibody neutralization activity detection

Neutralization experiments were performed with an initial concentration of 1ug/mL of antibody, from 1:2 serial dilutions were started, 4 sample wells were repeated for each dilution. The diluted antibodies were incubated with 100 TCID50 CH-HuB-2017 viruses for 1 hour at 37 ℃. Add 50. Mu.L IBRS-2 cells to 96 well plates at 37℃with 5% CO ₂ Is cultured for 72 hours and then the lesions of the cells are observed using an inverted microscope. Finally, the neutralizing antibody values were calculated by the Reed-Muench method.

The results show that: the live SVA was neutralized by an initial concentration of 1. Mu.g/ml of antibody, and after 2-fold serial dilutions, highly neutralizing antibodies were obtained (see FIG. 3 and Table 5).

TABLE 5 neutralization Activity detection results for SVA Single-domain antibodies

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Example 2 preparation of SVA neutralizing liquid phase blocking ELISA antibody detection kit

Preparation of SVA antigen

(1) IBRS-2 cells were cultured in a 37 ℃ incubator for 24 hours, inoculated with 1MOI SVA virus solution, thawed after 10 hours, repeatedly freeze-thawed three times and inactivated with 1.2% -1.4% bei 30 ℃ for 28 hours, followed by the addition of 4% blocker (50% sodium thiosulfate).

(2) The inactivated virus solution was centrifuged at 6000rpm for 1 hour, cell debris was removed, and concentrated to 80ml with a membrane pack.

The concentrated virus solution was split into super-isolation tubes, centrifuged at 35000rpm for 2.5h, the supernatant was discarded, resuspended in 2ml PBS, ground on ice for 2h, centrifuged at 6000rpm for 30min, and the pellet was discarded.

(3) Adding trichloroethylene into the supernatant at a ratio of 1:1, repeatedly blowing and mixing uniformly, centrifuging at 6000rpm for 30min, and reserving the supernatant.

(4) The supernatant is subjected to sucrose density gradient, sucrose density of 15% -45%, centrifugal 2.5h at 36000rpm, sub-packaging at 500 μl/tube, and OD measurement ₂₆₀ 。

(5) Identification of viral particles using Transmission Electron Microscope (TEM) analysis

And (3) absorbing SVA virus liquid after sucrose density gradient centrifugation on a carbon film coated copper net for 2.5min at room temperature, removing redundant liquid on the copper net by using filter paper, performing negative dyeing on the copper net for 5min by using 2% tungsten phosphate, removing redundant liquid by using the filter paper, and finally observing a sample under a transmission electron microscope.

HRP (horseradish peroxidase) labelling of VHH

Fc-VHH-27 was labeled using the HRP coupling kit (Abcam) according to the manufacturer's instructions.

LPB-ELISA design

The amino acid sequences of the variable regions CDR-1, CDR-2 and CDR-3 of the SVA-VHH-27 are as follows:

CDR-1:YIERHYCMG；(SEQ ID NO.27)

CDR-2:TVAYEGSTTYAESVKG；(SEQ ID NO.79)

CDR-3:RTTYFCTPRANDFTY。(SEQ ID NO.131)

the amino acid sequences of the heavy chain constant regions FR-1, FR-2 and FR-3 derived from camelid antibodies are as follows:

FR-1：WFRQAPGKEREGVA；(SEQ ID NO.157)

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

FR-3：WGQGTRVTVSS。(SEQ ID NO.159)

the amino acid sequence of the antibody used as LPB-ELISA was as follows:

EVQLVESGGGSVQAGGSLRLSCAVSGYIERHYCMGWFRQAPGKEREGVATVAYEGS TTYAESVKGRFTISRDNAKNTLYLQINSLKPEDTGIYYCAARTTYFCTPRANDFTYWGQG TRVTVS。(SEQ ID NO.160)

the optimal concentration of capture antibody (Fc-VHH-27) and detection antibody (Fc-VHH-27-HRP) was determined using a checkerboard titration method. Culture time and blocking buffer were optimized based on the ratio of negative (N) and positive (P) serum readings.

