Preparation Of African Swine Fever Virus P72 Protein Monoclonal Antibody And Establishment Of Double Antibody Sandwich ELISA Method

African swine fever virus(ASFV)is the pathogen of African swine fever(ASF),which infects pigs and causes symptoms such as high fever and reticular tissue bleeding,and the morbidity and mortality rate are extremely high,and the rate of death is fast,bringing great losses to the pig industry.Because ASF has a rapid onset and rapid spread,a rapid test is needed to detect outbreaks as quickly as possible and minimize harm.In this study,the anti-p72-N protein monoclonal antibody was prepared and the epitope region was identified by immunizing BALB/c mice by using prokaryotic expression of purified ASFV p72-N recombinant protein.At the same time,the recombinant protein was immunized to New Zealand rabbits,rabbit polyclonal antibody was prepared,and a double antibody sandwich ELISA detection method was established based on monoclonal antibody and rabbit polyclonal antibody.Based on the N-terminal 672 bp sequence of ASFV p72 gene sequence,p ET-28a(+)-p72-N recombinant expression plasmid was successfully constructed,and after IPTG induction of successful expression,p72-N protein with protein size of 27 k Da was purified by Ni+ column.After immunizing BALB/c mice with p72-N protein,splenocytes were collected and fused with SP2/0 cells,and two hybridoma cells(2C3and 1H5)were obtained after screening.The indirect ELISA test identified that monoclonal antibody 2C3 had the strongest binding capacity to p72-N protein,and the characteristics of 2C3 were identified by the subtype identification kit,and the results showed that monoclonal antibody 2C3 belonged to Ig M type,κ chain;Immunofluorescence detection can detect eukaryotic p72 protein well.The prokaryotic expression was used to truncate the expression of p72-N protein into FR1,FR2 and FR3 overlapping recombinant proteins,and then the epitope region of monoclonal antibody 2C3 was localized to the N-terminal part of FR3 by WB method.The epitope region of monoclonal antibody 2C3 was subsequently further truncated using the N-terminal fraction of the synthetic polypeptide FR3 to aa160-175(ITFALKPREEYQPSGH)by indirect ELISA.Homology analysis showed that the aa160-175 sequence in the epitope region was highly conserved in ASFV.3D structural analysis found that aa160-175 is located on the surface of the p72-N protein,possibly in the region where the linear epitope is located.Rabbit polyclonal antibody was prepared by immunizing New Zealand rabbits with p72-N protein,and a bi-antibody sandwich ELISA method was established based on rabbit polyclonal antibody and pre-prepared monoclonal antibody 2C3.By optimizing optimal conditions,determining cut-off values,identifying specificity and sensitivity,and repeatability,a bi-antibody sandwich ELISA method was established.The results showed that the optimal concentration of the capture antibody was4μg/100μL,and the optimal dilution ratio of the detection antibody was 1:3200-fold dilution;The optimal coating condition for capturing antibodies was 0.05mmol/L coating solution,first incubated at 37℃ for 1h,then coated at 4℃ for 12 h.The optimal blocking solution concentration is 3% skim milk powder;The optimal closing time is 2h;The optimal incubation time for antigens is 1 h;The optimal incubation time for detecting antibodies is 1 h;The optimal color rendering time is 15 min.The established double-antibody sandwich ELISA method was used to determine the critical value of 20 African swine fever-negative pig serum,and the test results were judged as positive when OD450≥0.153 and negative when OD450≤0.147.The intrabatch coefficient of variation was 3.16%-4.58%;The coefficient of variation between batches was 3.12%-9.14%,indicating that the method had good repeatability.Compared with the colloidal gold test strip and PCR test for ASFV pathogen detection,the test results were negative,and the results of the three detection methods were consistent.