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目的:制备高效价、高特异性的抗人胱抑素C鸡卵黄IgY抗体,并对其基本特性进行分析和鉴定。方法:以人胱抑素C为抗原免疫产蛋的罗曼鸡,采用水稀释-盐析法提取及纯化IgY抗体,采用蛋白质定量、SDS-PAGE、West ern印迹和ELISA法对IgY抗体进行分析和鉴定。结果:免疫后14 d即可从鸡冠血中检测出抗胱抑素C的特异性抗体,抗体效价在28 d达最高峰(1∶32 000),并可维持2个月以上;收集高效价时的免疫鸡蛋,制备鸡卵黄抗体IgY;还原性SDS-PAGE显示抗体IgY为相对分子质量分别为65×103和21×103的2条带,抗体纯度可达92%,得率为每个鸡蛋36.5 mg,抗体检出敏感度为15.63 ng/mL;Western印迹证明该抗体具有高度特异性。结论:制备了抗胱抑素C的高效价、高特异性IgY抗体。
OBJECTIVE: To prepare high titer and high specificity anti-human cystatin C chicken egg yolk IgY antibody, and to analyze and identify its basic characteristics. Methods: The IgY antibody was extracted and purified by water dilution-salting-out method with human cystatin C as antigen. IgY antibody was analyzed by protein quantification, SDS-PAGE, West ern blot and ELISA Identification. Results: Anti-cystatin C-specific antibodies were detected from chicken’s coronaria blood 14 d after immunization. The antibody titers peaked at 28 d (1:32 000) and maintained for more than 2 months. IgY antibody IgY was prepared. Reducing SDS-PAGE showed that the antibody IgY was 2 bands with relative molecular masses of 65 × 103 and 21 × 103, respectively, and the antibody purity was 92%. The yield was Eggs 36.5 mg, the detection sensitivity of the antibody was 15.63 ng / mL; Western blot showed that the antibody was highly specific. CONCLUSIONS: High titer and high specificity IgY antibodies against cystatin C were prepared.