DEVELOPMENT OF ELISA-BASED REVERSE TRANSCRIPTASE ASSAYS

逆转录酶(RT)活性的测量是一种常见的方法 人类逆转录病毒学领域使用的程序，到目前为止， 在人类的隔离和表征方面发挥了突出的作用 逆转录病毒。此外，组织培养标本的RT测量 从艾滋病患者那里获得的药物是各种治疗方案的辅助手段 用于治疗艾滋病。此第一阶段研究的目标是 建立一种快速、准确的基于酶联免疫吸附试验的RT检测方法，以允许 检测将更经济地进行，不要求 放射性同位素，并协助继续寻找更多的人类 逆转录病毒。 这项研究的第一阶段目标是开发一种RT检测方法，它将 将RT反应的性能和量化纳入 96孔微滴定板。人工合成的均聚物 (Poly[RC].ogo[DT]，作为RT反应所需的模板引物，将 被固定在用于进行RT的96孔微量板孔中 化验。这一反应将通过使用兔抗体进行量化， 与生物素结合，针对合成的DNA-RNA均聚体开发。 检测的免疫学部分将由标准的酶联免疫吸附试验完成。 利用链霉亲和素标记过氧化物酶的技术。比较级的 评价用于固定合成模板的各种方法-- 在优化最终RT分析的同时，还将进行引物操作 格式化。

The measurement of reverse transcriptase (RT) activity is a common procedure utilized in the field of human retrovirology and has, to date, played a prominent role int he isolation and characterization of human retroviruses. In addition, RT measurements of tissue culture specimens obtained from AIDS patients is an adjunct to various therapeutic regimes used in the treatment of AIDS. The goal of this Phase I study is to develop a rapid and accurate ELISA-based RT assay that would permit such assays to be performed more economically, with no requirement for radioisotopes, and to assist in the continued search for additional human retroviruses. The Phase I goals of this research are to develop an RT assay which would incorporate the performance and quantitation of RT reactions within the wells of 96-well microtiter plates. The synthetic homopolymers (poly[rC].oligo[dT], required as template-primers for the RT reaction, will be immobilized in wells of 96-well microtiter plates used to perform the RT assay. The reaction will be quantitated by the use of rabbit antibodies, conjugated to biotin, developed against synthetic DNA-RNA homopolymers. The immunologic portion of the assay will be completed by standard ELISA technology utilizing streptavidin conjugated to peroxidase. A comparative evaluation of various methodologies to immobilize the synthetic template- primers will be performed along with the optimization of the final RT assay format.