CN113563461A

CN113563461A - Competitive monoclonal antibody based on African swine fever virus CD2v protein, kit and application thereof

Info

Publication number: CN113563461A
Application number: CN202110729427.5A
Authority: CN
Inventors: 林祥梅; 冯春燕; 王彩霞; 吴绍强; 于浩洋; 刘晓飞; 仇松寅; 梅琳
Original assignee: Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Institute of Animal Science of CAAS
Priority date: 2021-06-29
Filing date: 2021-06-29
Publication date: 2021-10-29
Anticipated expiration: 2041-06-29
Also published as: CN113563461B

Abstract

The disclosure provides an antigen binding protein, an antibody or an active fragment specifically binding to African swine fever virus CD2v protein, an African swine fever virus competitive ELISA detection kit comprising the antigen binding protein, the antibody or the active fragment, and a preparation method and application thereof. The ELISA detection kit disclosed by the invention is convenient to produce, stable in effect, free of non-specific reaction and low in coating amount, and is beneficial to mass production of the kit; the African swine fever virus competitive ELISA method provided by the disclosure has the advantages of rapidness, sensitivity, simplicity, convenience, specificity and the like, and has wide application in clinical detection of African swine fever.

Description

Competitive monoclonal antibody based on African swine fever virus CD2v protein, kit and application thereof

Technical Field

The disclosure relates to the technical field of biology, and in particular relates to a competitive monoclonal antibody based on African swine fever virus CD2v protein, a kit and application thereof.

Background

African Swine Fever (ASF) is a virulent infectious disease of pigs, with mortality rates as high as 100%, caused by African Swine Fever Virus (ASFV) infection. The African swine fever virus is the only DNA entomovirus known at present, the virus has an envelope, is mostly positive in blood adsorption, and can adsorb pig red blood cells to form a characteristic red blood cell rosette, which is related to a blood adsorption protein CD2v coded by the virus.

The CD2v protein is encoded by EP402R gene, has a full length of 1083bp, and is a glycoprotein assembled by a signal peptide, a transmembrane region, an intracellular region and two extracellular immunoglobulin-like domains. Wherein, the two immunoglobulin-like domains are the binding sites of erythrocytes, and have certain sequence homology with mouse, rat and human CD2 molecules. The intracellular domain has no sequence homology to the cellular CD2 molecule and is highly conserved between different strains of ASFV. The CD2v protein is ASFV late expression protein, is ASFV virulence factor and immune evasion protein, and is a common target gene for developing gene deletion vaccine.

In recent years, many reports on the research of CD2v deletion vaccines exist, but gene deletion vaccines have a great safety hazard, and gene mutation or recombination may occur during the use process, so that new strains are prevalent, and therefore, a differential diagnosis research method for CD2v genes is urgently needed to be established. Although the titer of blood adsorption inhibition antibodies of most of recovered pigs infected with ASFV is low, a plurality of researches prove that the CD2v protein has good immunogenicity and can be used for preparing and researching ASF immune serological diagnostic reagents. For example, the recombinant CD2v antigen with strong immunogenicity is obtained by using a prokaryotic expression vector pET-ELP, such as Zhou Xiao Hui et al, and the antigen is proved to have good detection specificity through experiments. The ASFV CD2vN-Fe protein is successfully expressed in autumn equinox, and the protein is proved to have good immunogenicity. The Ningxian et al utilizes prokaryotic expression vector pET-28a to express CD2v protein and prepare polyclonal antibody, and lays foundation for further research on biological function of CD2v protein.

However, the sensitivity and specificity of existing antibodies based on CD2v protein are still poor.

Disclosure of Invention

The invention aims to provide a competitive monoclonal antibody for African swine fever virus CD2v protein, a competitive ELISA detection reagent containing the monoclonal antibody, a detection kit of the detection reagent, and application of the detection reagent or the detection kit in detection of African swine fever virus, so as to overcome the defects of the existing detection technology.

In a first aspect, the present disclosure provides an antigen binding protein that specifically binds to african swine fever virus CD2v protein, wherein the antigen binding protein comprises at least one heavy chain variable region having the amino acid sequence shown in SEQ ID No. 2, or a conservative variant obtained by one or more amino acid additions, deletions, substitutions or modifications to the amino acid sequence shown in SEQ ID No. 2, and at least one light chain variable region; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 3, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3.

Wherein the amino acid sequence shown in SEQ ID NO. 2 is: ESGSLIGDLGSLKLSGQSGLQLHVKPEVMLCATLVMAPFYRASGRLEWVATFTSYFWKEWSLGSMVSLRSEVKGRFAMYFGVRQTPEGAVPTSDINAKNTLGISRSLSCATEGGDLQMSDFSTGMSTVSYQVGFKV are provided.

The amino acid sequence shown in SEQ ID NO. 3 is: QYNAEGKGDIQSWGTPASFLSTISLGHQRSNKAGLNIMTLSLKPHASEIKKSASAVEGTQEGVVTVTSLCQMWGSSCRAALQNSEPYISLFSSEYLAKSLSYYASKSVLGGPCITFRSYQAIQTGTTPYTFGGEYLVTQFEQDFYK are provided.

In a second aspect, the present disclosure provides an antibody or an active fragment specifically binding to african swine fever virus CD2v protein, wherein the antibody or the active fragment comprises at least one heavy chain variable region and at least one light chain variable region, the heavy chain variable region has an amino acid sequence shown in SEQ ID No. 2, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence shown in SEQ ID No. 2; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 3, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3.

Preferably, the antibody or active fragment is a monoclonal antibody and/or a genetically engineered antibody; the genetic engineering antibody is selected from one of a single-chain antibody, a single-chain antibody fragment, a chimeric monoclonal antibody fragment, a modified monoclonal antibody and a modified monoclonal antibody fragment.

Further preferably, the antibody is murine monoclonal antibody 30H6, the heavy chain variable region of murine monoclonal antibody 30H6 is shown as SEQ ID NO:2, and the light chain variable region of murine monoclonal antibody 30H6 is shown as SEQ ID NO: 3.

In a third aspect, the present disclosure provides an african swine fever virus competition ELISA detection kit, wherein the kit comprises the antigen binding protein of the first aspect, or the antibody or active fragment of any one of the second aspects.

Preferably, the antigen binding protein, antibody or active fragment is an antigen binding protein, antibody or active fragment labeled with a label, preferably, the label is selected from at least one of an enzyme, a fluorescent group or a chemiluminescent group.

Preferably, the kit comprises a microplate coated with ASFV CD2v protein, an enzyme-labeled murine monoclonal antibody 30H 6; more preferably, the coating amount of the CD2v protein is 0.1-8 mug/mL; the enzyme-labeled mouse monoclonal antibody 30H6 is used by volume dilution of 1 (2000-40000), preferably, the CD2v protein is obtained by eukaryotic expression.

Further, the kit of the present disclosure may further include a sample diluent, a washing solution, a developing solution, a stop solution, and ASFV standard positive serum and negative serum.

In a fourth aspect, the present disclosure provides the use of an antigen binding protein of the first aspect, or an antibody or active fragment of any one of the second aspects, or a kit of any one of the third aspects, for detecting african swine fever virus in a sample, preferably, the sample is an environmental sample, a food sample, a serum sample, or a blood sample.

