DEVELOPMENT OF ELISA-BASED REVERSE TRANSCRIPTASE ASSAYS

THE MEASUREMENT OF REVERSE TRANSCRIPTASE (RT) ACTIVITY IS A COMMON PROCEDURE UTILIZED IN HUMAN RETROVIROLOGY AND HAS PLAYED A PROMINENT ROLE IN THE ISOLATION AND CHARACTERIZATION OF HUMAN RETROVIRUSES. IN ADDITION, RT MEASUREMENTS OF TISSUE CULTURE SPECIMENS OBTAINED FROM AIDS PATIENTS ARE AN ADJUNCT TO VARIOUS THERAPEUTIC REGIMES USED IN THE TREATMENT OF AIDS. THE GOAL OF THIS PHASE I STUDY ISTO 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 ARE TO DEVELOP AN RT ASSAY THAT WOULD INCORPORATE THE PERFORMANCE AND QUANTITATION OF RT REACTIONSWITHIN THE WELLS OF 96-WELL MICROTITER PLATES. THE SYNTHETIC HOMOPOLYMERS, REQUIRED AS TEMPLATE-PRIMERS FOR THERT 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, AND 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.