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

Abstract

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.

Detection of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using Aptamers as Probes in Affinity Capillary Electrophoresis

“…Typically, this was accomplished by performing a similar assay in which the RT is used, in conjunction with other primer/template oligonucleotides, to produce a target oligonucleotide later amplified by PCR. , Although innovative PCR-based CE/LIF work has been reported for HIV-1 detection, , the majority of laboratories specializing in HIV-1 research and testing typically use agarose gel electrophoresis to purify the PCR products, followed by detection and quantitation by Southern blot hybridized to a 32 P end-labeled oligonucleotide probe, entailing much time and effort. A colorimetric RT assay has been developed using 96-well microtiter plates . This test also utilizes template/primer oligonucleotides to polymerize new DNA with incorporated 5‘-bromodeoxyuridine 5‘-triphosphate(BrdUTP), usually requiring a polymerization period of 24 h. Quantification is obtained by adding alkaline phosphatase-conjugated anti-BrdUTP antibody, washing, and adding p -nitrophenyl phosphate to measure the bound antibody, with colorimetric determination taking place at specific times over an additional 24-hour period.…”

Correction: Association of pol Diversity with Antiretroviral Treatment Outcomes among HIV-Infected African Children

“…It should be noted, however, that the association between HIV diversity and treatment outcome in this study was most striking for the pol region. While antibodies that target products of the HIV pol gene have been described [30][31][32], those proteins are not primary targets of the humoral immune response to HIV infection. Alternatively, higher levels of pol diversity prior to ART initiation may reflect properties of the viral strain, such as faster replication.…”