Development Of Two Diagnostic Tests For Detection Of African Swine Fever Virus Antigens Based On Monoclonal Antibodies Against The P54 Protein

African swine fever(ASF)is a severe infectious disease of domestic pigs and wild boar infected by African swine fever virus(ASFV),which has caused huge economic losses to the pig production and related industries in China and other affected countries.Since ASF was first discovered in Liaoning Province in 2018,it has now broken out in 31 provinces,municipalities,and autonomous regions of China,as well as in Hong Kong and Macau Special Administrative Regions.The situation of prevention and control is extremely severe.At present,there is no vaccine or therapy for ASF.The prevention and control of ASF mainly relies on early diagnosis combined with strict biosecurity measures.Because the clinical signs of ASF are very similar to common swine diseases,it brings great difficulties to the identification and must rely on laboratory testing for accurate diagnosis.In order to effectively prevent and control ASF,early and rapid diagnosis is essential.Antigens can be detected earlier than antibodies in ASFV-infected pigs,and double-antibody sandwich ELISA(DAS-ELISA)is widely used in antigen detection.DAS-ELISA is suitable for disease screening and high-throughput detection;colloidal gold-based immunochromatographic assay(GICA)are simple and quick,and suitable for rapid diagnosis in the field.ASFV encodes more than 50 structural proteins.As one of the main structural proteins of ASFV,the p54 protein is widely used in the research and development of diagnostic technology because of its strong antigenicity and stability.In this study,the ASFV p54 gene sequence was optimized according to the E.coli codon tropism and cloned into the prokaryotic expression vector p ET-32a to obtain the recombinant expression plasmid p ET-32a-p54,which was transformed into E.coli Rosetta DE3 competent cells.The recombinant strains were induced to express by IPTG and purified,and further identified by SDS-PAGE and Western blotting.The results showed that the recombinant p54 protein was successfully expressed,with a molecular weight of approximately 37 k Da,which was consistent with the expected molecular weight.Subsequently,we immunized mice with the recombinant p54 protein,and selected the splenocytes of the mice with the highest antibody titer for fusion with myeloma cells.After subclonal screening and identification by indirect ELISA and indirect immunofluorescence test,five hybridoma cell lines that can stably secrete anti-p54 monoclonal antibodies(m Ab)were obtained and named 2E8,2E2,2A3,6D8,and 1B9.The hybridoma cells were injected into mice to prepare ascites and the antibodies were purified.Next,the DAS-ELISA was performed and successfully screened m Abs 2E8 and 6D8 that can capture the ASFV antigen.The DAS-ELISA was established using 2E8 as the capture antibody and horseradish peroxidase(HRP)labeled 6D8(HRP-6D8)as the detection antibody.The DAS-ELISA parameters,including the coating concentration of the capture antibody,dilutions of samples and the detection antibody,and the color development time were optimized.137 pig sera,including 31 ASFV DNA-positive sera and 106 ASFV DNA-and antibody-negative sera,were detected by the new DAS-ELISA,and the critical value was determined to be 0.14 by the software Med Calc.The new DAS-ELISA can detect 10³TCID₅₀/100?L ASFV and 64-fold diluted ASFV DNA positive serum,and the sensitivity of the test was consistent with that of the commercial DAS-ELISA kit(INGENASA DAS-ELISA).Next,both the new DAS-ELISA and INGENASA DAS-ELISA were used to detect 146clinical pig serum samples determined by the ASFV real-time quantitative PCR(q PCR)kit developed by our laboratory(Announcement No.409,the Ministry of Agriculture and Rural Affairs of the People's Republic of China),including 37 ASFV DNA-positive sera and 109 ASFV DNA-and antibody-negative sera.The results showed that the agreement rate of the new DAS-ELISA and the INGENASA DAS-ELISA was 92%(134/146).At the same time,GICA was developed using m Abs 2E8 and 6D8(the new GICA).The new GICA can detect 10³TCID₅₀/100?L ASFV,which was 10 times sensitive than that of the Spanish INGENASA immunochromatographic assay(INGENASA ICA);the new GICA was specific and did not react with common swine pathogens;when detecting 85 clinical swine samples(19 whole blood and 66 serum),the agreement rate of the new GICA and the q PCR was 85%(72/85),and that of the new GICA and INGENASA ICA was 63%(whole blood samples,12/19)or 86%(serum samples,57/66).In summary,this study successfully expressed the recombinant ASFV p54 protein,and identified 5specific m Abs against ASFV p54 protein.The DAS-ELISA and the GICA were established based on the m Abs and both the tests are sensitive and specific,and show high agreements with the q PCR and the INGENASA DAS-ELISA and ICA.The newly established assays will provide technical support for ASF surveillance and control.