In a fifth aspect, the present disclosure provides a method for in vitro detection of african swine fever virus in a sample, preferably, an environmental sample, a food sample, a serum sample, or a blood sample, using the antigen binding protein of the first aspect, or the antibody or active fragment of any of the second aspects, or the kit of any of the third aspects.

Preferably, the method may comprise the steps of: (1) adding the sample into an enzyme-labeled microporous plate; (2) adding the antigen binding protein of the first aspect or the antibody or the active fragment of the second aspect which is diluted by the diluent into an enzyme-labeled microplate, and incubating; (3) determination of OD after development_450nmCalculating the value and calculating the PI value; (4) and when the PI value is more than or equal to 50%, judging the sample to be a positive sample, when the PI value is less than or equal to 40%, judging the sample to be a negative sample, and when the PI value is more than 40% and less than 50%, judging the sample to be a suspicious sample.

Through above-mentioned technical scheme, this disclosure has following beneficial effect:

(1) the monoclonal antibody aiming at the African swine fever virus CD2v protein has high titer and stable property, can be prepared by mouse ascites, is suitable for mass production, and can be competitively combined with ASFV positive serum to coat antigen;

(2) the titer of the 30H6 antibody after enzyme labeling is high, the African swine fever competitive ELISA method established by the method has good repeatability, good specificity and high sensitivity, the positive and negative differentiation of the detection result is obvious, and the coincidence rate of the detection result can reach 100 percent compared with the imported similar commercial kit;

(3) in the African swine fever virus competitive ELISA detection kit provided by the disclosure, the coating antigen CD2v protein can be obtained through eukaryotic expression, the production is convenient, the effect is stable, non-specific reaction does not exist, the coating amount is low, and the kit is beneficial to mass production;

(4) the African swine fever virus competitive ELISA method provided by the disclosure has the advantages of rapidness, sensitivity, simplicity, convenience, specificity and the like, and has wide application in clinical detection of African swine fever.

In accordance with the disclosed embodiments, certain amino acid residues in an amino acid sequence can be conservatively substituted without altering the activity or function of the protein, as shown in Table I below:

TABLE I

Residue of	Conservative substitutions	Residue of	Conservative substitutions
				Ala	Ser	Leu	Ile；Val
Arg	Lys	Lys	Arg；Gln
				Asn	Gln；His	Met	Leu；Ile
Asp	Glu	Phe	Met；Leu；Tyr
				Gln	Asn	Ser	Thr；Gly
Cys	Ser	Thr	Ser；Val
				Glu	Asp	Trp	Tyr
Gly	Pro	Tyr	Trp；Phe
				His	Asn；Gln	Val	Ile；Leu
Ile	Leu；Val

Furthermore, because of the degeneracy of the bases, substitutions can be made to bases of a polynucleotide sequence without altering the activity or function of the polynucleotide sequence, see Table II below:

TABLE II

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 is an identification chart of SDS-PAGE electrophoretic detection of purified CD2v protein, wherein M is the relative molecular mass of the protein, and 1 is the purified CD2v recombinant protein;

FIG. 2 is a graph showing the results of immunofluorescence assay of supernatants from hybridoma 15C8 and hybridoma 30H 6.

Detailed Description

The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

Examples

1. Materials and sources

PCR SuperMix, E.coli DH5 alpha competent cells, DH10Bac competent cells were purchased from Kyoto Kogyo gold Biotech Ltd. The pCAGGS vector, the pFastbac1 vector and the 293F cell are stored in an animal inspection and quarantine laboratory of Chinese inspection and quarantine scientific research institute. Fetal bovine serum, DMEM, HAT and HT are all products of Gibco, USA. Inactivated positive serum of porcine circovirus type 2 inactivated vaccine (LG strain), inactivated positive serum of porcine reproductive and respiratory syndrome live vaccine (HuN4-F122 strain), inactivated positive serum of Seneca, inactivated positive serum of porcine pseudorabies live vaccine (Kartha-K61 strain) and 185 parts of ASFV-negative sera were preserved by the laboratory. 5 parts of inactivated African swine fever positive serum and 40 parts of inactivated serum to be detected are stored by the African swine fever national reference laboratory of the Poland national veterinary institute. The ID-Vet ASFV antibody detection kit is purchased from Shenzhen Qinghan coral science and technology Limited. Myeloma cell SP2/0 was stored in this laboratory. Trizol was purchased from Thermo, EcoRI, XhoI and rTaq from TaKaRa, and all other chemicals were analytically pure. HRP Conjugation Kit was purchased from abcam.

The Cloning Kit is available from Beijing Quanjin Biotechnology Ltd. QuickAntibody-Mouse5W was purchased from Biotechnology Ltd, Boolong, Beijing. The HRP-labeled goat anti-mouse IgG and IgG subclass identification kit was purchased from Sigma.

SPF-grade 6-8 week-old BALB/c mice were purchased from Beijing Wittingle laboratory animal technology, Inc.; SPF piglets are purchased from SPF pig breeding management center in Beijing.

2. Obtaining and purifying African swine fever CD2v antigen

2.1 recombinant plasmid construction

According to the BA71v strain (GenBank: NC-001659) CD2v gene sequence published by GenBank, the CD2v protein is subjected to physicochemical property, secondary structure and tertiary structure analysis and T, B lymphocyte epitope Prediction by using bioinformatics technologies such as ExPASy, SOMPA, PSIPRED Server, DNASTAR, Phyre, ABCcred Prediction, Scatch, Nectcle and IEDB, 6 dominant antigen epitope regions are screened and linked by a Linker, and two dominant antigen epitope regions of C-end 236-281 and 288-378 are folded to form 2 repeated sequences, so that the gene sequence SEQ ID NO 1 of the CD2v target gene sequence is obtained, wherein the specific sequence is as follows:

tatgataataatcgtagtGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCgattccccaactattacatataattgtactaattctttaataacatgtaaaaataataatgggacaGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCgttaatgatactaatggagatatccttaattattattggaatggtaataataattttGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCtcattgaatgaaacagaaaatataaatGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCaaacatgttgaagaaatagaaagtccaccaccctctgaatctaatgaagaagatatttctcacgatgacaccacttccatacatgaaccatctcccagagaaccattacttcctaagccttacagtcgttatcagtatGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCatgcgtccctcaacacaaccactcaacccatttcccctacctaaaccatgcccgccacctaaaccatgtcctccacccaagccatgcccgccacccaaaccatgtcctccacctaaaccgtgttctccacccaaaccgtgtcgtccacctaaaccatgtcctccacctaaaccatgtcctccacctaaaccatgtcctccacctaaaccatgtcctccatccaaaccatgtccttcacctgaatcctattctccacccaaaccactacctagtGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCaaacatgttgaagaaatagaaagtccaccaccctctgaatctaatgaagaagatatttctcacgatgacaccacttccatacatgaaccatctcccagagaaccattacttcctaagccttacagtcgttatcagtatGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCatgcgtccctcaacacaaccactcaacccatttcccctacctaaaccatgcccgccacctaaaccatgtcctccacccaagccatgcccgccacccaaaccatgtcctccacctaaaccgtgttctccacccaaaccgtgtcgtccacctaaaccatgtcctccacctaaaccatgtcctccacctaaaccatgtcctccacctaaaccatgtcctccatccaaaccatgtccttcacctgaatcctattctccacccaaaccactacctagt。

the sequence is synthesized by Beijing Optimalaceae Biotechnology Co., Ltd, EcoRI restriction site, XhoI restriction site, His tag and signal peptide region are respectively introduced at two ends of the sequence, the CD2v target gene sequence (hereinafter referred to as His-CD2v) is inserted into eukaryotic expression vector pCAGGS through the two restriction sites, pCAGGS-ASFV-CD2v recombinant plasmid is constructed, escherichia coli DH5 alpha competent cell is transformed, and the plate coating culture is carried out overnight. And (4) selecting positive clones for sequencing on the next day, adding glycerol into positive bacteria liquid with correct sequencing for bacteria preservation, and storing at-80 ℃ for eukaryotic expression.

