Patent EA-004880-B1 (EA)
Title: Reverse transcriptase assay kit and use thereof
Inventor: KALLANDER CLAS
IPC: C12Q1/42
Filed: 2000-06-16
Abstract: 1. Reverse transcriptase (RT) assay kit comprising one or several package (s) containing solid phase bound polyriboadenylic acid (prA) and/or polydeoxyadenylic acid (pdA) template (s) obtainable by contacting a polystyrene-based solid phase with a coupling solution comprising 1-methylimidazole, and prA and/or pdA, followed by incubation, washing with a wash buffer, drying and packaging, RT-type adapted separately packaged assay components selected from the group consisting of a mixture of or separately a buffer, pH [approximately equal to] 7 - 8, divalent metal ion, chelator, polyamine, RNase inhibitor, reducing agent, salt, stabilizing agent, and detergent, and a mixture of or separately lyophilized deoxynucleotide triphosphate, primer, protective agent and a concentrated washing buffer, and optionally lyophilized reference enzyme (s), and optionally components of a detection system comprising lyophilized alkaline phosphatase conjugated anti-BrdU monoclonal antibody, alkaline phosphatase substrate buffer and alkaline phosphatase substrate, and written instructions for use of the assay kit. 2. RT assay kit according to claim 1, wherein the solid phase is a microtiter plate and an aliquot of the coupling solution, which comprises 100 mM 1-methylimidazole, pH [approximately equal to] 5-7, and 0.5-2 mg/ml prA and/or pdA, is added to each well, followed by the incubation at a temperature of 10-60 degree C for 0.5-10 h, and washing each well for the removal of the 1-methylimidazole with the wash buffer, which comprises Bis-Tris propane, pH [approximately equal to] 5-7, and drying and packaging the plates. 3. RT assay kit according to claim 2, wherein 100 [mu]l of the coupling solution, which comprises 100 mM 1-methylimidazole, Ph [approximately equal to] 6.25, and 1 mg/ml prA and/or pdA, is added to each well, followed by the incubation at room temperature for-2 h, washing of each well with 2x300 [mu]l of the wash buffer, which comprises 10 mM Bis-Tris propane, pH [approximately equal to] 6.25, drying the plates at 37 degree C for 25 minutes and putting the plates in foil bags and vacuum sealing the bags. 4. RT assay kit according to any one of claims 1-3, wherein the assay components are selected from the group consisting of the buffers Tris and Hepes, pH [approximately equal to] 7-8, the divalent metal ions Mg<2+> and Mn<2+>, the chelators ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(beta-aminoethyl ether) N, N, N', N'-tetraacetic acid (EGTA) and, the polyamines spermine and spermidine, the RNase inhibitors heparin sulfate and dextran sulfate, the reducing agents dithiothretiol (DTT), dithioerythritol (DTE), and glutathione, the salts NaCI and KCI, the stabilizing agents newborn calf serum (NCS) and bovine serum albumin (BSA), the detergents Tween 20 and Triton X-100, the deoxynucleotide triphosphate BrdUTP, the primer oligo dT, and the protective agent agents ATP, GTP and CTP. 5. Use of an assay kit according to any one of claims 1-4 for the qualitative and quantitative analysis of RT activity in a biological sample. 6. Use according to claim 5, wherein the biological sample is selected from biological fluids and cell extracts. 7. Use according to claim 6, wherein the biological fluid is selected from plasma, serum, spinal fluid, synovial fluid and pleural fluid. 8. Use according to any one of claims 5-7 followed by evaluation of the status of a RT activity related disorder or disease based on the result of the analysis of the RT activity.