Abstract: 【Objective】 This study was aimed to establish an ELISA method for detecting antibodies against African swine fever virus (ASFV).【Method】 In this study,recombinant truncated p72 (p72s) protein was used as the detection antigen,and the optimal working concentration of antigen,serum to be detected and enzyme-labeled antibody were determined by square titration.The reaction conditions of ELISA were optimized,such as antigen coating,ELISA plate sealing,serum/enzyme-labeled antibody reaction and substrate color developing.The threshold evaluation criteria of receiver operating characteristic (ROC) curve were used to determine the negative and positive critical values of the method.The specificity,sensitivity and intra- and inter-batch reproducibility of the method were detected.Finally,a total of 124 serum samples were detected by the established ELISA method and commercial kits respectively.The positive detection rates were compared by the ELISA and the kit,and the ELISA results were verified by Western blotting.【Result】 The optimal antigen coating concentration of ELISA was 0.5 μg/mL,the best dilution ratios of serum and HRP-conjugated antibody were 1∶100 and 1∶5 000,respectively.The reaction conditions were as follows:The antigen was incubated at 37 ℃ for 1 h and then coated at 4 ℃ overnight;The ELISA plate was blocked at 37 ℃ for 1 h or at room temperature for 2 h;The reaction time of serum or the enzyme-labeled antibody were at 37 ℃ for 60 or 45 min,respectively;And the substrate was kept away from light for color developing at room temperature for 20 min.The determination criteria of negative and positive serum were as follows:When the D_{450 nm} value of the serum to be tested was ≥0.365,it was determined as positive;When that was ＜ 0.365,it was judged as negative.This method only specifically binds to anti-ASFV positive serum,but did not cross react with antiserums against Classical swine fever virus, Pseudorabies virus and Porcine reproductive and respiratory syndrome virus.The minimum amount of total protein detected in ASFV antiserum was 0.091 to 0.153 mg/mL,which was lower than that of commercial kit (0.110 to 0.554 mg/mL).The mean coefficients of variation for intra-batch and inter-batch repeatability tests were 4.70% and 5.125%,respectively.The positive rates of serum samples detected by the established method were 75.81% (94/124) and those did by commercial kit were 32.26% (40/124).Further verification showed that the ELISA results were consistent with those of Western blotting.【Conclusion】 An ELISA antibody detection method based on ASFV-p72s protein was established.This method was specific,sensitive and reproducible.It could be used for clinical diagnosis and epidemiological investigation of ASF,which had great potential for development and application.

Key words: African swine fever virus (ASFV); truncated p72 protein; prokaryotic expression; indirect ELISA