Figure 1. Cloning of Human CFH mAbs Specific to an Antigenic Peptide.

(A) Isolation of CFH-specific memory B cells that bind to the epitope of interest. PBMCs were obtained from patients positive for anti-CFH antibodies and used for sorting CFH antigen-binding memory B-cells via FACS. To minimize false-positives, streptavidin was labeled with Alexa Fluor 647 and Brilliant Violet 421. Labeling with each fluorophore was carried out on separate aliquots of streptavidin, which were then mixed together prior to interaction with biotinylated antigen peptide (CFH_1110–1124). Cells showing elevated fluorescence for both fluorophores, indicated by the outlined region (0.32% of the total), were sorted into single wells for recombinant mAb synthesis. The B cell expressing mAb7968 is designated in pink.

(B) Identification of cloned mAbs that bind a CFH epitope-containing peptide by ELISA. Supernatants from HEK293 cells transiently expressing pairs of V_H and V_L chains were collected, adjusted to 1 µg/ml IgG, and equal volumes tested for binding to epitope peptide bound to neutravidin (Pep-NA) or to neutravidin alone (NA). Bound antibody was detected with anti-human IgGγ-HRP and ABTS/H₂O₂ substrate. Sample 31 is an affinity purified anti-CFH autoantibody serving as a positive control.