猪流感抗体间接ELISA检测方法的建立

Establishment of an indirect ELISA for detection of antibodies against swine influenza virus

猪流感病毒A/Swine/Fujian/668/2001(H3N2)株感染的鸡胚尿囊液,经差速离心后,再经蔗糖密度梯度离心,提纯、纯化的猪流感病毒经NP-40处理并反复冻融,作为猪流感间接ELISA抗原,确立了间接ELISA检测方法。对29份HI试验猪流感为阴性的血清进行了检测,经统计学分析,确定间接ELISA判定标准,被检血清OD_(490nm)值≥0.20判定为阳性。该方法对猪瘟等11种猪疫病阳性血清无交叉反应,批内和批间重复试验的吸收变异系数分别在3.34%～8.12%和6.2%～9.04%之间。与HI的符合率达到92.8%,经卡方检验(P<0.01)比HI试验敏感。为猪流感抗体检测提供了快速、准确、简便的方法。

An indirect ELISA for detecting antibodies against swine influenza virus （SIV） was developed using antigen that was derived from allantoic fluid of chicken embryonated eggs infected with A/Swine/Fujian/668/2001 （H3N2）. The antigen was purified by differential centrifugation and sucrose density gradient centrifugation, followed by NP-40 treatment and 3 freeze-thaw cycles. Samples with an optical density value≥ 0.2 were scored positive based on the ELISA data from twenty-nine sera that are known SIV negative in hemagglutination inhibition （HI） test. There was no cross-reactivity detected against virus strains of other 11 swine infectious diseases including classical swine fever. The variant coefficient of optical density value in a batch and between batches varied from 3.34 % to 8.12 %, and from 6.2 % to 9.04 %, respectively. The coincidence rate was 92.8 % between indirect ELISA and HI test. The results showed that indirect ELISA is more sensitive than HI （P〈0.01） and could be used as an efficient and sensitive method to detect antibodies against SIV.