Tomita N, Miyahara M, Satoh H, Suzuki K, Kitajima K, Miyamoto K
Okayama Red Cross Blood Center, Japan.
Acta Med Okayama. 1995 Apr;49(2):69-73. doi: 10.18926/AMO/30390.
An enzyme-linked immunosorbent assay (ELISA) using biotin-labelled oligo-dT primer and digoxigenin (Dig)-dUTP was designed to measure the reverse transcriptase (RT) activity of human immunodeficiency virus type 1 (HIV-1). The ELISA system involves the selective detection step of a newly synthesized cDNA by two specific bindings, biotin-streptavidin binding and alkaline phosphatase (AP)-conjugated anti-Dig-Dig binding, and the enzymatic amplification step to increase coloring generated by AP. This method was used to measure the activity of RT in the culture supernatants of peripheral leukocytes obtained from four anti-HIV-1-positive persons cocultivated with those from four anti-HIV-1-negative persons. RT activity was detected in all of four anti-HIV-1-positive culture supernatants but not in those cultivated with anti-HIV-1-negative supernatants alone. Thus, our improved ELISA for detection of HIV-1 appears to be sensitive enough and useful for routine laboratory work. This non-radioactive method will also be useful for detecting other retroviruses and for screening of RT inhibitors.
设计了一种使用生物素标记的寡聚-dT引物和地高辛配基（Dig）-dUTP的酶联免疫吸附测定（ELISA）来检测1型人类免疫缺陷病毒（HIV-1）的逆转录酶（RT）活性。该ELISA系统包括通过生物素-链霉亲和素结合和碱性磷酸酶（AP）偶联的抗Dig-Dig结合这两种特异性结合来选择性检测新合成的cDNA的步骤，以及用于增强AP产生的显色反应的酶促放大步骤。该方法用于检测从4名抗HIV-1阳性者与4名抗HIV-1阴性者共同培养获得的外周血白细胞培养上清液中的RT活性。在所有4份抗HIV-1阳性培养上清液中均检测到RT活性，而单独与抗HIV-1阴性上清液共同培养的上清液中未检测到。因此，我们改进的用于检测HIV-1的ELISA似乎足够灵敏，适用于常规实验室工作。这种非放射性方法也将有助于检测其他逆转录病毒以及筛选RT抑制剂。
