Glycosylation analysis of a polyreactive human monoclonal IgG antibody derived from a human-mouse heterohybridoma.
Glycosylation of the human monoclonal IgG1 lambda antibody (mAb) CBGA1 was analysed by lectin blotting. The CBGA1 antibody binds to several antigens including donor self antigens, as detected by ELISA immunoblotting techniques and an erythrocyte binding assay. The mAb producing cell line was obtained by EBV transformation of peripheral blood lymphocytes of a healthy donor followed by fusion to the heteromyeloma cell line, CB-F7. The resulting heterohybridoma was cultivated in a hollow fibre bioreactor system. A bulk pool of 0.9 g antibody was produced. Fab and Fc fragments of the purified mAb were prepared and analysed. A noteworthy heterogeneity of CBGA1 and its fragments in SDS-PAGE and IEF was detected. We found glycosylation in the Fab fragment of CBGA1 in addition to the conserved glycosylation site in the Fc fragment at Asn 297. Fab glycosylation was detected in both the Fd region and the lambda-chain. The glycosylation pattern of the gamma-chain differs from that of the lambda-chain. Sequence analysis of the VH gene shows a potential N-glycosylation site located in framework III at position Asn 75.
Leibiger
H
H
Department of Medical Immunology, Medical School (Charité), Humboldt-University Berlin, Germany.
Hansen
A
A
Schoenherr
G
G
Seifert
M
M
Wüstner
D
D
Stigler
R
R
Marx
U
U
D000818:Animals; D018033:Antibodies, Bispecific / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D015151:Immunoblotting; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D037102:Lectins / Q000032:analysis; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology; D017421:Sequence Analysis
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D037102:Lectins; D011993:Recombinant Fusion Proteins