Suzuki K, Craddock B P, Kano T, Steigbigel R T
Biomedical Research Center, Olympus Corporation, East Setauket, New York 11733.
Anal Biochem. 1993 May 1;210(2):277-81. doi: 10.1006/abio.1993.1196.
A chemiluminescent assay for reverse transcriptase (RT) of the human immunodeficiency virus 1 was developed using biotin-labeled oligodeoxythymidylic acid (biotin oligo-dT) and digoxigenin-deoxyuridine triphosphate instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from biotin oligo-dT contained digoxigenin labels. This nucleotide was bound to a streptavidin-coated microtiter plate by the reaction to biotin. At the detection step, an alkaline phosphatase-conjugated antibody to digoxigenin was added, followed by the reaction of a chemiluminescent substrate for this enzyme. This method shows very close correlation with the isotopic assay using purified avian myeloblastosis virus reverse transcriptase (RT). This assay was also compared with the isotopic RT assay using lymphocytes infected in vitro with HTLV-IIIB and again demonstrated a close correlation. The total assay time after the RT reaction step was less than 100 min.
利用生物素标记的寡聚脱氧胸苷酸（生物素寡聚 - dT）和地高辛配基 - 脱氧尿苷三磷酸替代氚化胸苷三磷酸，开发了一种用于检测人类免疫缺陷病毒1逆转录酶（RT）的化学发光测定法。逆转录反应后，来自生物素寡聚 - dT的新聚合链含有地高辛配基标记。该核苷酸通过与生物素的反应结合到包被有链霉抗生物素蛋白的微量滴定板上。在检测步骤中，加入与地高辛配基偶联的碱性磷酸酶抗体，随后加入该酶的化学发光底物进行反应。该方法与使用纯化的禽成髓细胞瘤病毒逆转录酶（RT）的同位素测定法显示出非常密切的相关性。该测定法还与使用体外感染HTLV - IIIB的淋巴细胞的同位素RT测定法进行了比较，再次证明了密切的相关性。逆转录反应步骤后的总测定时间少于100分钟。