The optimal conditions for the LPB-ELISA were: SVA antigen 1. Mu.g-The mixture of the mL and the serum is coated on a 96-well microplate at 37 ℃ for 1 hourNY, USA) at 4℃overnight at 0.16. Mu.g/mL, detection antibody was used at 37℃for 1 hour at 0.1. Mu.g/mL, blocking solution was 5% milk, 37℃for 1 hour, development time was 15 minutes (all dilution buffers were PBST).

Step 1: and (5) coating the plate. 96-well microwell plates were coated overnight with diluted Fc-VHH-150 in carbonate/bicarbonate buffer (pH 9.6) at 4℃to provide capture antibodies. Subsequently, PBST was washed 3 times, 200. Mu.L of 5% milk was added to each well, and blocked at 37℃for 1 hour, and washed 3 times with PBST.

Step 2, antigen and serum are mixed. 50. Mu.L of diluted test serum was mixed with 50. Mu.L of SVA 1. Mu.g/mL antigen in 96-well microwell plates and incubated for 1 hour at 37 ℃. Thereafter, 100. Mu.L of these mixtures were transferred to an already coated capture antibody 96-well microplate.

Step 3, detecting the antibody. 50. Mu.L/well of diluted Fc-VHH-150-HRP (0.1. Mu.g/mL) was added, incubated for 1 hour at 37℃and washed 3 times with PBST.

And 4, a substrate. The reaction was terminated by adding 50. Mu.L/well of tetramethylbenzyl substrate (TMB), incubating for 15 minutes at 37℃and finally adding 2M sulfuric acid.

And 5, determining the result. OD measurement Using an automated microplate reader (BioTek, USA) ₄₅₀ Wavelength in nm.

And 6, controlling the setting. Positive control serum samples with known titers (reference serum samples with high titers) and negative control serum samples (from unvaccinated pigs) were measured simultaneously on ELISA plates as references. The positive serum should be diluted in the range of 1:4 to 1:512, while the negative serum should be diluted in the range of 1:4 to 1:32 for detection. Antigen control was performed using four wells, which should exhibit 100% reactivity. The threshold of these control wells was half the Optical Density (OD) of the four antigen control wells. Antibody titers were expressed as the reciprocal of serum dilution (log 10).

And 7, determining the result. An antigen control was considered effective when its average OD value was about 1.65. The titer (log 10) of the strong positive serum control should be 2.25±0.3 and the titer (log 10) of the negative serum control should be less than 0.9.

3. Other solutions of the kit are prepared:

(1) sample dilution: phosphate Buffer (PBS) containing 0.01mol/L of tween-20 at a volume concentration of 0.1% and ph=7.2-7.4;

(2) washing liquid: 1mL of Tween-20 (Tween-20) was added to 1000mL of 0.01M PBS solution;

(3) stop solution: 54.34mL of 98% concentrated sulfuric acid is added with distilled water to 1000 mL.

4. Data and statistical analysis

Subject operating characteristics (ROC) curve analysis is used to determine the cut-off value, sensitivity, pearson coefficient test and specificity of assays by using serum from young pigs and SVA positive porcine serum from experimentally infected or immunized animals. P <0.05 is considered statistically significant.

5. Results

(1) Production and characterization of SVA antigens

SVA supernatants were concentrated and purified in a 10% -50% (wt/vol) sucrose linear gradient.

As shown in FIG. 4A, 10mL of the sample was uniformly distributed in 20 test tubes, and OD was measured _260/280 Values to produce peaks. The 12 th tube where the peak was located was taken out, and round particles having a diameter of 30nm were observed by a transmission electron microscope. Dynamic light scattering technique further determined that the particle size of the sample tube was 28nm (fig. 4B). Finally, SDS-PAGE was used to identify VP2, VP3 and VP1 bands of SVA (FIG. 4C).

(2) Sensitivity and specificity analysis:

192 serum samples with different antibody titers were tested by established ELISA method, with a specificity of 100% and a sensitivity of 98.85%.

(3) Sensitivity and specificity of LPB-ELISA

A total of 87 SVA negative sera and 105 SVA infected or immunized sera confirmed by previous studies were selected to determine the cutoff value of the LPB-ELISA. When LCut-off value of PB-ELISA 1.65log ₁₀ Optimum sensitivity and specificity values are obtained when. Sensitivity and specificity were 100% and 99.04%, respectively (fig. 5A and 5C). The area under ROC curve (AUC) of the LPB-ELISA was calculated as 0.9996 (standard error [ SE)]= 0.0005261). The 95% confidence interval is 0.9986-1.000 (fig. 5B).

(4) Correlation of LPB-ELISA with VNT

Antibodies from 68 serum samples obtained from SVA-infected or immunized pigs were tested using established LPB-ELISA and VNT experiments to determine if the established method was able to detect NA. As shown in FIGS. 6A and 6B, all 68 serum samples were positive, and the LPB-ELISA and VNT assays were consistent (R ² ＝0.83，P<0.0001 Pearson correlation confirms this.

NA change kinetics of SVA infected or immunized pigs were analyzed to further verify the effectiveness of the established methods. NA level changes were monitored in two groups of SVA-VLP vaccine 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 (fig. 7C-7H). Both vaccine batches had 100% protection against porcine aggressive SVAs; thus, when log2>6.49 (1.65 log as described herein) ₁₀ ＝6.49log ₂ ) Full protection can be achieved (table 6).

TABLE 6 correlation of LBP-ELISA, VNT and detection protection rate

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Claims

1. A liquid phase blocking ELISA kit for a neutralizing antibody of a porcine Seebeck virus comprises a porcine Seebeck virus single domain antibody and a matched detection reagent.

2. The porcine saikovirus neutralizing antibody liquid phase blocking ELISA kit of claim 1, wherein the porcine saikovirus single domain antibody can also be an antigen binding fragment of a single domain antibody.

3. The liquid phase blocking ELISA kit of porcine sai virus neutralizing antibodies according to claim 1, characterized in that the single domain antibodies are selected from one or more of the group 52, the heavy chain variable region of each group of single domain antibodies or antigen binding fragments comprises CDR-1, CDR-2 and CDR-3, the amino acid sequence of the heavy chain variable region CDR-1 (VH-CDR-1) of the group 52 single domain antibodies or antigen binding fragments is shown as SEQ ID No.1 to SEQ ID No.52, the amino acid sequence of the heavy chain variable region CDR-2 (VH-CDR-2) is shown as SEQ ID No.53 to SEQ ID No.104, and the amino acid sequence of the heavy chain variable region CDR-3 (VH-CDR-3) is shown as SEQ ID No.105 to SEQ ID No.156, respectively.

4. The kit of claim 1, wherein the heavy chain constant region of the single domain antibody or antigen binding fragment thereof is selected from the group consisting of porcine IgG, human IgG and/or chicken IgY.

5. The kit of claim 1, wherein the heavy chain constant region IgG is selected from one or more of IgG1, igG2a, igG2b and IgG 4.

6. The kit of claim 1, wherein the heavy chain variable region has an amino acid sequence of 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; variants having 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%, at least 99% sequence identity or no more than 8 amino acids.

7. The kit of claim 1, wherein the porcine sai virus neutralizing antibody liquid phase blocking ELISA kit further comprises porcine sai virus antigen as a mixture of antigen and serum.

8. Application of a single domain antibody of porcine sai virus in preparing neutralizing antibody liquid phase blocking ELISA reagent.

9. The use of a single domain antibody of porcine Seika virus according to claim 8 in the preparation of a neutralizing antibody liquid phase blocking ELISA reagent comprising a reagent for detecting a single domain antibody or an antigen binding fragment of a single domain antibody of porcine Seika virus, said single domain antibody being selected from one or more of the group 52, the heavy chain variable region of each of said group of single domain antibodies or antigen binding fragments of said single domain antibody comprising CDR-1, CDR-2 and CDR-3, the amino acid sequence of the heavy chain variable region CDR-1 (VH-CDR-1) of said group 52 single domain antibody or antigen binding fragment being shown as SEQ ID NO.1 to SEQ ID NO.52, the amino acid sequence of the heavy chain variable region CDR-2 (VH-CDR-2) being shown as SEQ ID NO.53 to SEQ ID NO.104, the amino acid sequence of the heavy chain variable region CDR-3 (VH-CDR-3) being shown as SEQ ID NO.105 to SEQ ID NO.156, respectively.

10. Use of a single domain antibody of porcine sai virus according to claim 8 for the preparation of a neutralizing antibody liquid phase blocking ELISA reagent, wherein the reagent may be a kit.