A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase.
A sensitive non-isotopic assay for specific detection of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is described using 5-bromo-2'-deoxyuridine triphosphate (BrdUTP) instead of tritiated thymidine triphosphate. After the RT reaction the template primer is degraded by alkaline hydrolysis. Single-stranded poly.(BrdU) is detected in an immunoenzymometric assay using monoclonal anti-BrdU antibodies. The specificity of the assay is demonstrated by the isolation of RT from virus lysate by an insolubilised monoclonal anti-HIV-1 RT antibody prior to the RT reaction. Immunological RT binding leads to a tenfold increase in analytical sensitivity since substances inhibiting the RT reaction can be removed. This non-isotopic assay is some 30 times more sensitive than the classical radioisotopic RT assay. In terms of RT determination, however, there is a good correlation between these tests (r = 0.96). Several filtrations are no longer necessary to remove non-incorporated nucleotides. The test can be adapted to microtitre plates and hence is easy to automate.
Porstmann
T
T
Department of Medical Immunology, Medical School (Charité), Humboldt University Berlin, F.R.G.
Meissner
K
K
Glaser
R
R
Döpel
S H
SH
Sydow
G
G
D000911:Antibodies, Monoclonal / Q000276:immunology; D003856:Deoxyuracil Nucleotides; D015497:HIV-1 / Q000201:enzymology*; D006868:Hydrolysis; D007124:Immunoenzyme Techniques; D012194:RNA-Directed DNA Polymerase / Q000276:immunology / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D013936:Thymidine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003856:Deoxyuracil Nucleotides; C032549:bromodeoxyuridine triphosphate; D012194:RNA-Directed DNA Polymerase; D013936:Thymidine