Lennerstrand J, Rytting A S, Orvell C, Gronowitz J S, Kallander C F
The Research Unit of Replication Enzymology, Department of Medical Genetics, Uppsala University, Bromma, Sweden.
Anal Biochem. 1996 Mar 15;235(2):141-52. doi: 10.1006/abio.1996.0106.
Detection of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity in crude specimens was greatly enhanced using a novel capture RT assay. Eighteen different monoclonal antibodies (Mabs) raised against purified HIV-1 RT were tested for their ability to bind to HIV-1 RT without affecting its activity. The anti-HIV-1 RT Mabs were immobilized on plastic macrobeads and used as solid carriers in the capture RT assay. The assay system first involved RT's adherence to the immobilized Mabs. Nonspecific enzymes and other impurities were removed by a simple wash after which the RT reaction mixture was added. Substrate and product were finally separated by a wash of the beads. Practically all radioactivity incorporated into DNA (>98%) was recovered on the bead. The Michaelis-Menten constants and the saturation velocity values for the nucleotide substrate were similar for free and immobilized RT. The reaction mechanism for the immobilized RT is discussed. When comparing the function of this assay with more conventional soluble RT assays for samples consisting of recombinant HIV-1 RT mixed with an extract of peripheral blood lymphocytes (PBL), an almost 100-fold higher sensitivity was found. The capture RT assay had the capacity to recover approximately 80% of the RT activity added to an extract of 1 x 10(7) PBL cells/ ml. A strong correlation (r = 0.947) between the results obtained with this assay and a HIV-1 p24 enzyme-linked immunosorbent assay was found, when samples from a collection of 16 HIV strains propagated in cell culture were analyzed.
使用一种新型捕获逆转录酶（RT）检测法可大大提高在粗制标本中检测1型人类免疫缺陷病毒（HIV-1）逆转录酶（RT）活性的能力。针对纯化的HIV-1 RT产生的18种不同单克隆抗体（Mab），检测了它们在不影响其活性的情况下与HIV-1 RT结合的能力。抗HIV-1 RT Mab被固定在塑料大珠上，并用作捕获RT检测法中的固体载体。该检测系统首先涉及RT与固定化Mab的结合。通过简单洗涤去除非特异性酶和其他杂质，然后加入RT反应混合物。最后通过洗涤珠子分离底物和产物。实际上，掺入DNA中的所有放射性（>98%）都在珠子上回收。游离RT和固定化RT的核苷酸底物的米氏常数和饱和速度值相似。讨论了固定化RT的反应机制。当将该检测法的功能与用于由重组HIV-1 RT与外周血淋巴细胞（PBL）提取物混合组成的样品的更传统的可溶性RT检测法进行比较时，发现灵敏度提高了近100倍。捕获RT检测法能够回收添加到1×10⁷个PBL细胞/ml提取物中的约80%的RT活性。在分析来自在细胞培养中繁殖的16种HIV毒株集合的样品时，发现该检测法获得的结果与HIV-1 p24酶联免疫吸附测定法之间存在强相关性（r = 0.947）。
