ANTIBODIES TO UV IRRADIATED DNA: THE MONITORING OF DNA DAMAGE BY ELISA AND INDIRECT IMMUNOFLUORESCENCE

Abstract The enzyme-linked immunosorbant assay (ELISA) was modified to (1) characterize antibodies raised in rabbits against UV-irradiated single-stranded DNA (UVssDNA) complexed with methylated BSA and (2) directly detect pyrimidine dimers in irradiated DNA. The antisera specifically bound to UVssDNA, UVpoly(dT) and to a limited extent to UVdsDNA and UVpoly(dC) immobilized on protamine sulfate coated microliter wells. Fifty percent of the maximum antibody binding was observed at a 1-5000 dilution against UVssDNA. Binding to ssDNA and poly(dT) was observed only at much higher concentrations of antibody (1:500 dilution), whereas no binding to double stranded DNA (dsDNA) was observed. The extent of binding of the antibody was dependent on the dose of UV radiation to DNA, as well as, to the concentration of antigen immobilized on the plate. Specific binding to DNA irradiated with 5.0 J/m² was detected with as little as 10 ng of DNA. The sensitivity was further extended to less than 1 J/m² by using higher concentrations (100 ng) of UVssDNA. The ability of various irradiated molecules, DNA, homopolymers and linkers to act as inhibitors of antibody binding establish that the antigenic determinants are mainly thymine homodimers with lower affinity for cytosine dimers. Potential usefulness of the antibodies to directly quantitate pyrimidine dimers in cells exposed to UV radiation was determined by indirect immunofluorescence. Flow cytometric analysis of immunostained human lymphocytes irradiated with 254 nm radiation indicated that greater than 50% of the population had significantly higher fluorescent intensity than unirradiated control cells.