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以纯化的兔抗传染性胰坏死病病毒(IPNV)VP2重组蛋白多克隆抗体为包被抗体,抗IPNV VP2单克隆抗体为检测抗体建立了IPNV双抗体夹心ELISA方法。优化反应条件为:兔抗IPNV VP2重组蛋白多克隆抗体包被浓度为1.28μg/mL,抗IPNV VP2单克隆抗体的工作浓度为1.34μg/mL,酶标二抗稀释比例为1∶2 000,以P/N>2,且OD490 nm>0.101 494作为阳性判定标准。该方法的重复性变异系数均小于10%,与传染性造血器官坏死病毒(IHNV)、病毒性出血败血症病毒(VHSV)、鲤春病毒血症病毒(SVCV)、轮状病毒(HRV)无交叉反应。对41份虹鳟肝脏样品分别进行双抗体夹心ELISA和RT-PCR检测,结果双抗体夹心法与RT-PCR法检测符合率为97%,表明本实验建立的双抗体夹心ELISA检测方法检测IPNV具有较高的敏感性和特异性,可用于IPNV的病原学检测。
IPNV double antibody sandwich ELISA was established by using purified polyclonal antibody of VP2 recombinant protein of rabbit anti-infectious pancreatic necrosis virus (IPNV) as coating antibody and anti-IPNV VP2 monoclonal antibody as detection antibody. The optimized reaction conditions were as follows: the concentration of polyclonal antibody against IPNV VP2 recombinant protein was 1.28μg / mL, the concentration of anti-IPNV VP2 monoclonal antibody was 1.34μg / mL, the dilution of enzyme labeled secondary antibody was 1: 2000, With P / N> 2, and OD490 nm> 0.101 494 as a positive decision criteria. The method had reproducibility of less than 10% and had no cross-correlation with IHNV, VHSV, SVCV and HRV reaction. The results showed that the coincidence rate of double antibody sandwich method and RT-PCR method was 97%, which indicated that the double antibody sandwich ELISA assay established in this experiment could detect IPNV more High sensitivity and specificity, can be used for the etiological detection of IPNV.