摘要
目的采用异硫氰酸荧光素标识Ⅳ型胶原单克隆抗体,为直接或间接荧光方法检测人体肝脏或血液中Ⅳ型胶原含量奠定基础。方法采用直接透析方法进行荧光素标识,标识后通过Sephadex G25凝胶过滤,除去多余的荧光素和其他杂质,获得标识荧光抗体纯品。经SDS-PAGE和双向免疫扩散凝胶电泳,对标识抗体进行纯度,荧光特性,抗体特异性进行检测鉴定。结果经全自动化学发光荧光图像分析仪检测SDSPAGE电泳结果,激发波490nm,发射波510nm,在150KD处有一条黄绿荧光带,其他处未见条带出现,凝胶图像分析仪检测纯度达96%;免疫琼脂双向扩散电泳结果显示,标识抗体与未标识抗体与抗原之间均有乳白色沉淀线,且沉淀物含量一致,荧光分析仪检测标识抗体与中心孔之间有黄绿色荧光条带。结论采用本研究方法制备的荧光抗体具有纯度高、荧光性好、特异性强等特点,为临床检测人体Ⅳ型胶原含量提供便利。
Objective To lay the foundation of detecting the content of Ⅳ type collagen in human liver or blood through direct or indirect fluorescent method, Identifying monoclonal antibody of collagen type Ⅳ by using the fluorescein isothiocyanate. Methods Using a direct dialysis method to identification of fluorescein. The purity of the identifying fluorescent antibody were obtained by Sephadex G-25 gel filtration column, which remove excess fluorescein and other impurities. The purity, fluorescence characteristic and specificity of antibody were measured with SDS-PAGE and double immune diffusion gel electrophoresis. Results The results of SDS-PAGE electrophoresis were measured by fully automatic chemiluminescence fluorescence image analyzer. Eexcitation wave 490nm and emission wave 510nm. The purified of antibody were shown only one strap of yellow with green fluorescence and the molecular weight （MW） of them were estimated to be approximately 150kD, its purity was 96%, there were not any other strap. The result of double agar gel immune-diffusion test showed that both identify antibodies and not identify antibody were combination with antigen, all of product were formed milky white precipitation lines of antigen-antibody complexes with antigen. And the sediment content was consistent. There was a strap of yellow with green fluorescence in the center hole by fluoroanalyzer. Conclusion In this study, the fluorescent antibody against human collagen type Ⅳ with high purity and strong specificity was produced by separation and purification methods, which provide convenience for clinical detection of the human bodyⅣtype collagen.
出处
《中国医药科学》
2015年第1期37-39,共3页
China Medicine And Pharmacy
基金
辽宁省沈阳市科技计划项目(F10-191-8-00)
关键词
Ⅳ型胶原
荧光抗体
荧光素
FITC
Collagen type Ⅳ
Fluorescent antibody
Fluorescein isothiocyanate
FITC
