Suzuki K, Craddock B P, Kano T, Steigbigel R T
Biomedical Research Center, Olympus Corporation, East Setauket, NY 11733.
J Virol Methods. 1993 Jan;41(1):21-8. doi: 10.1016/0166-0934(93)90159-o.
A colorimetric assay for reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) was developed using a double labelled (biotin and digoxigenin) deoxyuridine triphosphate mixture instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from oligodeoxythymidylic acid (oligo-dT) contained both biotin and digoxigenin labels. This nucleotide was bound to streptavidin-magnetic beads by the reaction to biotin. At the detection step, an alkaline phosphatase conjugated antibody to digoxigenin was added, followed by the reaction of a colorimetric substrate for this enzyme. This RT assay was comparable to the isotopic RT assay using purified AMV-RT and two strains of HIV grown in cell lines. In addition it was equivalent to the isotopic RT assay for analysis of the time course of in vitro infection of human peripheral blood lymphocytes by HIV-1. The total assay time after the RT reaction step was less than one hour.
利用双标记（生物素和地高辛配基）三磷酸脱氧尿苷混合物而非氚标记的三磷酸胸腺嘧啶核苷，开发了一种用于检测1型人类免疫缺陷病毒（HIV-1）逆转录酶（RT）的比色测定法。RT反应后，来自寡聚脱氧胸苷酸（oligo-dT）的新聚合链同时含有生物素和地高辛配基标记。该核苷酸通过与生物素的反应与链霉抗生物素蛋白磁珠结合。在检测步骤中，加入与地高辛配基偶联的碱性磷酸酶抗体，随后该酶的比色底物发生反应。这种RT测定法与使用纯化的禽成髓细胞瘤病毒逆转录酶（AMV-RT）以及在细胞系中培养的两株HIV的同位素RT测定法相当。此外，它等同于用于分析HIV-1体外感染人外周血淋巴细胞时间进程的同位素RT测定法。RT反应步骤后的总测定时间不到一小时。
