A SENSITIVE ASSAY FOR THE QUANTIFICATION OF REVERSE-TRANSCRIPTASE ACTIVITY-BASED ON THE USE OF CARRIER-BOUND TEMPLATE AND NON-RADIOACTIVE-PRODUCT DETECTION, WITH SPECIAL REFERENCE TO HUMAN-IMMUNODEFICIENCY-VIRUS ISOLATION

A non-radioactive 96-well microtitre plate reverse transcriptase (RT) assay, based on the use of covalently bound riboadenosine homopolymer in the wells and 5-bromodeoxyuridine 5'-triphosphate (BrdUTP) as dNTP, is described. The whole assay is performed in a single well, includin g the quantitative detection of incorporated BrdU, which is performed immunologically using alkaline phosphatase-conjugated anti-BrdU antibo dy and colorometric reading, The system also allows the use of variabl e amounts of primer. The kinetics and characteristics of the assay usi ng BrdUTP is similar to the use of [H-3]dTTP. The sensitivity of the a ssay can be varied either by altering the duration of RT assay time an d/or by prolonging the alkaline phosphatase reaction, Thus the assay c an detect <0,02 pg of recombinant human-immunodeficiency-virus (HIV) t ype I RT <0,005 munit of avian-myeloblastosis-virus RT or <0.02 munit of recombinant Moloney-murine-leukaemia-virus RT The assay was found t o be useful with various types of cell-culture material, and a compara tive study of 16 HIV-infected lymphocyte cultures, using 10 mu l of su pernatant medium for RT assay and 22.5 mu l for p24 antigen assay show ed that tile new RT assay was at least 25-fold more sensitive than the p24 antigen assay, The results also show a good correlation between t he Ri activities found and the p24-antigen level detected, with except ion for HIV2 isolates, as they only became positive in the Ri assay, T he technical performance and the capacity of the test compared with ot her available RT kits is discussed, as well as its use for other appli cations.