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。 该方法用于人类逆转录病毒学领域，迄今为止， 在隔离和描述人类的过程中扮演了重要的角色 逆转录病毒 此外，组织培养标本的RT测量 从艾滋病患者中获得的药物是各种治疗方案的辅助药物 用于治疗艾滋病。 本I期研究的目标是 开发一种快速准确的基于ELISA的RT检测方法， 更经济地进行分析，不需要 放射性同位素，并协助继续寻找更多的人类 逆转录病毒 本研究的第一阶段目标是开发一种RT测定法， 将RT反应的性能和定量纳入 96孔微量滴定板的威尔斯孔。 合成均聚物 （聚[rC].寡聚[dT]，作为RT反应的模板引物，将 固定在用于进行RT的96孔微量滴定板的威尔斯孔中 比色法 将通过使用兔抗体对反应进行定量， 与生物素缀合，针对合成DNA-RNA均聚物开发。 该试验的免疫学部分将通过标准ELISA完成 利用与过氧化物酶结合的链霉亲和素的技术。 比较 评价各种方法来合成模板- 引物将沿着最终RT测定的优化 格式.

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.