双抗原夹心ELISA检测抗HCV总抗体

Double-antigen sandwich ELISA for detection of total antibodies against hepatitis C virus

目的建立检测抗HCV抗体的双抗原夹心ELISA法,评价其可行性。方法将与His或GST融合表达的HCV基因工程抗原,分别用作ELISA的包被和酶标记抗原,建立用于抗HCV总抗体检测的双抗原夹心ELISA。用此方法检测1 968份临床血清标本,并以间接ELISA(北京万泰试剂)与之对照;此外,用套式RT-PCR检测部分血清的HCV RNA。结果有1 761份血清2种ELISA检测均为阴性,有190份血清均为阳性,两种方法符合率为99.1%;有17份血清的检测结果不相符,间接法阳性而本法阴性的14份,其中HCV RT-PCR阳性1份;本法阳性而间接ELISA阴性的3份,其中RT-PCR阳性2份。双抗原夹心ELISA与间接ELISA的敏感性分别为99.48%、98.96%,特异性分别为99.94%、99.27%。结论新研制的检测抗HCV总抗体的双抗原夹心ELISA具有较高的敏感性和特异性,值得作进一步的深入研究。

Objective To establish a double-antigen sandwich ELISA （S-ELISA） for detection of total antibodies against hepatitis C vires （HCV）. Methods Recombinant HCV proteins fusion-expressed with His-tag and GST-tag were separately used as coating and HRP-labeling antigen of the S-ELISA, Serum samples were tested with both the S-ELISA and another commercial indirect ELISA （I-ELISA） kit （ Beijing Wantai Biological Pharmacy Enterprise Co. Ltd. ）. HCV RNA in some of the samples were tested by RT-nested PCR. Resuits Among 1 968 tested samples, 190 （9.7%） were total anti-HCV-positive while 1761（89.5%） were negative by both of the S-ELISA and I-ELISA, with a resultant concordance rate of 99.1% of the two ELISA assays. However, the results of 17 （0.9%） were not consistent in the two assays, in which 14 were S-ELISA negative but I-ELISA positive and 3 were S-ELISA positive but I-ELISA negative. One of the 14 samples （0.7%） with S-ELISA negative was detected as HCV RNA-positive, while 2 of the 3 （66.7%） samples with S-ELISA positive were detected as HCV RNA-positive. In addition, HCV RNA was detectable in 23 of 31 （74.19%） samples which were total anti-HCV positive in both S-ELISA and I-ELISA. Taking the PCR data together into account, the sensitivity of the S-ELISA and I-ELISA were 99.48% and 98.96% and specificity were 99.94% and 99.27% , respectively. Conclusions The estab-lished S-ELISA in this study may provide a novel means for detection of HCV antibody with high sensitivity and specificity.