摘要
目的探讨以多聚赖氨酸为载体的聚合物标记物在丙型肝炎病毒(Hepatitis C virus,HCV)核心抗原检测中的应用。方法以多聚赖氨酸作为载体,将HCV核心抗原单克隆抗体与辣根过氧化物酶(HRP)偶联形成聚合物标记物,同时制备过碘酸钠标记物,比较两种标记物中的抗体相对含量及抗体反应性;将两种标记物分别应用于ELISA和Western blot法检测HCV核心抗原中,比较二者的检测结果,并与市售试剂盒进行比较。结果通过竞争抑制和直接结合试验结果显示,当达到50%抑制率时,聚合物标记物和过碘酸钠标记物的游离抗体浓度分别为0.0019和0.0011mg/ml;当A403值为1.0时,聚合物标记物的稀释度为1:200000左右,而传统标记物为1:20000左右。聚合物标记物和过碘酸钠标记物应用于ELISA法,检测HCV核心抗原的最低检测限分别为2.0和10pg/ml,试验内、试验间变异系数均<10%,特异性良好,临床血清标本检测结果与市售试剂盒的符合率分别为97.0%和98.6%;在Western blot检测中,与过碘酸钠标记物相比,聚合物标记物至少可以提高检测灵敏度10倍。结论以多聚赖氨酸为载体的聚合物标记物可应用于HCV核心抗原的ELISA和Western blot检测,且能提高酶、抗体的比例,进而提高检测的灵敏度,防止漏检。
Objective To investigate the application of polymer conjugate using polylysine as a carrier in the detection of hepatitis C virus（HCV）core antigen.Methods The McAb against HCV core antigen was conjugated with HRP using polylysine as a carrier.Meanwhile,the conjugate with sodium periodate was prepared,and the relative contents and reactivities of antibodies in the two conjugates were compared.Both the conjugates were used for detection of HCV core antigen by ELISA and Western blot.The detection results by the two methods were compared,both of which were compared with those by commercial kit.Results Competitive inhibition and direct binding tests showed that,when the inhibition rate reached 50%,the free antibody concentrations in polymer and sodium periodate conjugates were 0.001 9 and 0.001 1 mg/ml respectively.When the A403 was 1.0,the dilution of polymer conjugate was about 1：200 000,while that of traditional conjugate was about 1：20 000.The minimum detection limits of HCV core antigen by ELISA with polymer and sodium periodate conjugates were 2.0 and 10 pg/ml respectively.Both the intra-and inter-coefficients of variation of detection results with the two conjugates were less than 10%.Both the conjugates showed high specificities.The coincidence rates of detection results of clinical serum samples by polymer and sodium periodate conjugates with that by commercial kit were 97.0% and 98.6% respectively.The sensitivity of Western blot using polymer conjugate increased by at least 10 folds as compared with that using sodium periodate conjugate.Conclusion The polymer conjugate using polylysine as a carrier may be used for the detection of HCV core antigen by ELISA and Western blot,and may increase the ratio of enzyme to antibody thus increase the sensitivity of detection and prevent miss detection.
出处
《中国生物制品学杂志》
CAS
CSCD
2010年第11期1239-1243,共5页
Chinese Journal of Biologicals
基金
广东省科技计划项目(2008A030201025)
深圳市科技计划项目(200712)
深圳大学青年基金(200835)
