抗HPV16L1 IgY抗体的制备及活性检测

目的 制备出高特异性的抗HPV16L1 IgY抗体,为HPV16L1蛋白的检测提供一种方法.方法 用纯化HPV16 L1蛋白免疫母鸡,分别收集鸡蛋,4℃下保存备用;经聚乙二醇法提取鸡蛋黄中IgY抗体;用酶联免疫吸附法检测IgY抗体效价的测定;采用免疫组织化学法通过对转染pcDNAEGFP-HPV16L1(含EGFP-HPV16L1融合基因)质粒的CHO细胞中L1蛋白的检测评价IgY抗体的特异性.结果 3次免疫之后,IgY抗体效价达到1:10 240,同时可特异性与转染pcDNAEGFP-HPV16L1(含EGFP-HPV16L1融合基因)质粒的CHO细胞中EGFP-HPV16L1 结合.结论 采用HPV16L1蛋白免疫母鸡,成功获得了较高效价的特异性IgY抗体,并可用于HPV16L1蛋白的细胞学检测,为IgY抗体在免疫组织化学检中的应用提供实验依据.

Preparation and activity detection of chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein

OBJECTIVE: To prepare highly specific chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein (HPV16L1) for detection of HPV16L1. METHODS: Purified HPV16L1 protein was used to immunize the hens, from which the eggs were collected since one week after the first immunization. The egg yolk was separated and the IgY antibody purified by PEG-6000 method. The bioactivity of the antibody was tested using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed to detect the HPV16L1 in the CHO cells transfected with the recombinant pcDNA-EGFP-HPV16L1 plasmid (containing EGFP-HPV16L1 fusion gene) for assessing the specific affinity of IgY to HPV16L1. RESULTS: After 3 immunizations of the hens, the titer of the purified IgY antibody against HPV16L1 from the egg yolk reached 1:10240. The IgY bound specifically to the EGFP-HPV16L1 protein expressed in the transfected CHO cells. CONCLUSION: High titer IgY can be prepared by immunization of the hens with HPV16L1 protein, and the prepared IgY can be used for HPV16L1 detection at the cellular level.