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Titolo:
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
Autore:
EKSTRAND DHL; AWAD RJK; KALLANDER CFR; GRONOWITZ JS;
Indirizzi:
UNIV UPPSALA,CTR BIOMED,DEPT MED GENET,RES UNIT REPLICAT ENZYMOL,BOX 584 S-75123 UPPSALA SWEDEN UNIV UPPSALA,CTR BIOMED,DEPT MED GENET,RES UNIT REPLICAT ENZYMOL S-75123 UPPSALA SWEDEN
Titolo Testata:
Biotechnology and applied biochemistry
, volume: 23, anno: 1996,
parte:, 2
pagine: 95 - 105
SICI:
0885-4513(1996)23:<95:ASAFTQ>2.0.ZU;2-B
Fonte:
ISI
Lingua:
ENG
Soggetto:
DNA-POLYMERASE; HIV-1; PURIFICATION; ANTIBODIES; SERUM; RT;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
25
Recensione:
Indirizzi per estratti:
Citazione:
D.H.L. Ekstrand et al., "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", Biotechnology and applied biochemistry, 23, 1996, pp. 95-105

Abstract

A non-radioactive 96-well microtitre plate reverse transcriptase (RT)assay, based on the use of covalently bound riboadenosine homopolymerin the wells and 5-bromodeoxyuridine 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 performedimmunologically 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 [H-3]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 munit of avian-myeloblastosis-virus RT or <0.02 munitof 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 mu l of supernatant medium for RT assay and 22.5 mu l for p24 antigen assay showed 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 the Ri activities found and the p24-antigen level detected, with exception for HIV2 isolates, as they only became positive in the Ri 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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/12/20 alle ore 16:08:15