兔抗金黄地鼠酶标抗体的制备及应用
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刘先菊,女,副主任技师,研究方向:实验动物质量控制,E-mail: xianjuliu@263.net。

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中央级公共性科研院所基本科研业务费专项资金项目(项目编号:DWS200906)。


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

    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。

    Abstract:

    ObjectiveTo purify IgG from hamster serum, and to prepare the rabbit anti-hamster IgG-HRP, to be used for detection of the antibody from Sendai virus (SV)-infected hamsters. MethodsUsing 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 Sendai virus-infected hamster by enzyme immunoassay (IEA). ResultsThe 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 1:5000 measured by direct ELISA, and 1:2000 measured by Western blot. The IEA titer was 1:2000. ConclusionsThe 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.

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刘先菊,林树柱,杨帆,佟巍,刘云波.兔抗金黄地鼠酶标抗体的制备及应用[J].中国实验动物学报,2012,(1):84~87.

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