Recombinant anti-P proteins antibodies isolated from human autoimmune library: reactivity, specificity and epitope recognition

Introduction

The ribosomal phosphoproteins P0, P1 and P2 are targeted by autoantibodies in SLE. The presence in the patient sera of the anti-P antibodies is highly specific for the disease and correlates with psychiatric, renal and liver involvement. In order to better characterize these autoantibodies (reactivity, specificity and epitope recognition), recombinant anti-P monoclonal antibodies were isolated from an human SLE patient derived phage display library.

Methods

Two synthetic peptides were used to select the recombinant anti-P antibody fragments: a synthetic peptide representing the C-22 common immunogenic region of the three P proteins and the multiple antigenic peptide (MAP) carrying four copies of the last 13 residues of the C-22. The human library was derived from the bone marrow lymphocytes of an anti-P positive SLE patient. The selected anti-P antibodies were tested for reactivity with the C-22, the MAP and a control panel of recombinant autoantigens in an ELISA assay. Specificity of the selected antibodies was further analyzed by immunoblotting and immunoprecipitation assays using Jurkat total cell extract. Indirect immunofluorescence staining on HEp-2 cells was also performed. Using different synthetic peptides derived from the C-22 peptide epitope recognition was further characterized in an ELISA assay. Sequencing of the selected antibody fragments was performed and the antibody sequence was compared to the nearest germ-line sequence. In all the experiments human anti-P positive control sera were included.

Results

Six recombinant anti-P antibodies were isolated from the human library when using the C-22 synthetic peptide. Some of the isolated antibodies reacted specifically with the C-22 antigen in ELISA, others recognized the ribosomal P proteins on Western blot, immunoprecipitated the P proteins from the Jurkat cell extract and showed cytoplasmic staining on HEp-2 cells in an immunofluorescence assay. The selected antibodies exhibited features similar to serum antibodies of the patients with respect to their reactivity, specificity and epitope recognition.

Conclusions

The phage display technology proofs once again to be a very useful technique for the production of human monoclonal autoantibodies and for the characterization of the reactivity and specificity of these autoantibodies.