PROCESS AND TEST KIT FOR NON-RADIOACTIVE ENZYMATIC DETECTION OF REVERSE TRANSCRIPTASE

摘要

The process disclosed facilitates routine non-radioactive and enzymatic detection of reverse TRANSCRIPTASE. This is done using magnetic particles and reverse transcription primers and templates immobilised on those particles, specifically in the form of a so-called particle-primer/template complex (PPP complex) combined with biotin labeling of the newly synthesised cDNA in a PPP-cDNA complex, followed by detection using streptavidin conjugate in a PPP-cDNA conjugate complex following addition of the substrate. The process and test kit are characterised by the magnetic handling of the above-mentioned particle-ligand complexes using a microtitre magnet separator which facilitates successive separation and dispersion of the particle ligand complexes out of/in solution and thus also washing operations, exchange of solutions, reaction start/stop as well as photometric/fluorimetric or luminometric quantification. The combination of magnetic handling of the particle-ligand complexes with biotin/streptavidin labeling results in an at least ten-fold improvement in sensitivity (0.05 mU/test for the first time) and widens the linear measurement range of 0.05-10.0 mU, as well as making kinetic investigations possible. The magnetic handling and the strong bonding of the biotin/streptavidin system make the process very rapid (three hours), simple and reproducible. In addition, as a result of using its own magnet separator, the process can be carried out in microtitre format and thus makes it possible to process a large number of samples simultaneously in a short time. The process can be used under appropriately optimised reaction conditions with all species of retroviruses and reverse transcriptases and can be used for the direct detection of retroviruses in cases of viral disease, in transfusion and transplant medicine, in virological research and development and in biological quality control of biopharmaceutical and biotechnology products.