Figure 3.

Recombinant antibodies representing clonal lineages bind citrullinated antigens. The CCP3.1 ELISA was used to measure anti-CCP antibody binding A, in RA subject plasma and B, among the expressed recombinant antibodies. Using the manufacturer’s protocol, activity units and plasma activity cutoff (blue dotted line) were determined. For recombinant antibodies, the activity cutoff (red dotted line) was set to three standard deviations above the average binding activity of negative control antibodies. Data presented as mean ± SEM from duplicates. C, Recombinant antibodies were evaluated for epitope specificity using an RA antigen microarray that includes ~350 citrullinated and native proteins/peptides. Microarrays were probed with recombinant antibodies representing clonal families/lineages and singletons derived from plasmablasts binding to citrullinated-peptide tetramers. The presented heatmap depicts median fluorescent intensities (MFI) from quadruplicate print spots. Gray indicates an antibody-antigen combination for which data could not be obtained. Each antibody is labeled according to the subject (S) and timepoint (T) from which it is derived. The recombinant antibodies derived from a plasmablast that stained for the citrullinated-peptide tetramers during sorting are marked in red. Further, similar peptide sequences to those used to generate the tetramers are marked with a blue asterisk.