Cloning the CD2v target gene fragment into a pFastBac1 vector by the same method to construct a pFastBac1-ASFV-CD2v recombinant plasmid, transforming the positive recombinant plasmid pFastBac1-ASFV-CD2v with correct sequencing into an escherichia coli DH10Bac competent cell, carrying out blue-white spot screening, culturing at 37 ℃ for 48h, selecting a white colony, identifying by using an M13 universal primer, carrying out amplification culture, extracting the recombinant plasmid by using a plasmid extraction kit, and naming the recombinant plasmid as Bacmid-ASFV-CD2v for recombinant baculovirus.

2.2 expression and purification of His-CD2v protein in 293F cells

Using 293-SIM medium (product M293II of China Bio Inc.) and 5% CO₂Suspending at 37 deg.CHEK-293F cells were cultured. A mixture of 2mg of pCAGGS-ASFV-CD2v plasmid and 12mg of PEI was prepared in 1L of the cell culture medium, and the cell density was 2X 10⁶cells were transfected with the mixture at cell/ml. 48h after the transfection, the culture solution is centrifuged for 20min under the centrifugation condition of 1000 Xg and the supernatant of the transfected cells is collected, impurities are removed by using a 0.22 μm filter, then the obtained supernatant is added into pretreated Ni-NTA beads, the purification is carried out according to the operation instructions of the kit, the effluent of the target protein and the protein eluent are collected and concentrated by a 10Ku ultrafiltration tube, 10 μ L samples are taken for SDS-PAGE detection, and the result is shown in figure 1, and the purified protein has a dispersion band between 35KDa and 80KDa and can be His-CD2v protein. Meanwhile, the protein concentration is measured to be 3.5mg/mL by using a BCA protein assay kit, and the separated mixture is stored at the temperature of 80 ℃ below zero for later use.

2.3 antigenic characterization of the His-CD2v protein

The optimal coating concentration of the His-CD2v protein is determined to be 1 mu g/ml by adopting a chessboard titration method, and the coating condition is used for carrying out His-CD2v protein coating. The method comprises the steps of detecting an African swine fever inactivated positive serum sample (5 parts) and an inactivated negative serum sample (185 parts) by using indirect ELISA, detecting a blue-ear disease (PRRS) inactivated positive serum sample, detecting a Sailon-Va (SVA) inactivated positive serum sample, and detecting a Pseudorabies (PR) inactivated serum sample.

Detecting 5 parts of inactivated positive serum sample and 185 parts of inactivated negative serum sample of non-epidemic area by using indirect ELISA method, wherein the negative control is pig negative serum purchased from Hyclone, and the P/N ratio (more than or equal to 2.1) is used as the positive and negative judgment standard. The specific operation steps are as follows: diluting CD2v protein with antigen coating solution, adding 100 μ L per well, and coating overnight at 4 deg.C; PBST is washed for 3 times, 100 mu L of sealing liquid is added into each hole, and sealing is carried out for 1h at 37 ℃; PBS washing 3 times; all samples are diluted and detected at a ratio of 1:100, and meanwhile, the purchased negative serum of the non-immune pig is set as a negative control, and the samples are incubated for 1h at 37 ℃; PBST wash 3 times, PBS wash 2 times; mu.L of diluted rabbit anti-porcine HRP antibody (1:2000) was added to each well and incubated at 37 ℃ for 1 h; PBST wash 3 times, PBS wash 2 times; adding 100 μ L of TMB substrate solution into each well, reacting at room temperature for 15min, and adding 50 μ L of 2mol/L H₂SO₄Stopping the reaction with the stop solution, and measuring OD at 450nm with an enzyme-linked immunosorbent assay₄₅₀The value is obtained.

The results are shown in table 1: the detection results of 5 inactivated positive serum samples are positive, and in 185 inactivated negative serum samples, 183 detection results are negative, and 2 detection results are false positive. According to the calculation formula (table 1): DSe ═ a/(a + C) and DSp ═ D/(B + D), calculated with CD2v protein as coating antigen, DSe was 100% and DSp was 98.9%.

In addition, the specific experiment result shows that the His-CD2v protein has no cross reaction with a blue ear disease (PRRS) inactivation positive serum sample, a Saint Geranium (SVA) inactivation positive serum sample and a Pseudorabies (PR) inactivation serum sample. These results indicate that the His-CD2v protein expressed in this example has very good antigenicity.

TABLE 1 antigenic determination test results of CD2v protein

	Positive sample	Negative sample
			Positive test (copy)	5(A)	2(B)
Negative test (copy)	0(C)	183(D)

3. Preparation and sequencing of monoclonal antibodies against CD2v antigen

3.1 animal immunization and cell fusion

The purified His-CD2v protein was used as an immunogen, and the His-CD2v protein was diluted to 2-fold final concentration with physiological saline according to the adjuvant instructions. After the adjuvant is fully mixed, the required dosage (50 mul per injection) is taken out under the aseptic condition, and the adjuvant and the antigen are rapidly mixed according to the volume ratio of 1: 1. Mice are immunized by two modes, namely intramuscular injection of hind legs and shanks, multipoint injection of the back and the like, and each mouse is injected with 100 mu L. The immunization was boosted 1 time 21 days after the first injection in the same manner. And collecting blood from submaxillary vein on day 35, and collecting serum for indirect ELISA detection. Taking the spleen cell of the immune mouse with the highest titer and the bone marrow of the mouse with good growth state, and fusing the tumor cell SP2/0 according to the conventional method.

3.2 establishment of Indirect ELISA detection method

The optimal working concentration of coating antigen and antibody for indirect ELISA was determined by matrix titration. Diluting purified His-CD2v protein to 1mg/mL, diluting with coating buffer solution (0.05mol/L, pH9.6 carbonate buffer solution) at 1:400, 1:600, 1:800, 1:1000, 1:1200 and 1:1400 respectively, coating ELISA plate with 100 μ L of antigen dilution solution per well, overnight at 4 deg.C, diluting BALB/c mouse positive serum and negative serum at 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200 times respectively, setting up sp2/0 cell culture supernatant and blank control, selecting OD_450nmThe value is about 1.0, and the antigen dilution concentration with the maximum P/N ratio is the optimal working concentration. The results show that the optimal dilution of antigen is 1:1000 (final concentration of protein 1. mu.g/mL).

3.3 establishment of monoclonal antibody (m Ab) hybridoma cell line against CD2v protein

The His-CD2v protein expressed by eukaryotic cells is taken as a coating antigen, and the established indirect ELISA method is adopted to detect the specific antibody in the supernatant of the fusion cells. And selecting cells with high antibody titer, single clone growth and good cell state for subclone culture, and performing amplification culture until the antibody secretion is stable and the proportion of positive holes is more than 95%.

