USE OF DRUG-SPECIFIC ANTIBODIES TO IDENTIFY ETHIDIUM ADDUCTS PRODUCEDIN TRYPANOSOMA-BRUCEI BY PHOTOAFFINITY-LABELING

A photoreactive azido analog of the trypanocide ethidium bromide, 3-am ino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, attached covale ntly to calf thymus DNA (CT DNA) by photoaffinity labeling, was used t o generate antibodies for the drug analog. The specificity of the anti serum was rested using enzyme-linked immunoadsorbant assays (ELISA) ag ainst immobilized antigen (photoaffinity labeled DNA) and by both the avidin-biotin peroxidase reaction and indirect immunofluorescence perf ormed on smears of drug treated trypanosomes. The reaction of the anti serum with the covalently bound drug adduct was diminished effectively by prior incubation with an excess of ethidium monoazide, ethidium di azide, and ethidium bromide, and to a lesser extent by the DNA-ethidiu m complex, the diazide-DNA or RNA adduct, and the monoazide-RNA adduct . DNA which had been photoaffinity labeled with either the propidium o r the acridine moiety did not react. The antiserum recognition of DNA photoaffinity labeled with ethidium monoazide was based on the substit uted phenanthridinium ring system of the parent ethidium, as evidenced by competition binding studies involving the free monoazido analog (E A1), the diazido analog (EA2), and the parent compound, ethidium bromi de (EB). This approach and the sensitivity it provides should prove us eful for identifying the distribution and fate of covalently bound dru gs resulting from antiparasitic drug treatment, and for studying their roles in antiparasitic action.