A sandwich ELISA was generated to detect viral hemorrhagic septicemia virus (VHSV) using MAb IP5B11 as capture antibody and rabbit polyclonal antibody to VHSV as detection antibody.The reaction conditions were optimized and the coating and blocking agents were confirmed to be buffer bicarbonate at 4 ℃ for 12 h and 10％ fetal bovine serum at 37 ℃ for 2 h,respectively.The optimal reaction time and temperature of antigen were 20 ℃ for 90 min.The specificity of the sandwich ELISA was tested with several common fish virus and no cross reaction was observed.The coefficients of variation of the inter-and intra-assay were 0.046 and 0.064,respectively.Application of this sandwich ELISA to 40 eel tissue homogenate mixed with VHSV and 40 eel and 5 platichthys stellatus homogenate without virus as negative control revealed that the sandwich ELISA had the same accuracy as the RT-PCR,the coincidence rate was 100％.The development and application of this test used for entry and exit fish quarantine and epidemic surveillance of domestic farms were promising.%为建立病毒性出血性败血症病毒(VHSV)的夹心ELISA方法,本研究以VHSV单克隆抗体(MAb)IP5B11为捕捉抗体,兔抗VHSV多抗为检测抗体,经反应条件优化建立了VHSV夹心ELISA检测方法.结果表明,该方法与几种常见鱼病病毒无交叉反应,具有较好的特异性;该方法组内和组间变异系数平均值分别为0.046和0.064,重复性良好.采用夹心ELISA和RT-PCR同时检测添加VHSV病毒悬液的40份鳗鱼组织匀浆和未添加病毒的40份阴性对照鳗鱼以及5份星斑川鲽模拟组织样品,结果两种方法检测符合率为100％.本研究建立的夹心ELISA方法可以用于出入境鱼类疫病的检测和国内养殖场的疫情监控.
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