Molecular analysis of IgM rheumatoid factor binding to chimeric IgG.
To localize regions on IgG bound by rheumatoid factors (RF), we studied IgM RF binding to chimeric IgG antibodies consisting of murine V regions fused to human constant regions. Using a modified RF ELISA, we showed that polyclonal RF from rheumatoid arthritis patients bound IgG1, 2, and 4 strongly; IgG3 was also bound, although less well. The majority of 18 monoclonal RF from patients with Waldenstrom's macroglobulinemia bound IgG1, 2, and 4 only. In contrast to RF from RA, 14 of 18 monoclonal RF did not react with IgG3. Only 3 of 18 monoclonal RF bound IgG3 well. By shuffling C region domains between IgG3 and IgG4, we showed that sequence variation in the CH3 domain is responsible for the differential binding of monoclonal RF to IgG3 and IgG4. Hybrid IgG3/IgG4 antibodies containing the CH3 domain of IgG4 were bound by monoclonal RF, whereas those containing the CH3 domain of IgG3 were not. To evaluate the contribution of the N-linked carbohydrate moiety at Asn-297 to RF binding sites on IgG, we measured RF binding to aglycosylated IgG antibodies produced by mutating Asn-297 to another amino acid. Glycosylated and aglycosylated IgG1, 2, and 4 were bound identically by monoclonal and polyclonal RF. Aglycosylated IgG3, however, was bound better than glycosylated IgG3 by polyclonal RF and by IgG3-reactive monoclonal RF.
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
S E
SE
Artandi
S M
SM
Canfield
M H
MH
Tao
K L
KL
Calame
S L
SL
Morrison
V R
VR
Bonagura
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000918:Antibody Specificity; D001172:Arthritis, Rheumatoid / Q000276:immunology; D003619:Dansyl Compounds / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D006031:Glycosylation; D006241:Haptens / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D007132:Immunoglobulin Isotypes / Q000378:metabolism; D007075:Immunoglobulin M / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012217:Rheumatoid Factor / Q000378:metabolism*; D008258:Waldenstrom Macroglobulinemia / Q000276:immunology
D000911:Antibodies, Monoclonal; D003619:Dansyl Compounds; D000939:Epitopes; D006241:Haptens; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D011993:Recombinant Fusion Proteins; D012217:Rheumatoid Factor