title: A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase.
abstract: 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.
authors.affiliation: Department of Medical Immunology, Medical School (Charité), Humboldt University Berlin, F.R.G.
authors.forename: T
authors.initials: T
authors.lastname: Porstmann
authors.forename: K
authors.initials: K
authors.lastname: Meissner
authors.forename: R
authors.initials: R
authors.lastname: Glaser
authors.forename: S H
authors.initials: SH
authors.lastname: Döpel
authors.forename: G
authors.initials: G
authors.lastname: Sydow
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D003856:Deoxyuracil Nucleotides; C032549:bromodeoxyuridine triphosphate; D012194:RNA-Directed DNA Polymerase; D013936:Thymidine