基于非洲猪瘟病毒截短p72蛋白的间接ELISA抗体检测方法的建立

【目的】建立非洲猪瘟病毒（African swine fever virus，ASFV）ELISA抗体检测方法。【方法】以重组截短p72（p72s）蛋白作为检测抗原，通过方阵滴定法，确立ELISA的抗原、待检血清和酶标抗体的最佳工作浓度，优化抗原包被、酶标板封闭、血清/酶标抗体反应及底物显色等工作条件；应用受试者工作特征（receiver operating characteristic，ROC）曲线阈值评价标准，确定方法的阴阳性临界值；检测方法的特异性、敏感性、批内与批间重复性；最后分别用建立的ELISA方法和商品试剂盒检测124份血清，比较两者的阳性检出率，并通过Western blotting验证ELISA的检测结果。【结果】ELISA方法的抗原最佳包被浓度为0.5 μg/mL，待检血清与酶标抗体的最适稀释度分别为1∶100和1∶5 000；反应条件为：抗原于37 ℃孵育1 h后经4 ℃包被酶标板过夜、于37 ℃封闭1 h或室温封闭2 h、待检血清和酶标抗体分别于37 ℃反应60和45 min及加入底物后于室温避光显色20 min；阴阳性血清的判定标准为：当待检血清的D_{450 nm}值≥0.365时，判定为阳性；当D_{450 nm}值＜0.365时，判定为阴性。该方法只与ASFV阳性血清发生特异性结合，与猪瘟病毒、伪狂犬病病毒、猪繁殖与呼吸综合征病毒等病毒抗血清均无交叉反应；能检测到ASFV抗血清中的总蛋白量为0.091~0.153 mg/mL，低于商品试剂盒的最低检测量（0.110~0.554 mg/mL）；批内和批间重复性试验的平均变异系数分别为4.70%与5.125%。对临床血清样本检测时，建立方法和商品试剂盒的阳性检出率分别为75.81%（94/124）和32.26%（40/124）。进一步验证表明，Western blotting与ELISA的检测结果完全一致。【结论】建立了基于ASFV-p72s蛋白的ELISA抗体检测方法，该方法特异、敏感和重复性稳定，能够用于ASF临床诊断与流行病学调查，具有极大的开发应用潜力。

Establishment of Indirect ELISA Method for Detection of African Swine Fever Virus Antibodies Based on Truncated p72 Protein

【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.