| 1. |
- Gronowitz, J S, et al.
(författare)
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Carrier bound templates for single tube reverse transcriptase assays and for combined purification and activity analyses, with special reference to HIV
- 1991
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 13:1, s. 127-142
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Tidskriftsartikel (refereegranskat)abstract
- Polyriboadenosine (prA) was coupled to polycarbonate macrobeads or magnetic beads. The efficiency of the beads and of prA-Sepharose, after priming with odT, as templates in activity assays of purified AMV- and HIV-reverse transcriptase (RT), using [125I]iododeoxyuridine-triphosphate as substrate, was studied. Although the use of immobilized templates, compared with soluble template, resulted in a decreased total molar turnover, it did not affect the sensitivity of the assay for detecting RT. The utility of the new assay was analyzed by mixing purified AMV- or HIV-Rt with different dilutions of the untreated clinical specimen. This showed that RT activity was unaffected by 100 microliters of an extract of whole blood cells resuspended to their original blood volume and diluted 1/64, and also by 100 microliters of serum diluted 1/64. To improve the utility of the assay at the inhibitory concentrations of clinical specimens, the following procedure was adopted: the sample to be analyzed was incubated with the carrier bound template in order to allow the RT to bind, the carrier was washed to remove inhibitory factors, and the reaction components were then added to determine the amount of bound RT. This procedure greatly enhanced the recovery of RT activity from crude specimens and made the direct detection of HIV-RT possible. The assay is easily automated and useful for RT determination in multiple samples and for determining RT-inhibiting substances such as substrate analogs and antibodies.
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| 2. |
- Neumüller, M, et al.
(författare)
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HIV-1 reverse transcriptase inhibiting antibody titer in serum : relation to disease progression and to core-antibody levels.
- 1992
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Ingår i: Journal of Medical Virology. - : Wiley. - 0146-6615 .- 1096-9071. ; 36:4, s. 283-291
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Tidskriftsartikel (refereegranskat)abstract
- A new assay for detecting inhibition of reverse transcriptase activity (the RT-i REA) was developed. This assay was standardized for screening serum samples for reverse transcriptase inhibiting antibodies (RT-iAb). High specificity (100%) and sensitivity (greater than 98%) were achieved with samples from HIV-negative individuals and HIV-infected individuals. The RT-i REA was also used in a study of the titers of RT-iAb in serum samples obtained from 33 HIV-infected homosexual men. The results confirmed the relation between decreasing RT-iAb levels and progression to late stages of the disease. Furthermore, a falling RT-iAb titer was observed in 14 of 15 individuals experiencing periods of severe clinical symptoms attributed to HIV-activity. In 7 of the patients the decline in RT-iAb titer began prior to severe clinical symptoms. The fall in RT-iAb titer also correlated with a reduction in core Ab level. The core Ab level has previously been reported to be a disease progression marker with considerable prognostic value. However, whereas all patients were positive for RT-iAb, 8 of the 33 patients did not have detectable core Ab. The use of RT-iAb titer as a marker of disease progression is discussed.
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