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Titolo:
Colorimetric capture assay for human-immunodeficiency-virus-I reverse transcriptase activity
Autore:
Rytting, AS; Akerblom, L; Gronowitz, JS; Kallander, CFR;
Indirizzi:
Cavidi Tech, Staben, SE-75183 Uppsala, Sweden Cavidi Tech Uppsala SwedenSE-75183 ch, Staben, SE-75183 Uppsala, Sweden Uppsalaweden, BMC, Dept Genet & Pathol, Med Genet Sect, SE-75123 Uppsala, S Uppsala Univ Uppsala Sweden SE-75123 Med Genet Sect, SE-75123 Uppsala, S Natl Vet Inst, Dept Virol, SE-75007 Uppsala, Sweden Natl Vet Inst Uppsala Sweden SE-75007 pt Virol, SE-75007 Uppsala, Sweden
Titolo Testata:
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
, volume: 29, anno: 1999,
parte:, 3
pagine: 241 - 250
SICI:
0885-4513(199906)29:<241:CCAFHR>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
ACTIVITY-BLOCKING ANTIBODY; RT ACTIVITY; HIV; SUBTYPE; PLASMA; QUANTIFICATION; PURIFICATION; QUANTITATION; CAMEROON; SERUM;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
27
Recensione:
Indirizzi per estratti:
Indirizzo: Kallander, CFR Cavidi Tech, Staben, Uppsala Sci Pk, SE-75183 Uppsala, Sweden Cavidi Tech Uppsala Sci Pk Uppsala Sweden SE-75183 Sweden
Citazione:
A.S. Rytting et al., "Colorimetric capture assay for human-immunodeficiency-virus-I reverse transcriptase activity", BIOT APP B, 29, 1999, pp. 241-250

Abstract

The development of a colorimetric capture assay for HIV-1 reverse transcriptase (RT) activity is described. This assay consisted of three basic steps: enzyme purification, RT reaction and product detection, which were all performed in the same microtitre plate. Mouse monoclonal anti-RT antibodies of subclass G2a were bound by polyclonal goat anti-(mouse IgG2a) immobilizedin the wells of a microtitre plate. The monoclonal antibodies (mAbs) were selected for their ability to bind HIV-1 RT without hampering the polymerase activity. The assay system first involved the RT's adherence to the immobilized mAbs, Non-specific enzymes and other impurities were removed by a simple wash, after which an RT reaction mixture containing BrdUTP as nucleotide substrate was added. After the RT reaction substrate and product had been separated by washing of the plate, the amount of BrdUMP-DNA in the wells was finally detected with alkaline-phosphatase-conjugated mouse anti-BrdU antibodies of subclass IgGI. The background signal in this system was similar to the signals obtained with control wells coated with BSA only. A detection limit of 1,2 mu-units of RT activity, corresponding to 0.3 pg of RT protein, was obtained for the capture assay when applying colorimetric productdetection, The assay detected RTs from HIV-1 subtypes A and B and one of the two D type isolates tested. None of the five non-HIV-1 RTs tested was found positive. At least 50 mu l of human serum or plasma per sample could beincluded in the capture assay without adverse effects on the recovery of the RT activity.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/12/20 alle ore 16:53:05