兔抗金黄地鼠酶标抗体的制备及应用

目的 纯化金黄地鼠血清IgG,制备兔抗金黄地鼠酶标抗体(IgG-HRP),开展金黄地鼠仙台病毒的初步检测.方法 采用亲和层析纯化法纯化金黄地鼠IgG,用SDS- PAGE电泳测定IgG纯度并制备兔抗金黄地鼠IgG抗体(second antibody,Ab2)；用免疫双扩散法检测抗血清效价后,再用亲和层析纯化抗血清IgG( Ab2)；采用改良过碘酸钠标记法制备兔抗金黄地鼠酶标抗体( rabbit anti-hamster IgG-HRP)；用直接ELISA和Western-blot法对兔抗金黄地鼠IgG酶标抗体进行工作浓度测定；应用金黄地鼠酶标抗体对金黄地鼠仙台病毒进行酶免检测(IEA).结果 金黄地鼠血清IgG纯度达95％；兔抗金黄地鼠IgG抗体(Ab2)免疫双扩散效价为1(:)64;兔抗金黄地鼠IgG -HRP经直接ELISA和Western-Blot测定工作浓度分别为1∶5000和1∶2000；酶免(IEA)效价为1:2000.结论 高效快速纯化了金黄地鼠IgG,制备了金黄地鼠IgG-HRP,为金黄地鼠病原微生物的血清学检测提供了条件.

Preparation and application of the rabbit anti-hamster IgG-HRP

Objective To purify IgG from hamster serum, and to prepare the rabbit anti-hamster IgG-HRP, to be used for detection of the antibody from Sendal virus （ SV）-infected hamsters. Methods Using HiTrap Protein-A affinity chromatography column to purify the IgG from hamster serum. The purity of the hamster-IgG was identified by SDS-PAGE analysis. To produce the hamster against rabbit IgG, and to measure the titer of rabbit anti-hamster IgG by double immunodiffusion. To purify the rabbit anti-hamster IgG by affinity chromatography and prepare the rabbit anti-hamster HRP-conjugated antibodies. The working concentration of the rabbit anti-hamster IgG-HRP was detected by direct ELISA and Western blot, and detect the antibody from Sendal virus-infected hamster by enzyme immunoassay （IEA）. Results The hamster IgG was purified up to 95% determined by SDS-PAGE analysis, and the titer of the antisrum was up to 1：64 assessed by double immunodifusion. The working concentration of the rabbit anti-hamster IgG was up to l ：5000 measured by direct ELISA, and 1：2000 measured by Western blot. The IEA titer was 1：2000. Conclusions The method has been established for efficient purification of IgG fraction from hamster serum, and the polyclonal antibody of the hamster-IgG and the rabbit anti-hamster IgG-HRP are prepared, which may provide a basis for development of serological test system of microbial pathogens of hamster.