UBC Theses and Dissertations
A non-radioactive method of detecting nucleic acids Kalmar, Gabriel Bela
Monoclonal antibodies (MCAs) specific for iodinated cytidine were produced and characterized for their possible application in the development of a serological, non-radioactive assay for nucleic acids. The method requires the modification of single-stranded ribonucleic acid (ss-RNA) by addition of an iodine to the C-5 position of cytidine via the Commerford reaction.. The level of iodination was followed by ultraviolet (UV) spectroscopy and X-ray diffraction (XRF) and was found to be higher for polyC than for cellular RNA or ss-RNA of southern bean mosaic and cowpea chlorotic mottle virus. No evidence was obtained for iodination of double-stranded (ds) RNA of turnip yellow mosaic virus. Only one hybridoma, (Rl), of 2,500, tested secreted MCAs specific for iodinated cytidine. The specificity and affinity of this MCA for iodinated and non-iodinated synthetic polynucleotides and iodinated cellular ss-RNA were assessed by solid-phase and inhibition assays, using the enzyme-1 inked immunosorbent assay (ELISA). Seven other hybridomas produced MCAs with varying specificities for iodinated nucleotide-carrier combinations. 'The binding of MCA Rl to iodocytidine-carrier was inhibited by iodocytidine or iodocytidine-5'-phosphate and by adenosine or adenosine-5'-phosphate (20 and 200 times less so, respectively) but not by guanosine, thymidine, and uridine, or their monophosphates. As little as 5 ng of iodocytidine-carrier bound to a nitrocellulose membrane could be detected by MCA Rl in an immune-blot assay whereas no reaction was observed with 1-1000 ng of cytidine-carrier. The lower levels of detection for iodinated cellular nucleic acid and iodinated polyC were 50 and 25 ng, respectively, in the same assay. The method may have applications in the development of a serological, non-radioactive assay for nucleic acids.