3.4 preparation of ascites and determination of potency

Co-screening by indirect ELISA method to obtain 3 hybridoma cells, selecting 2 hybridoma cells (15C8 and 30H6) with higher titerCulturing, reserving cell supernatant, performing conventional treatment on cells, performing intraperitoneal injection on Balb/c mice treated by incomplete Freund's adjuvant to prepare ascites, and measuring the prepared ascites by using an indirect ELISA method, wherein the results show that the titer of the prepared ascites can reach 1:10⁶. mAb in the ascites was purified by the octanoic acid-ammonium sulfate method.

3.5 immunofluorescence identification of monoclonal antibodies

Transfecting Bacmid-ASFV-CD2v to SF9 cells, packaging recombinant baculovirus, infecting Hi5 cells with P3 virus, laying a 96-well plate according to the cell number, continuously culturing for 48h, removing culture medium supernatant, washing twice with PBS, and fixing the cells with 4% paraformaldehyde at room temperature for 15 min; washing with PBS for 3 times, adding 100 μ L goat serum into each well, and sealing at 37 deg.C for 1 h; removing the blocking solution by suction, adding 100 mu L of cell supernatant into each hole for incubation, and incubating for 1h at 37 ℃ by taking mouse negative serum as a negative control; PBS washing 3 times; adding 1:1000 diluted Alexa Fluor488 labeled goat anti-mouse IgG into each well, and incubating for 1h at 37 ℃ in the dark; DAPI staining was performed after 3 PBS washes, and the results were observed under a fluorescent microscope. As shown in fig. 2, green fluorescence was clearly observed in cell supernatants of 15C8 and 30H6, while no green fluorescence was observed in the negative control, indicating that both monoclonal antibodies specifically recognize CD2v protein.

3.6 stability and subtype identification of monoclonal antibodies

And measuring the titer of culture supernatants of 10 th generation, 20 th generation, 30 th generation, 40 th generation and 50 th generation of the screened 15C8 and 30H6 hybridoma cells by using an indirect ELISA method to determine the stability of the hybridoma cells. The result shows that the two hybridoma cells can stably secrete the monoclonal antibody, and the titer is basically kept at the same level.

The subclass of IgG of the monoclonal antibody ascites is identified by using a subclass identification kit of Sigma company, and the specific operation is carried out according to the kit instruction. The subclass of the 15C8 monoclonal antibody is IgG2b/k type, and the subclass of the 30H6 monoclonal antibody is IgG1/k type.

Establishment of competitive ELISA method for ASFV CD2v monoclonal antibody

4.1 reagent preparation

(1) Coating liquid: 0.05mol/L, pH 9.6.6 carbonate buffer;

(2) washing liquid: pH7.4, 0.1M PBS, 0.05% Tween-20;

(3) sealing liquid: 5% BSA solution prepared with washing solution;

(4) diluting liquid: namely the confining liquid;

(5) TMB: solution A: 0.02% H₂O₂Diluting with 0.1M citric acid-0.2M disodium hydrogen phosphate solution (pH5.0);

and B, liquid B: dissolving 0.4 ‰ TMB-HCl in 50mM sodium citrate solution (pH 2.8);

mixing the 50 μ L A solution and the solution B, and storing in dark place (or using commercial TMB developing solution);

(6) stopping liquid: 2M of H₂SO₄And (3) solution.

4.2 preparation of Standard ASFV Positive serum and Standard ASFV negative serum

In order to reduce errors of the ELISA kit caused by different operators and different detection batches in the actual detection process, the standard ASFV antibody positive control serum and the standard ASFV antibody negative control serum are prepared in this embodiment and used for optimization of the detection method and determination of the result judgment standard.

4.2.1 preparation of Standard Positive serum

SPF piglets of 6 weeks old were selected and the skin of the injection site was disinfected with iodine. For the first immunization, 5mL of Freund's Complete Adjuvant (FCA) and an equal amount of purified eukaryotic expression His-CD2v antigen (3.5mg/mL) are sucked by a syringe for emulsification, and the piglet is subjected to multi-point immunization by intramuscular injection. After 10-14 days of interval, a second immunization is carried out, and 8ml of Freund incomplete adjuvant is taken and emulsified with an equal amount of purified eukaryotic expression His-CD2v antigen, and then the piglets are injected with multiple points of muscle in the same way. After 7-10 days, collecting blood from the anterior vena cava, separating serum, detecting serum titer by ELISA method, collecting blood from heart when antibody titer can reach above 1:10000, adding thimerosal into the separated serum for antisepsis, subpackaging with 0.5ml, and storing at-20 deg.C for use.

4.2.2 preparation of Standard negative serum

Collecting qualified SPF pig, collecting blood from heart, separating to obtain negative serum, and adding thimerosal for antisepsis. 0.5ml of the extract was dispensed into a sterile tube and stored at-20 ℃ for further use.

4.3 preparation and screening of enzyme-labeled antibodies

Purified CD2v monoclonal antibodies 15C8 and 30H6 are subjected to HRP labeling by using HRP conjugation Kit of abcam company according to Kit operation steps to obtain the required enzyme labeled antibodies 15C8-HRP and 30H 6-HRP.

Eukaryotic expression of His-CD2v antigen at 1 g/mL coating concentration per well 100 u L, 4 degrees C coated overnight; pouring off the coating solution the next day, washing with the washing solution for 3 times, drying, adding 200 μ L of 5% BSA solution prepared with the washing solution into each hole, sealing at 37 deg.C for 1h, and washing with the washing solution for 3 times; diluting standard negative and positive serum with blocking solution at a ratio of 1:10, incubating at 37 deg.C for 1h with 100 μ L per well, and washing with washing solution for 3 times; diluting HRP-labeled 15C8-HRP and 30H6-HRP by 1:10000 times and 1:20000 times respectively, adding 100 μ L of the diluted solution into each well, incubating for 1H at 37 ℃, washing with washing solution for 3 times, and draining; adding 100 μ L of TMB color developing solution into each well, and developing at room temperature for 15 min; the reaction was stopped by adding 50. mu.L of stop solution to each well and reading at 450nm with a microplate reader. And calculating the N/P value of different enzyme-labeled antibodies.

As shown in Table 2, the best competitive enzyme-labeled antibody was selected as the antibody having the highest N/P value. As can be seen from Table 2, the N/P value of 30H6-HRP at 1: 10000-fold dilution and 1: 20000-fold dilution is higher than that of 15C8-HRP, therefore, 30H6-HRP is selected for optimization and establishment of CD2v competitive ELISA detection method.

TABLE 2 screening test results for enzyme-labeled antibodies

4.4 determination of optimal coating concentration of antigen and optimal dilution of sample to be tested

According to a chessboard titration method, coating concentrations of eukaryotic expression His-CD2v antigen of 0.1 mu g/mL, 0.5 mu g/mL, 1.0 mu g/mL, 2.0 mu g/mL, 4.0 mu g/mL and 8.0 mu g/mL are respectively adopted, 100 mu L of antigen coating solution is coated in each hole, the coating solution is poured out after the night at 4 ℃, and the washing solution is washed for 3 times and is dried; adding 200 μ L of 5% BSA solution prepared from washing solution into each well, sealing at 37 deg.C for 1h, and washing with washing solution for 3 times; diluting standard negative and positive serum by multiple times from 0, 1:2 to 1:32, respectively adding into 96-well enzyme-labeled microporous plate from top to bottom, incubating at 37 deg.C for 1h with each well of 100 μ L, and washing with washing solution for 3 times; then 100 mu L of enzyme-labeled antibody diluted at the ratio of 1:20000 is added into each hole, the incubation is carried out for 1h at the temperature of 37 ℃, and washing liquid is washed for 3 times and is dried by beating; adding 100 μ L of TMB color developing solution into each well, and developing at room temperature for 15 min; the reaction was stopped by adding 50. mu.L of stop solution to each well and reading at 450nm with a microplate reader. And selecting the optimal antigen coating concentration and the optimal enzyme-labeled antibody dilution according to the N/P value.

