摘要
禽流感病毒（ＡＩＶ）感染的鸡胚尿囊液经差速离心后，再经蔗糖密度梯度离心，提纯ＡＩＶ，纯化的ＡＩＶ经ＮＰ４０处理并反复冻融，即为ＡＩＥＬＩＳＡ抗原，用该抗原包被聚苯乙烯微量反应板。将健康鸡ＩｇＧ提纯后免疫兔，制备兔抗鸡ＩｇＧ，用过碘酸钠法制备辣根过氧化物酶标记的兔抗鸡ＩｇＧ。确立了间接酶联免疫吸附试验（ＥＬＩＳＡ）检测禽流感抗体的最适工作条件，即：抗原包被浓度１．９～３．８μｇ／ｍｌ，每孔１００μｌ，４℃冰箱过夜；用含０．５％牛血清白蛋白（ＢＳＡ）的磷酸盐缓冲液，３７℃封闭６０分钟；待检血清最佳稀释度为１８０～１６４０，作用６０分钟；酶标抗体作１２０００稀释，作用６０分钟。根据对５２份ＳＰＦ鸡血清的检测结果制定了判定标准。
Avian influenza virus (AIV) was purified from infected allantoic fluid by centrifugation.Fractions with high hemagglutination titer was examined by electron microscopy,and numerous AIV particles were observed with no obvious debris contamination.The antigen for enzymelinked immunosorbent assay(ELISA) was prepared from the purified AIV.Rabbit antichicken (RAC)IgG was prepared and conjugated with horseradish peroxides (HRP).Optimum for detection of specific antibodies to AIV were investigated employing the AIV antigen and HRPcoupled RAC IgG.Polyst yrene chloride microtitration plates were coated by the purified ELISAantigen at the amount of 019～038μg in 100μl 005M sodium bicarbonate buffer (pH96) per well.The coated plates were blocked by 05% bovine serum albumin in 001M phosphate buffered saline solution.The working concentration of HRPlabeled RAC was 1∶2000 dilution,and that of serum samples was 1∶80～1∶640 dilution.All the incubation steps above were done at 37℃ for 60 min.The samples with optical dense≥0274 wrer judged as positive based on the ELISA data from 52 sera of specificpathogenfree chickens.
