应用斑点金免疫渗滤试验快速同步检测抗HIV-1，HIV-2 IgG抗体

应用斑点金免疫渗滤试验(dotimmunogoldfiltrationassay,DIGFA)建立了一种同步快速检测四种抗HIV-1/2IgG抗体的HIV诊断试纸。通过基因工程技术在大肠杆菌中表达了5种HIV抗原蛋白片段(P24,GP41,GP36,GP120V3,GP120C)。这5种抗原蛋白首先被固定在硝酸纤维素膜上,然后滴加待测血清,其中的病毒抗体通过免疫反应与抗原结合,再加胶体金标记的葡萄球菌蛋白A(SPA),待其渗过膜片后,洗涤,即可形成肉眼可见的红色斑点。用已确证的21份HIV阳性血清(其中包括1份HIV-1标准阳性血清和1份HIV-2标准阳性血清)和30份阴性血清进行了试验,结果表明该快速检测方法与ELISA方法无显著差异。该检测方法不需任何仪器,仅凭肉眼即可判定结果,整个检测过程不超过5分钟。与传统的的ELISA法相比,具有方便快速,成本低廉,应用范围广等优点。同时,此HIV快速诊断试纸可以同步检测并区分针对HIV-1和HIV-2感染的不同检测标志物(抗P24、GP41、GP120和GP36抗体),这对提高快速检测的灵敏度和准确性,以及对判断HIV感染者是否临近或已进入AIDS期有着较高的应用价值。

Rapid and Simultaneous Detection of IgG Antibodies to HIV-1 and HIV-2 by Dot Immunogold Filtration Assay

A simple rapid detection of HIV-1 and HIV-2 antibodies was developed by using dot immunogold filtration assay. Five recombinant proteins derived from HIV env and gag regions of HIV-1 and HIV-2 (P24, GP41, GP120C, GP120V3 and GP36) were expressed in E. coli. The proteins were purified, immobilized onto the nitrocellulose membrane and used for detecting anti-HIV IgG antibodies Human IgG was used as internal control. Anti-HIV antibodies detected in sera by these antigens were observed with the naked eye with the aid of colloidal gold labeled SPA. A total of 51 sera were tested by ELISA and by our rapid HIV assay. A total of 21 HIV positive samples (containing one HIV-1 positive control serum and one HIV-2 positive control serum) confirmed by Western blots exhibited a positive reaction when tested by our assay. HIV negative sear (30 samples) were also negative by our test. These results suggested that our method can be used for detecting anti-HIV antibodies and has the advanteage of quickness, simplicity and cost effectiveness. In addition, simultaneous detection and discrimination among four HIV markers (anti-P24, GP41, GP120 and GP36 antibodies) by the rapid HIV test would be useful for increasing the sensitivity and accuracy of this rapid test and for evaluating and monitoring infected individuals and identifying the risk of imminent progression to AIDS.