Ma Yi, Ni Chao, Dzakah Emmanuel E, Wang Haiying, Kang Keren, Tang Shixing, Wang Jihua, Wang Jufang
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
National Engineering Laboratory of Rapid Diagnostic Tests, Guangzhou Wondfo Biotech Co., Ltd., Guangzhou 510663, China.
Biomed Res Int. 2016;2016:6743904. doi: 10.1155/2016/6743904. Epub 2016 Mar 16.
Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed in E. coli strain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection.
1型人类免疫缺陷病毒（HIV-1）p24蛋白是HIV-1中含量最丰富的病毒蛋白。在HIV-1感染的早期阶段，这种蛋白会大量分泌到血清中，使其成为早期诊断的生物标志物。在本研究中，建立了一种胶体金免疫层析法（GICA），使用小鼠单克隆抗体（mAb）检测p24蛋白。HIV-1 p24蛋白在大肠杆菌BL21菌株中表达，纯化后的蛋白用于免疫小鼠。经过ELISA筛选和有限稀释亚克隆后，获得了分泌抗p24单克隆抗体的稳定杂交瘤细胞系。通过一种新型抗体捕获间接夹心ELISA筛选出34对不同的捕获和标记单克隆抗体对，然后将其应用于GICA检测p24蛋白。该GICA方法的检测限（LOD）为25 pg/mL，能够检测从HIV-1 p24抗原国家参考品中获得的所有10个阳性样本中的p24蛋白。在检测的153个阴性样本中，有3个出现假阳性结果。该检测的总体特异性为98.03%。该方法良好的敏感性和特异性使其成为一种合适的替代方法，可为HIV感染的早期诊断提供更方便、高效的工具。
