Monospecific antibody against 5-methyl-cytidine for the structural analysis of nucleic acids.

Anti 5-methyl-cytidine antibodies might be useful agents for the detection and localization of 5-methyl-cytidine of nucleic acids, but only if the antibodies recognize this nucleoside with sufficient specificity. A conjugate containing 18 moles of 5-methyl-cytidine per mole of BSA was prepared and antibodies directed against this nucleoside hapten were produced by immunization of rabbits (as determined by gel diffusion in agar containing excessive amounts of the carrier). A slight crossreaction of cytidine-BSA was eliminated by adsorption on the cross-reacting antigen. Further purification of the antibodies was effected by chromatography on DEAE-Sephadex A-50 and a method for the rapid quantitation of the antibodies showed that 12.7% of the IgG protein are monospecific against 5-methyl-cytidine-BSA. Hydrolysis of antibodies with insolubilized papain produced monovalent Fab fragments which were identified by SDS-Disk-electrophoresis. A two stage method for cross linking the immunoproteins to ferritin by glutaraldehyde was used. The isolation of immunoferritin conjugates by Bio-Gel A 1.5 m column chromatography is described. The identification of the effluents was made by glycerin density gradient ultracentrifugation. The results were visualized by electron microscopy after the treatment of immunoferritin conjugates with (methylated and unmethylated) denaturated DNA, fractionation on the glycerine density gradient, and the spreading by a modification of drop technique.