Characterization of anti‐P monoclonal antibodies directed against the ribosomal protein–RNA complex antigen and produced using Murphy Roths large autoimmune‐prone mice

Summary Autoantibodies, including anti‐ribosomal P proteins (anti‐P), are thought to be produced by an antigen‐driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti‐P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti‐P monoclonal antibody (mAb) that recognized the conserved C‐terminal tail sequence common to all three P proteins. We also obtained two P0‐specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti‐P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non‐phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non‐phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor‐2 (eEF‐2)‐coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C‐terminal sequence common to all three P proteins, but not the peptide that lacked the last three C‐terminal amino acids, mostly prevented the mAb‐induced inhibition of GTPase activity. Thus, at least two types of anti‐P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C‐termini, particularly that of the last three C‐terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus.