摘要
目的建立检测SIVp27抗原的双抗体夹心ELISA方法并进行初步应用。方法使用ProteinA亲和层析柱纯化腹水中的SIVp27单抗,用过碘酸钠法对单抗进行辣根过氧化物酶标记。经抗体配对实验确定包被抗体和检测抗体,再用棋盘滴定法确定抗体工作浓度后,探索构建检测SIVp27抗原的双抗体夹心ELISA法。应用建立的双抗体夹心ELISA法,对SIV模型猴血浆标本和SHIV模型猴病毒分离培养上清标本进行了检测,并将其结果与Coulter公司试剂盒检测结果进行比较。结果以稀释至20ug/mL的2E12为包被抗体、1:400稀释的酶标3G3为检测抗体为ELISA最优体系。模型猴血浆标本和病毒分离培养上清标本的检测结果表明自建体系可以用于SIVp27抗原的检测,特异性良好,灵敏度略低于Coulter公司试剂盒。结论检测SIVp27抗原的双抗体夹心ELISA方法初步建成,为研制有独立知识产权的检测试剂奠定了一定的基础。
Objectives To establish a double-antibody sandwich ELISA assay for p27 antigen of SIV,and to detect samples from rhesus macaques using this method. Methods McAbs were purified by Protein A affinity chromatography, then labeled with HRP by sodium iodide method. A sandwich ELISA with double-antibody to detect SIV p27 antigen was established, in which the coating antibody and detective antibody were determined and chosen by antibody match test and chessboard titration. The plasmas and culture supernatants, from SIV and SHIV model macaques respectively, were detected with the established double-antibody sandwich ELISA. Results The coating antigen concentration 20μg/mL and the working concentration of HRP-labeled antibody 1：400 were chosen to establish ELISA assay. Blood and supernatant samples of model macaques were detected by this method, and the result indicated the method could be used for detection of SIV p27 antigen with high specificity, but sensitivity was lower than that of the Coulter kit. Conclusion A double-antibody sandwich ELISA assay to detect p27 antigen of SIV was established, which provided a basis for developing reagents with intellectual property right.
出处
《中国动物检疫》
CAS
北大核心
2007年第4期32-35,共4页
China Animal Health Inspection
