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Rheumatoid-factor-reactive sites on CH2 established by analysis of overlapping peptides of primary sequence
1Department of Medicine, University of Florida School of Medicine, Gainesville 32610.
Polyclonal IgM rheumatoid factors (RF) from ten patients with rheumatoid arthritis (RA) isolated on IgG affinity columns were studied for their reactivity with overlapping 7-mer peptides representative of solvent accessible regions of the CH2 domains of IgG using a pin-ELISA assay. A panel of nine monoclonal RF from RA patients' B cells were studied in parallel. Four peptides SVFLFPP (239-245), KFNWYVD (274-280), NSTYRVVSV (297-305) and VLTVLHQNWL (305-314) reacted with most RF. Glycine substitution showed that tryptophanes at 277 and 313, tyrosine at 278, valines at 279 and 305, and leucine 314 represented important residues for RF reactivity. Assays using monoclonal IgM RF produced from RA synovial B cells or peripheral blood B cells frequently showed a restricted spectrum of reactivity for CH2 epitopes, which often were identical to those binding to polyclonal IgM RF. Combinations of synthetic 7-mer peptides representing RF-reactive CH2 or CH3 epitopes produced as much as 60-90% inhibition of RF binding to IgG when peptides were preincubated with RF in free solution before completion of the reaction of RF with IgG on the ELISA plate.
Polyclonal IgM rheumatoid factors (RF) from ten patients with rheumatoid arthritis (RA) isolated on IgG affinity columns were studied for their reactivity with overlapping 7-mer peptides representative of solvent accessible regions of the CH2 domains of IgG using a pin-ELISA assay. A panel of nine monoclonal RF from RA patients' B cells were studied in parallel. Four peptides SVFLFPP (239-245), KFNWYVD (274-280), NSTYRVVSV (297-305) and VLTVLHQNWL (305-314) reacted with most RF. Glycine substitution showed that tryptophanes at 277 and 313, tyrosine at 278, valines at 279 and 305, and leucine 314 represented important residues for RF reactivity. Assays using monoclonal IgM RF produced from RA synovial B cells or peripheral blood B cells frequently showed a restricted spectrum of reactivity for CH2 epitopes, which often were identical to those binding to polyclonal IgM RF. Combinations of synthetic 7-mer peptides representing RF-reactive CH2 or CH3 epitopes produced as much as 60-90% inhibition of RF binding to IgG when peptides were preincubated with RF in free solution before completion of the reaction of RF with IgG on the ELISA plate.