Recombinant anti-P protein autoantibodies isolated from a human autoimmune library: reactivity, specificity and epitope recognition

Abstract

The ribosomal P proteins are specific and important autoantigens in patients affected by systemic lupus erythematosus. In this study, we describe for the first time the selection and characterization of recombinant human monoclonal anti-P protein (auto)-antibody fragments from an autoimmune patient-derived phage display antibody library. The selected recombinant anti-P antibodies specifically recognize the P proteins in immunofluorescence assays on HEp-2 cells and in immunoblotting assays, and they immunoprecipitate the P proteins under native conditions. Using both anti-P-positive patient sera and the selected recombinant anti-P antibodies, the immunodominant epitope was determined and shown to be located at the C-terminal end of the P proteins (amino acids 111-115). Inhibition of in vitro protein translation demonstrated that interaction of the monoclonal patient-derived anti-P antibodies with their native epitope functionally inhibits the activity of the P proteins on the ribosome, confirming the notion that patient autoantibodies are often directed to the functional centre of their autoantigenic target.

Cloning and characterization of two human Ro52-specific monoclonal autoantibodies directed towards a domain associated with congenital heart block

“…An average to high level of mutation was identified in S3A8 and M4H1 V H clones, indicative of an antigen-driven response. Similar observations have been reported for other autoantibodies [24,26,37], though the previously described Ro52 specific IgM monoclonals contained no mutations and two replacements together with two silent mutations, respectively [38].…”

“…Selections were performed with (a) a library derived from SLE patient S339, and (b) a mix of the five libraries derived from autoimmune patients diagnosed with MCTD, SLE and UCTD (Mix) [24].…”

Selective Blockade of Trypanosomatid Protein Synthesis by a Recombinant Antibody Anti-Trypanosoma cruzi P2β Protein

“…Thus, the ribosomal stalk is directly involved in the translocation step of protein synthesis [9]. It has been previously shown that antibodies against the C-terminal region of ribosomal P proteins (markers of systemic lupus erythematosus in humans) and their scFv recombinant forms posses the ability to block in vitro translation in a rabbit reticulocyte lysate system [10], [11]. In chronic Chagas' heart disease, antibodies against the C-terminal region of T. cruzi ribosomal P proteins have been also detected [12], [13].…”

A Novel Subtractive Antibody Phage Display Method to Discover Disease Markers

“…Autoimmune patient libraries of SLE (patients D5, D18, D181, O11, and Z5), systemic sclerosis (SSc) (patients B92, H248, J70, S185, and T5), and rheumatoid arthritis (RA) (patients Du, He, and Wy) patients were available in pHenIX-VSV vector format (23)(24)(25). The synthetic library made by Nissim et al (26) was used for test selections.…”

“…Libraries of RA, SSc, and SLE patients (23)(24)(25) were mixed using equal amounts of phages from each library, and phages…”