Class-specific monoclonal antibodies for the detection of O⁶-alkyl-2'-deoxyguanosines

• Main Author: McPhillips, Fiona
• Published: University of Edinburgh 1994
• Subjects: 572.85
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.657131

The reaction of alkylating reagents with DNA yields a variety of potential mutagenic and carcinogenic lesions. One such group, the O<SUP>6</SUP>alkyl-2'-deoxyguanosines (O<SUP>6</SUP>alkyl dGs) are known to induce guanine to adenine transitions, which may lead to the activation of oncogenes. Antibodies with predefined specificities can be employed in the detection and quantification of such lesions. Four O<SUP>6</SUP>alkyl dG analogues (methyl, ethyl, n-propyl and hydroxyethyl) have been synthesised. Each was made into a suitable immunogen by i) conjugation to BSA and/or CγG, and ii) incorporation into an oligonucleotide. An O<SUP>6</SUP>methylbenzoate dG derivative has also been synthesised and conjugated to BSA and CγG. All conjugates and oligonucleotides have been characterised. Each O<SUP>6</SUP>alkyl dG immunogen has been used for immunisation. Hybridoma technology was employed to give monoclonal antibody producing cell lines. Screening assays were developed for the detection of a cell line producing a monoclonal antibody specific for O<SUP>6</SUP>alkyl dG. Three monoclonal antibodies have been produced. The relative affinities of each antibody for each O<SUP>6</SUP>alkyl dG have been determined. The highest affinity antibodies have been immobilised on sepharose. Immunoaffinity columns have been constructed and employed in the extraction of O<SUP>6</SUP>alkyl dG from a pool of nucleosides.