Determining reverse transcriptase for diagnosing HIV etc. - using hapten-labelled or substd. nucleotide tri:phosphate in solid phase immunoassay, avoiding use of radio-labelled nucleotide

摘要

Process for determining the activity of reverse transcriptase (RT) comprises using a hapten-labelled or substd nucleotide triphosphate (I) and specific antibodies or other specific binding agents for (I). (I) is polymerised into a chain, and the degree of incorporation of (I) is measured in a binding test. Pref. the process is carried out in a double-sided assay, using identical antibodies as the solid phase antibody and as the labelled antibody for the chain. Part of the chain formed is therefore bound to the solid phase and the rest is determined by immunoassay. The labelled antibody that is not incorporated in the chain is separated through substrate optimisation by reaction of the RT excess or is measured in a double-sided, two part assay, so that the chain is not bound to the solid phase by the labelled antibody. The measurement is made after a washing process. A test kit comprises (a) a solid phase with immobilised anti-HIV 1-RT antibodies; (b) a solid phase as in (a) with insolubilised antibodies for binding a single or double chain; (c) labelled antibodies to the haptenised nucleotide; (d) RT-buffer concentrate with the corresp. haptenised nucleotide; (e) a substrate mixt. for the enzyme reaction; (f) a stop reagent for the enzyme reaction; (g) diluents; (h) washing buffer; and (i) control material (NaOH and citrate-phosphate buffer for single strand prodn.) USE/ADVANTAGE- For determining RT activity of HIV-1 and other retroviral infections. The process avoids the use of radiolabelled markers and so does not require specialised equipment. The process can be automated and can therefore be used widely and -easily.