As shown in Table 3, it can be seen from Table 3 that the N/P value is the largest and 10 or more when the antigen coating concentration is 1. mu.g/mL and the serum dilution is 1:2, and therefore it is determined that the antigen coating concentration is 1. mu.g/mL optimally and the sample dilution is 1:2 optimally.

TABLE 3 antigen coating concentration and sample dilution optimization test results

TABLE 3 antigen coating concentration and dilution of sample optimization test results (Table continuation)

4.5 optimization of the optimal dilution of enzyme-labeled antibodies

The same method as 4.4, the enzyme-labeled antibody is diluted 1:2000, 1:5000, 1:10000, 1:20000, 1:30000 and 1: 40000. And (3) performing an optimization experiment of the optimal enzyme-labeled antibody dilution under the same other conditions by using standard negative and positive serum according to the determined optimal antigen coating concentration and the optimal dilution of the sample to be detected, reading a light absorption value of 450nm by using an enzyme-labeling instrument after the reaction is finished, calculating an N/P value, and determining the optimal dilution of the enzyme-labeled antibody to be 1:30000 according to the N/P value.

As shown in Table 4, it is understood from Table 4 that the N/P value is the largest and 10 or more when the dilution of the enzyme-labeled antibody is 1:30000, and thus the optimum dilution of the enzyme-labeled antibody is determined to be 1: 30000.

TABLE 4 dilution of enzyme-labeled antibody optimization test results

4.6 determination of optimal blocking solution

The method is the same as the above, 10% bovine serum, 5% BSA, 5% skimmed milk powder and 1% gelatin are respectively used as blocking liquid to be blocked for 1h at 37 ℃, other conditions are tested according to the determined optimal conditions, standard negative and positive serum samples are detected, after the reaction is finished, a light absorption value of 450nm is read by using an enzyme labeling instrument, the N/P value is calculated, and the condition when the N/P value is the highest is determined as the optimal blocking condition.

As shown in Table 5, it can be seen from Table 5 that the N/P value is the largest and 10 or more when the blocking solution is 5% BSA, and therefore the optimal blocking solution was determined to be 5% BSA.

TABLE 5 optimized test results for optimal confining liquids

4.7 determination of optimal color development time

And (3) performing a test according to the determined optimal reaction condition, adding a TMB color development solution, performing color development in a dark place at room temperature for 5min, 10min, 15min and 20min, respectively, detecting standard negative and positive serum samples under the condition that other conditions are not changed, reading a light absorption value of 450nm by using an enzyme labeling instrument after the reaction is finished, calculating an N/P value, and determining the reaction condition when the N/P value is the highest as the optimal color development time.

As shown in Table 6, it can be seen from Table 6 that the N/P value is the largest and 10 or more when the development time is 15min, and therefore the optimum action time of TMB was determined to be 15min at room temperature.

TABLE 6 optimization of TMB development time test results

4.8 determination of the criteria for determination of negative and positive sera by competitive ELISA

The limit of negative and positive sera of the c-ELISA of the present disclosure was determined by using the established monoclonal antibody c-ELISA method to test 5 known positive sera and 100 negative sera 3 times, respectively, and calculating the inhibition ratio (PI) of each known background serum sample according to the following formula.

The detection result shows that the PI% values of 5 positive serums are all more than 50%, and the PI% values of 100 negative serums are all less than 40%. Therefore, the determination criteria for determining the monoclonal antibody c-ELISA negative and positive serum samples established in the present study are: when the serum PI is more than or equal to 50 percent, the serum PI can be judged to be positive; when the serum PI is less than or equal to 40 percent, the serum PI can be judged to be negative; when the range of serum PI is 40% < PI < 50%, it can be judged as suspicious.

4.9 repeatability test

The stability of the c-ELISA method was evaluated on the basis of the intra-and inter-batch coefficient of variation. Error in batch: and (3) detecting 2 positive serums by using 3 enzyme-labeled plates coated in the same batch, setting two repeated holes in each serum, and calculating the variation coefficient of the two repeated holes to express the error in the batch. Batch-to-batch error: taking the ELISA plates coated for 3 times at different time, simultaneously detecting 2 positive serums, arranging two repeated holes on each plate, and calculating the inter-batch variation coefficient to express the inter-batch error. The results are shown in Table 7.

TABLE 7 results of the repeatability tests

The results of the repeated experiments in batches show that the coefficient of variation values are 1.27% and 1.19% respectively; the results of the batch-to-batch repeated experiments showed that the coefficient of variation was 3.82% and 3.66%. The intra-batch variation coefficient and the inter-batch variation coefficient of the two samples are both less than 10%, so that the monoclonal antibody c-ELISA method determined by the embodiment has better repeatability.

4.10 evaluation of the specificity of monoclonal antibody c-ELISA

According to the determined c-ELISA operation program, ASFV standard positive serum, pcv2 vaccine inactivated positive serum, PRRSV vaccine inactivated positive serum, seneca inactivated positive serum and PRV vaccine inactivated positive serum are respectively detected, the inhibition rate is calculated according to the color development result and the formula, and the specificity of the monoclonal antibody c-ELISA is evaluated according to the inhibition rate.

The detection results are shown in table 8, and it can be seen from table 8 that the PI% values corresponding to the pcv2 vaccine inactivated positive serum, the PRRSV vaccine inactivated positive serum, the seneca inactivated positive serum, and the PRV vaccine inactivated positive serum are all lower than 40%, the PI% corresponding to the ASFV standard positive serum is higher than 50%, and is much higher than the PI% values corresponding to the foregoing several sera, so that it can be determined that the 30H6 monoclonal antibody competitive ELISA method of the present embodiment can distinguish several common diseases of swine from ASFV, and thus has better specificity.

TABLE 8 monoclonal antibody c-ELISA specificity test results

Kind of serum	Inhibition ratio (PI%)
		ASFV standard positive serum	91.35
Pcv2 vaccineInactivation positive serum	9.75
		PRRSV vaccine inactivated positive serum	10.22
Inactivated positive serum of intracapsin	10.31
		PRV vaccine inactivated positive serum	8.79
ASFV standard negative serum	0

4.10 evaluation of sensitivity of monoclonal antibody c-ELISA

The ASFV standard positive serum and the ASFV standard negative serum are respectively diluted in a gradient multiple ratio of 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280, and the detection is carried out by using optimized c-ELISA. And simultaneously, the ID-Vet kit is also adopted to detect the serum diluted by times, and the sensitivity of the two ELISA kits is compared. The detection results are shown in table 9, and it can be seen from table 9 that the lowest antibody recognition concentrations of the two detection kits determined in this example are both diluted 1:640 times, and it can be seen that the 30H6 monoclonal antibody competitive ELISA method of this example has better sensitivity.

