Measurement of levels of human immunodeficiency virus type 1 reverse transcriptase (RT) and RT activity-blocking antibody in human serum by a new standardized colorimetric assay.

Standardization and calibration of a new colorimetric assay for detection of reverse transcriptase (RT) was carried out for optimal detection of RT activity-blocking antibody (RTb-Ab) in serum. A total of 99 of 100 Swedish and 54 of 54 African human immunodeficiency virus type 1 (HIV-1) antibody-positive individuals had RTb-Ab. The one RTb-Ab-negative HIV-1 serum sample from a Swedish individual was obtained early during seroconversion. Five of 615 HIV-1-negative sera from tumor patients, pregnant women, patients undergoing routine viral diagnostics, and blood donors gave false-positive results. In addition, 3 of 126 HIV-1-negative African serum samples and 2 of 91 serum samples selected because of false reactivity in other commercially available HIV antibody assays were positive for RTb-Ab. RT activity and RTb-Ab were measured in sera from newly HIV-1-infected individuals during seroconversion. Peak RT activity was usually detected between days 8 and 13 after the onset of symptoms of primary infection. In addition, HIV-1 RTb-Ab was detected in the same recently infected individuals in most cases within 1 month and in some cases as early as 10 to 12 days after the onset of symptoms. A cross-reactivity study involving HIV-1 and HIV-2 RTb-Abs and their homologous RT showed HIV-1 RTb-Ab to be highly type specific. None of 10 serum samples from HIV-1-infected individuals showed cross-reacting RTb-Ab toward HIV-2 RT, whereas 4 of 10 serum samples from HIV-2-infected patients showed cross-reactivity toward HIV-1 RT; however, the cross-reactivity toward HIV-1 RT was 3,000 times lower than that toward its homologous RT. Future uses for the assay with reference to the recent World Health Organization proposal for other methods instead of Western blotting (immunoblotting) for confirming HIV-1 infection and for methods for the diagnosis of infection as follow-up in vaccine trials are also discussed.

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-bromodeoxyuridined 5'-triphosphate (BrdUTP) as dNTP, is described. The whole assay is performed in a single well, including the quantitative detection of incorporated BrdU, which is performed immunologically using alkaline phosphatase-conjugated anti-BrdU antibody and colorometric reading. The system also allows the use of variable amounts of primer. The kinetics and characteristics of the assay using BrdUTP is similar to the use of [3H]dTTP. The sensitivity of the assay can be varied either by altering the duration of RT assay time and/or by prolonging the alkaline phosphatase reaction. Thus the assay can detect < 0.02 pg of recombinant human-immunodeficiency-virus (HIV) type I RT, < 0.005 m unit of avian-myeloblastosis-virus RT or < 0.02 m unit of recombinant Moloney-murine-leukaemia-virus RT. The assay was found to be useful with various types of cell-culture material, and a comparative study of 16 HIV-infected lymphocyte cultures, using 10 microliters of supernatant medium for RT assay and 22.5 microliters for p24 antigen assay showed that the new RT assay was at least 25-fold more sensitive than the p24 antigen assay. The results also show a good correlation between the RT activities found and the p24-antigen level detected, with exception for HIV2 isolates, as they only became positive in the RT assay. The technical performance and the capacity of the test compared with other available RT kits is discussed, as well as its use for other applications.