Immunogenicity of free histones and of histones complexed with RNA

Histone antibodies have been obtained by immunizing rabbits with histones H1, H2A, H2B, H3, H4 and triacetylated H4, uncomplexed to RNA. The reactivity of these antibodies was investigated by ELISA using as antigen isolated histones and chromatin as well as thirty-five different synthetic peptides covering the entire sequence of the four core histones, two peptides of H1 and two acetylated peptides of H4. The binding of these antibodies to histones was also measured in immunoblotting and in microcomplement fixation (MCF) tests. In parallel experiments using the same assays the various antigens were tested with antisera raised against histones complexed with RNA. Antibodies induced in the absence of RNA did not react with histones in MCF tests nor with chromatin in ELISA but reacted with the histones in ELISA, although the antibody titers were somewhat lower than in the case of antisera to histone-RNA complexes. Antibodies to RNA-histone complexes reacted with histones in both ELISA and MCF tests. When they were tested with peptide-coated microtiter plates in a direct binding ELISA format, antibodies induced with uncomplexed histones recognized very few fragments which were mainly located in the N- and C-terminal ends of the histones.