TABLE 9 monoclonal antibody c-ELISA sensitivity test results

4.11 detection of clinical samples

And (3) detecting 40 parts of inactivated pig serum to be detected by using the optimized c-ELISA reaction conditions, and comparing the detection result with the detection result of a c-ELISA kit of IDvet company.

The results are shown in Table 10. The number of positive samples detected by the monoclonal antibody c-ELISA is 3, the number of negative samples is 37, 3 positive samples and 37 negative samples are detected by the c-ELISA kit of the IDvet company, the detection results of the two detection methods are consistent, and the coincidence rate reaches 100%. Therefore, the CD2vc-ELISA kit of the embodiment can achieve the same detection effect as the existing commercial kit.

TABLE 10 sample detection results of the disclosed c-ELISA and IDvet kits

4.12 preparation of enzyme-labeled plate

Diluting the His-CD2v antigen expressed by eukaryotic cells with coating solution according to the optimal coating concentration (1 mug/mL), adding 100 mug L of the coating solution into a 96-hole enzyme-labeled micropore plate per hole, and standing overnight at 4 ℃; the next day the wells were drained, the wash washed 3 times, and the last beat dried, after which 200. mu.L of blocking solution (5% BSA) was added to each well and blocked for 1h at 37 ℃. Washing with the washing solution for 3 times, drying, and vacuum packaging.

4.13 procedures of ASFV CD2v c-ELISA detection kit

4.13.1 diluting the sample to be detected with the sample diluent 1:2, adding 50 μ L of diluted serum to be detected into each hole, adding 50 μ L of enzyme-labeled monoclonal antibody diluted with 1:10000, setting standard negative and positive controls, incubating at 37 ℃ for 30min, washing with washing solution for 5 times, adding 100 μ L of TMB substrate developing solution into each hole, developing at room temperature for 15min, and developing at 2mol/L H₂SO₄Color development was stopped at 50. mu.L/well and OD was measured_450nmValues and serum inhibition rates were calculated according to the formula.

4.13.2 judging the result: when the PI of the detected serum is more than or equal to 50 percent, the result can be judged to be positive; when the PI of the detected serum is less than or equal to 40 percent, the detected serum can be judged to be negative; when the range of PI of the serum to be tested is 40% < PI < 50%, it can be judged as suspicious.

Determination of 5CD2v competitive monoclonal antibody sequence

5.1 extraction of hybridoma cell RNA

When CD2v is competitiveWhen the hybridoma cells of the clone antibody 30H6 grew to a monolayer, the culture solution was discarded and blown down with 3mL of sterile PBS and counted, and 1X 10 cells were collected⁶And (3) putting the cells into a 1.5mL centrifuge tube, centrifuging for 5min at the speed of 800r/min, discarding the supernatant, and extracting the RNA of the hybridoma cells by using an RNA extraction kit according to the operation steps of the instruction.

5.2 RT-PCR amplification and identification of RNA extracted from hybridoma cells

14 degenerate primers (among them, VH-F4, VH-R2, VL-F6, VL-R2) were designed for a total of 20 pairs.

TABLE 11 heavy and light chain RT-PCR amplification primers for monoclonal antibodies

Primer and method for producing the same	Sequences (5 'to 3')	SEQ ID NO:
			mVL-F1	ATGGAGACAGACTCCTGCTAT	4
mVL-F2	ATGGATTTTCAGGTGTTTTCAG	5
			mVL-F3	ATGRAGTCACAKACGGTCTTYRTA	6
mVL-F4	ATGAGGKCCCHGCTYTYCTKGGR	7
			mVL-F5	ATGAAGTTGCCTGTGCTGTTG	8
mVL-F6	ATGATGAGTCCTGCCTTCC	9
			mVL-R1	ACTGGATGGTGGGAGGA		10
VL-R2	CCCAAGCTTACTTGGGAAGATGGA	11
			mVH-F1	ATGGRATGSAGCTGMATSCTCTT	12
mVH-F2	ATGRACTTCGGGYCTKGGTTTT	13
			mVH-F3	ATGGCTGTCTTGGGGCTCTTCT	14
mVH-F4	ATGGRCAGTACHTYY					15
			mVH-R1	AYCTCCACACRCCAGTGGATAGAC	16
VH-R2	CCCAAGCTTRCCARKGGATRA	17

Note: r is A/G, S is C/G, Y is C/T, M is C/A, K is T/G, H is A/T, D is G/T/A

Using the extracted RNA as a template, primers are shown in Table 11, and the reaction system is 50. mu.L (10 Xone Step RNA PCR Buffer 5. mu.L, MgCl)₂10 μ L (25mM), 10 μ L of dNTP mix (10mM), 1 μ L of RNase Inhibitor (40U/μ L), 1 μ L of AMV RTase XL (5U/μ L), 1 μ L of AMV-Optimized Taq (5U/μ L), 1.5 μ L of F-equivalent mixed primer, 1.5 μ L of R-equivalent mixed primer, 4 μ L of template, ddH₂O15. mu.L), reaction procedure (reverse transcription at 50 ℃ for 30min, pre-denaturation at 94 ℃ for 2min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, amplification for 35 cycles, and final extension at 72 ℃ for 10 min). Finally, 8 microliter of product is used for observing the RT-PCR amplification result by 1 percent agarose gel electrophoresis, and proper primers are selected for carrying out large-scale amplification on VH and VL after identification (the step is repeated twice). The band of interest was recovered by 2% agarose gel electrophoresis, and then VH and VL genes of interest were recovered using a gel recovery kit.

5.3 ligation transformation and characterization of VH and VL

Recovering purified VH and VL target genes

Connecting, wherein the reaction system comprises the following steps: 1 μ L

The purified PCR product was recovered in 4. mu.L. The reaction was carried out at room temperature for 30 min.

50 mu L of each Trans 5 alpha competent cell is taken to melt on ice, the ligation product is added and mixed evenly, the mixture is ice-cooled for 30min, heat shock is carried out on the mixture in water bath at 42 ℃ for 45s, and ice bath is carried out for 2-3 min. Adding 600 mu L of LB liquid culture medium, performing shake recovery culture at 37 ℃ and 220r/min for 60min, uniformly coating 100 mu L of bacterial liquid on an LB solid culture medium (AMP resistance), culturing in an incubator at 37 ℃ for 12-15 h, and observing the appearance of transformed single colonies. 4-6 single colonies were picked into a 1.5mL centrifuge tube (600. mu.L of AMP-resistant LB liquid medium), incubated at 37 ℃ in a shaker at 220r/min, and controls were set up.

After culturing for 4 hours at 37 ℃, 2.0 mu L of bacterial liquid is taken from each centrifugal tube for PCR identification of bacterial liquid. Reaction system 20 μ L: rTaq enzyme (5U/. mu.L) 0.2. mu.L, 10 XPCR buffer 2.0. mu.L, dNTP (2.5mM) 2.0. mu.L, M13-F (10. mu.M) 1.0. mu.L, M13-R (10. mu.M) 1.0. mu.L, bacterial suspension 2.0. mu.L, ddH₂O9.8 mu L; reaction procedure: pre-denaturation at 95 ℃ for 4min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, amplification for 32 cycles, and final extension at 72 ℃ for 10 min. And finally, taking 8 mu L of product, observing the PCR amplification result by using 1% agarose gel electrophoresis, and selecting positive clone bacterial liquid to send to sequencing.

