| Abstract |
By employing two different immunogens and two different antibody-screening strategies, we established four mouse hybridoma clones producing monoclonal antibodies against N epsilon -acetyllysine. Three different protocols were used in this study; i.e., mice were (1) immunized with an N epsilon -acetyllysine-containing peptide, Gly-Lys(Ac)- epsilon -aminocaproic acid (Aca)-Cys, conjugated to KLH, and the hybridoma clones were screened for their reactivity to a histone H3 peptide containing five acetyllysines; (2) immunized as in "1" and screened with chemically acetylated bovine serum albumin (BSA); (3) immunized with chemically acetylated keyhole limpet hemocyanin (KLH) and screened with chemically acetylated BSA. Antibodies produced by the four different hybridomas established here all reacted with acetyllysine residues, but their reactivity was not the same when evaluated with enzyme-linked immunosorbent assay (ELISA), Western blotting, and resonant mirror sensor analyses. Among the three protocols examined, protocol "3" was especially useful to obtain hybridomas producing anti-N epsilon -acetyllysine antibodies that could detect not only the acetylated histones but also other acetylated proteins. By cloning and sequencing the cDNAs encoding the variable regions of the antibodies, we found that their framework sequences were almost the same, which suggests that some framework amino acids in addition to their complementarity determining regions (CDRs) directly contribute to their recognition function.
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