(中国医学科学院医学实验动物研究所,北京 100021)
R-33
北京市科技计划(Z161100000216154);国家重点研究计划(2017YFA0105201)
(Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical Collage (PUMC), Beijing 100021, China)
目的 狨猴血清中纯化IgG 抗体,制备兔抗狨猴抗血清,并纯化抗血清中IgG,HRP(辣根过氧化物酶)标记兔抗狨猴IgG?方法 HiTrap TM Protein G 亲和层析纯化狨猴和兔抗狨猴血清IgG,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS?PAGE)进行纯度鉴定,免疫琼脂双扩散法(double immunodiffusion assay)测定制备的兔抗狨猴抗血清效价,改良“简易过碘酸钠标记法”制备兔抗狨猴IgG?HRP 标记抗体,酶联免疫吸附测定法(ELISA)及蛋白免疫印迹法(Western blotting)对兔抗狨猴IgG?HRP 标记抗体进行工作浓度测定及特异性鉴定?结果 狨猴和兔抗狨猴血清纯化IgG 纯度分别大于95%?97%;兔抗狨猴IgG 抗血清的效价为1∶ 64?ELISA 和Western blotting 鉴定了兔抗狨猴IgG?HRP 酶标抗体参考工作浓度为1∶ 256 000?1∶ 15 000,特异性明显?结论 制备狨猴IgG?HRP 标记抗体并初步鉴定,包括ELISA 及Western blotting 的特异性及使用浓度,为狨猴病原体免疫学检测体系及分子免疫学检测体系储备了资源?
Objective To purify marmoset serum IgG, prepare and identify the antiserum and the rabbit anti?marmoset antibody IgG?HRP (horseradish peroxidase). Methods Using SDS?PAGE analysis to identify the serum IgG from HiTrap TM Protein G. The antiserum titer was determined by double immunodiffusion assay. The rabbit anti?marmoset IgG was labeled with HRP by improved sodium periodate method. ELISA and western blotting were used to evaluate the concentration and specificity of rabbit anti?marmoset IgG?HRP. Results The purity of purified marmoset serum IgG determined by SDS?PAGE was higher than 95%, and the anti?serum titer of the anti?marmoset IgG polyclonal antibody was 1∶ 64. The concentration of rabbit anti?marmoset IgG?HRP identified by direct ELISA was 1∶ 256 000, and that by western?blotting was 1∶ 15 000, with a strong specificity. ConclusionsThe IgG?HRP marker antibody is prepared and the specificity and concentration are identified by ELISA and western blotting. It reserves the resources for the detection system of marmoset pathogens and the molecular immunological testing system.
丛日旭,刘先菊,滕永康,向志光,佟巍,张丽芳,阮研硕,刘云波.兔抗狨猴IgG⁃HRP 标记抗体的制备及初步鉴定[J].中国比较医学杂志,2018,28(6):101~105.
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