Selecting 3 positive clones identified by the light and heavy chain gene clones of the 30H6 monoclonal antibody as sequencing samples to obtain the nucleotide sequence of the variable region of the antibody, and comparing by using DNAStar and IgBLAST software, wherein the results show that: the 3 light chain variable region genes are 408bp long and encode 136 amino acids; the 3 heavy chain variable region gene sequences are 438bp long and encode 146 amino acids. The heavy chain variable region has an amino acid sequence shown in SEQ ID NO:2 below:

ESGSLIGDLGSLKLSGQSGLQLHVKPEVMLCATLVMAPFYRASGRLEWVATFTSYFWKEWSLGSMVSLRSEVKGRFAMYFGVRQTPEGAVPTSDINAKNTLGISRSLSCATEGGDLQMSDFSTGMSTVSYQVGFKV。

the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 3:

QYNAEGKGDIQSWGTPASFLSTISLGHQRSNKAGLNIMTLSLKPHASEIKKSASAVEGTQEGVVTVTSLCQMWGSSCRAALQNSEPYISLFSSEYLAKSLSYYASKSVLGGPCITFRSYQAIQTGTTPYTFGGEYLVTQFEQDFYK。

6 comparative experiment

6.1 construction of recombinant CD2v plasmid

The 6 dominant antigen epitope regions screened out in 2.1 are directly constructed into an expression vector pCAGGS after being linked by a Linker, and two dominant epitopes at the C end are not repeatedly folded to obtain a nucleotide sequence shown as SEQ ID NO. 18:

tatgataataatcgtagtGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCgattccccaactattacatataattgtactaattctttaataacatgtaaaaataataatgggacaGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCgttaatgatactaatggagatatccttaattattattggaatggtaataataattttGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCtcattgaatgaaacagaaaatataaatGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCaaacatgttgaagaaatagaaagtccaccaccctctgaatctaatgaagaagatatttctcacgatgacaccacttccatacatgaaccatctcccagagaaccattacttcctaagccttacagtcgttatcagtatGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCatgcgtccctcaacacaaccactcaacccatttcccctacctaaaccatgcccgccacctaaaccatgtcctccacccaagccatgcccgccacccaaaccatgtcctccacctaaaccgtgttctccacccaaaccgtgtcgtccacctaaaccatgtcctccacctaaaccatgtcctccacctaaaccatgtcctccacctaaaccatgtcctccatccaaaccatgtccttcacctgaatcctattctccacccaaaccactacctagt。

the sequence is synthesized by Beijing Optimalaceae Biotechnology Limited, EcoRI and XhoI restriction sites, His labels and signal peptide regions are respectively introduced at two ends of the sequence, the gene sequence is inserted into eukaryotic expression vector pCAGGS through the two restriction sites to construct pCAGGS-ASFV-CD2v (S) recombinant plasmid, protein expression and purification are carried out according to the operation steps in 2, the purified protein is used for preparing monoclonal antibody, 2 monoclonal antibodies 20G9 and 32B8 with competitive effect are screened, competitive ELISA (subsequently 20G9 competitive ELISA and 32B8 competitive ELISA) is respectively established, the optimized competitive ELISA method has different enzyme antibody concentrations, 20G9-HRP and 32B8-HRP are both diluted by 1:10000, the result judgment mode is consistent, and the two competitive ELISA competitions and the competitive ELISA (30H 6 for short) established by 30H6 are used for sensitivity competition, Comparison of specificity and detection of clinical samples.

6.2 comparison of the specificity of the three competitive ELISAs

Three competitive ELISA detection methods are respectively used for detecting ASFV standard positive serum, pcv2 vaccine inactivated positive serum, PRRSV vaccine inactivated positive serum, seneca inactivated positive serum and PRV vaccine inactivated positive serum, the inhibition rate is calculated according to the color development result and the formula, and the specificity of the monoclonal antibody c-ELISA is evaluated according to the inhibition rate.

As shown in table 12, it can be seen from table 12 that the inhibition rate of the 30H6 competitive ELISA of the present disclosure on the ASFV standard positive serum is significantly higher than that of the 20G9 competitive ELISA and that of the 32B8 competitive ELISA, but the inhibition rate on the remaining positive sera is significantly lower than that of the 20G9 competitive ELISA and that of the 32B8 competitive ELISA, and thus, compared with the 20G9 competitive ELISA and the 32B8 competitive ELISA, the 30H6 competitive ELISA of the present disclosure can better distinguish several common diseases of swine from ASFV, and thus the 30H6 competitive ELISA of the present disclosure has better specificity.

TABLE 12 monoclonal antibody c-ELISA specificity test results

6.3 comparative sensitivity assays for three competitive ELISAs

The ASFV standard positive serum and the ASFV standard negative serum are respectively diluted in a gradient multiple ratio of 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280, and three types of c-ELISA are used for detection.

As shown in table 13, it can be seen from table 13 that the 20G9 competitive ELISA can detect serum at the lowest 1: 320-fold dilution, the 32B8 competitive ELISA can detect serum at the lowest 1: 160-fold dilution, and the 30H6 competitive ELISA can detect serum at the lowest 640-fold dilution of serum at the 1: 160-fold dilution, and thus, the sensitivity of both the 20G9 competitive ELISA and the 32B8 competitive ELISA is lower than that of the 30H6 competitive ELISA of the present disclosure.

TABLE 13 monoclonal antibody c-ELISA sensitivity test results

6.4 clinical sample detection by three competitive ELISAs

And (3) detecting 40 parts of inactivated pig serum to be detected by using three optimized c-ELISA reaction conditions, and comparing the coincidence rate of the detection results of the three competitive ELISA and the detection result of the ID-Vet kit.

The results are shown in Table 14. The number of positive samples detected by the 30H6 competitive ELISA is 3, the number of negative samples is 37, 2 positive samples are detected by the 20G9 competitive ELISA kit, 38 negative positive samples are detected by the 20G9 competitive ELISA kit, 2 positive samples are detected by the 32B8 competitive ELISA kit, and 38 negative positive samples are detected by the 20G9 competitive ELISA kit. The detection results of 20G9 and 32B8 competitive ELISA are consistent, the coincidence rate of the detection results with the ID-Vet kit is 97.5%, and the coincidence rate of the detection results of the 30H6 competitive ELISA and the ID-Vet kit is 100%, so that the 30H6 competitive ELISA disclosed by the invention has higher detection accuracy.

TABLE 14 sample detection results of the disclosed c-ELISA and IDvet kits

By combining the sensitivity and specificity of the three competitive ELISA detection methods and the accuracy of the detection result of the clinical sample, compared with the 30H6 competitive ELISA disclosed by the invention, the competitive ELISA method is more advantageous, and the working efficiency of the enzyme-labeled antibody is higher.

