HBsAg人源噬菌体单链抗体的筛选及其在临床诊断中的应用

Screening and application of human-derived HBs Ag bacteriophage single chain antibody in clinical diagnosis

目的 筛选乙型肝炎病毒 (HBV)表面抗原 (HBsAg)的人源噬菌体单链抗体 ,并探讨其在临床治疗和诊断中的应用价值。方法 以HBsAg阳性血清超速离心纯化的HBsAg为固相抗原 ,从噬菌体单链可变区半合成抗体库中经过 5轮“吸附 洗脱 扩增”筛选过程 ,获得特异性较强的HBsAg人源单链可变区抗体 (ScFv) ;用该抗体对 10例石蜡包埋的乙型肝炎患者肝组织进行免疫组化鉴定。结果 酶联免疫吸附法 (ELISA)结果表明 ,制备的HBV人源单链抗体能与HBsAg抗原特异性结合 ;免疫组化结果表明 ,该抗体能够特异性识别乙型肝炎患者肝组织中的HBsAg抗原 ,与正常肝组织及丙型肝炎病毒 (HCV)的抗原均无交叉反应。结论 此法制备的单链抗体亲和性好 ,特异性强 ,且制备方法简便 ,周期短 ,为HBV病原的检测提供了新的有效试剂 ,为今后HBsAg人源抗体的研究和应用奠定了基础。

Objective To identify human single chain Fv antibody (ScFv) against hepatitis B viral surface antigen. Methods The recombinant phages were panned by HBsAg which was coated in a microtiter plate, after five rounds of biopanning,56 phage clones were identified specific to HBsAg.The specificity of ScFv was evaluated by ELISA and immunohistochemistry, respectively. Results The data of HBsAg ScFv DNA digestion and DNA sequencing showed that the ScFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis B surface antigen has a specific combination character with hepatitis B surface antigen of different sources and paraffin embedded patients tissue specimens,it did not react with normal liver tissue and HCV. Conclusion The application of HBsAg specific ScFv in immunohistochemistry was successfully achieved.