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 P 0, phosphorylated P 1 and P 2 and a 28 S r RNA fragment covering the P 0 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 (m A b) that recognized the conserved C ‐terminal tail sequence common to all three P proteins. We also obtained two P 0‐specific monoclonal antibodies, but no antibody specific to P 1, P 2 or r RNA fragment. Two types of m A bs were found among these anti‐ P antibodies: one type (e.g. 9 D 5) reacted more strongly with the phosphorylated P 1 and P 2 than that with their non‐phosphorylated forms, whereas the other type (e.g. 4 H 11) reacted equally with both phosphorylated and non‐phosphorylated forms of P 1/ P 2. Both 9 D 5 and 4 H 11 inhibited the ribosome/eukaryotic elongation factor‐2 (eE F ‐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 GTP ase 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.