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Sequence listing

<110> scientific research institute of Chinese inspection and quarantine

<120> competitive monoclonal antibody based on African swine fever virus CD2v protein, kit and application thereof

<130> 20046-K-CAIQ-WCX

<160> 18

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attacatata attgtactaa ttctttaata acatgtaaaa ataataatgg gacaggcggc 120

ggcggcggcg gcggcggcgg cggcgttaat gatactaatg gagatatcct taattattat 180

tggaatggta ataataattt tggcggcggc ggcggcggcg gcggcggcgg ctcattgaat 240

gaaacagaaa atataaatgg cggcggcggc ggcggcggcg gcggcggcaa acatgttgaa 300

gaaatagaaa gtccaccacc ctctgaatct aatgaagaag atatttctca cgatgacacc 360

acttccatac atgaaccatc tcccagagaa ccattacttc ctaagcctta cagtcgttat 420

cagtatggcg gcggcggcgg cggcggcggc ggcggcatgc gtccctcaac acaaccactc 480

aacccatttc ccctacctaa accatgcccg ccacctaaac catgtcctcc acccaagcca 540

tgcccgccac ccaaaccatg tcctccacct aaaccgtgtt ctccacccaa accgtgtcgt 600

ccacctaaac catgtcctcc acctaaacca tgtcctccac ctaaaccatg tcctccacct 660

aaaccatgtc ctccatccaa accatgtcct tcacctgaat cctattctcc acccaaacca 720

ctacctagtg gcggcggcgg cggcggcggc ggcggcggca aacatgttga agaaatagaa 780

agtccaccac cctctgaatc taatgaagaa gatatttctc acgatgacac cacttccata 840

catgaaccat ctcccagaga accattactt cctaagcctt acagtcgtta tcagtatggc 900

ggcggcggcg gcggcggcgg cggcggcatg cgtccctcaa cacaaccact caacccattt 960

cccctaccta aaccatgccc gccacctaaa ccatgtcctc cacccaagcc atgcccgcca 1020

cccaaaccat gtcctccacc taaaccgtgt tctccaccca aaccgtgtcg tccacctaaa 1080

ccatgtcctc cacctaaacc atgtcctcca cctaaaccat gtcctccacc taaaccatgt 1140

cctccatcca aaccatgtcc ttcacctgaa tcctattctc cacccaaacc actacctagt 1200

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Glu Ser Gly Ser Leu Ile Gly Asp Leu Gly Ser Leu Lys Leu Ser Gly

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Gln Ser Gly Leu Gln Leu His Val Lys Pro Glu Val Met Leu Cys Ala

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Val Ala Thr Phe Thr Ser Tyr Phe Trp Lys Glu Trp Ser Leu Gly Ser

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Tyr Lys

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atggattttc aggtgttttc ag 22

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<213> Artificial Sequence (Artificial Sequence)

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atgragtcac akacggtctt yrta 24

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<213> Artificial Sequence (Artificial Sequence)

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atgaggkccc hgctytyctk ggr 23

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<213> Artificial Sequence (Artificial Sequence)

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atgaagttgc ctgtgctgtt g 21

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atgatgagtc ctgccttcc 19

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<213> Artificial Sequence (Artificial Sequence)

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actggatggt gggagga 17

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<213> Artificial Sequence (Artificial Sequence)

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cccaagctta cttgggaaga tgga 24

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggratgsa gctgmatsct ctt 23

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgracttcg ggyctkggtt tt 22

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atggctgtct tggggctctt ct 22

<210> 15

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atggrcagta chtyy 15

<210> 16

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ayctccacac rccagtggat agac 24

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<212> DNA

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cccaagcttr ccarkggatr a 21

<210> 18

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<213> Artificial Sequence (Artificial Sequence)

<400> 18

tatgataata atcgtagtgg cggcggcggc ggcggcggcg gcggcggcga ttccccaact 60

attacatata attgtactaa ttctttaata acatgtaaaa ataataatgg gacaggcggc 120

ggcggcggcg gcggcggcgg cggcgttaat gatactaatg gagatatcct taattattat 180

tggaatggta ataataattt tggcggcggc ggcggcggcg gcggcggcgg ctcattgaat 240

gaaacagaaa atataaatgg cggcggcggc ggcggcggcg gcggcggcaa acatgttgaa 300

gaaatagaaa gtccaccacc ctctgaatct aatgaagaag atatttctca cgatgacacc 360

acttccatac atgaaccatc tcccagagaa ccattacttc ctaagcctta cagtcgttat 420

cagtatggcg gcggcggcgg cggcggcggc ggcggcatgc gtccctcaac acaaccactc 480

aacccatttc ccctacctaa accatgcccg ccacctaaac catgtcctcc acccaagcca 540

tgcccgccac ccaaaccatg tcctccacct aaaccgtgtt ctccacccaa accgtgtcgt 600

ccacctaaac catgtcctcc acctaaacca tgtcctccac ctaaaccatg tcctccacct 660

aaaccatgtc ctccatccaa accatgtcct tcacctgaat cctattctcc acccaaacca 720

ctacctagt 729

Claims

1. An antigen binding protein which specifically binds to African swine fever virus CD2v protein, wherein the antigen binding protein comprises at least one heavy chain variable region and at least one light chain variable region, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 2, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence shown in SEQ ID NO. 2; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 3, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3.

2. An antibody or an active fragment specifically binding to African swine fever virus CD2v protein, wherein the antibody or the active fragment comprises at least one heavy chain variable region and at least one light chain variable region, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 2, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence shown in SEQ ID NO. 2; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 3, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3.

3. The antibody or active fragment of claim 2, wherein the antibody or active fragment is a monoclonal antibody and/or a genetically engineered antibody; the genetic engineering antibody is selected from one of a single-chain antibody, a single-chain antibody fragment, a chimeric monoclonal antibody fragment, a modified monoclonal antibody and a modified monoclonal antibody fragment.

4. The antibody or active fragment according to claim 3, wherein the antibody is murine monoclonal antibody 30H6, the heavy chain variable region of murine monoclonal antibody 30H6 is set forth as SEQ ID NO:2, and the light chain variable region of murine monoclonal antibody 30H6 is set forth as SEQ ID NO: 3.

5. An African swine fever virus competition ELISA test kit, wherein the kit comprises the antigen binding protein of claim 1, or the antibody or active fragment of any one of claims 2-4.

6. The kit according to claim 5, wherein the antigen binding protein, antibody or active fragment is an antigen binding protein, antibody or active fragment labeled with a label, preferably the label is selected from at least one of an enzyme, a fluorescent group or a chemiluminescent group.

7. The kit of claim 5 or 6, wherein the kit comprises a microplate coated with the ASFV CD2v protein, an enzyme-labeled murine monoclonal antibody 30H 6;

preferably, the coating amount of the CD2v protein is 0.1-8 mug/mL; the enzyme-labeled mouse monoclonal antibody 30H6 is used by volume dilution of 1 (2000-40000), preferably, the CD2v protein is obtained by eukaryotic expression.

8. Use of the antigen binding protein of claim 1, or the antibody or active fragment of any one of claims 2 to 4, or the kit of any one of claims 5 to 7, for detecting african swine fever virus in a sample, preferably the sample is an environmental sample, a food sample, a serum sample or a blood sample.

9. Method for the in vitro detection of African swine fever virus in a sample, preferably an environmental sample, a food sample, a serum sample or a blood sample, using an antigen binding protein according to claim 1, or an antibody or active fragment according to any one of claims 2 to 4, or a kit according to any one of claims 5 to 7.

10. The method according to claim 9, wherein the method comprises the steps of:

(1) adding the sample into an enzyme-labeled microporous plate;

(2) adding the antigen binding protein of claim 1 or the antibody or the active fragment of any one of claims 2 to 4 which is diluted by a diluent into an enzyme-labeled microplate, and incubating;

(3) determination of OD after development_450nmCalculating the value and calculating the PI value;

(4) and when the PI value is more than or equal to 50%, judging the sample to be a positive sample, when the PI value is less than or equal to 40%, judging the sample to be a negative sample, and when the PI value is more than 40% and less than 50%, judging the sample to be a suspicious sample.