兔抗麻雀IgY酶标抗体的制备

Preparation of HRP-labeled Rabbit IgG against Sparrow IgY

目的制备辣根过氧化物酶(HRP)标记的兔抗麻雀IgY抗体,为禽类血清学检测体系的建立提供技术储备。方法硫酸铵盐析法粗提麻雀血清IgY,进一步在SDS-PAGE上分离后,切下带有目的条带的凝胶作为免疫原,免疫实验兔制备抗血清,Protein-A柱亲和纯化兔抗IgY血清IgG,,使用改良过碘酸钠法制备酶结合物。ELISA检测酶标抗体的工作浓度,western blotting检测酶标抗体的特异性。结果硫酸铵盐析法粗提IgY,可去除部分杂蛋白,SDS-PAGE上分离后切下带有目的条带的凝胶,可以得到足够纯度的抗原,将带有IgY的凝胶作为抗原免疫后获得的抗血清经Protein-A纯化后,二抗在SDS-PAGE上鉴定,纯度达到99%以上。改良的过碘酸钠法标记获得的抗体浓度为1.008 mg/mL,ELISA检测酶标抗体效价为1∶1000。Western blotting鉴定抗体具有特异性。结论获得了优质可靠的兔抗麻雀IgY酶标抗体。

Objective To obtain horseradish peroxidase （HRP） labeled rabbit anti-sparrow antibody for infectious disease testing. Methods From the sparrow serum IgY was precipitated by ammonium sulfate and then separated on SDS-PAGE. The gel containing the targeting protein（IgY） was sliced and was to be used as antigen to immunize the rabbit in a standard immunization procedure. HRP-labeled rabbit anti-sparrow. IgY was prepared using the improved method of NalO4. The titre and the optimal working dilution of the labeled antibody were determined by direct ELISA. The specificity of the labeled antibody was tested by western blot. Results The crucial IgY was precipitated from serum with ammonium sulfate and the purified IgY used as antigen was obtained by SDS-PAGE. The rabbit anti-sparrow antibody was produced 2 months after immunization and the purity was up to 99% on SDS-PAGE after Pretein-A affinity chromatography. The purified rabbit IgG was labeled with HRP, the HRP-labled and rabbit anti-spin＇row IgG was prepared and the titer of labeled antibody is 1 ： 1000 for ELISA and western blot. Conclusion The useful rabbit anti- sparrow IgY antibody and the HRP-labeled rabbit anti- sparrow IgY antibody were prepared.