High resolution functional analysis of antibody-antigen interactions.
A comprehensive mutational analysis was used to analyze the side-chains on human growth hormone (hGH) important for binding 21 different anti-hGH mouse monoclonal antibodies (MAbs) whose equivalent concentrations for 50% binding (EC50) ranged from approximately 10(7) to 3 x 10(10) M-1. A combination of homolog- and alanine-scanning mutagenesis coupled with a robot-aided enzyme-linked immunosorbent assay were used to create high resolution "functional epitopes" for each MAb. Every functional epitope mapped to at least two polypeptide segments of hGH that were close together in the folded protein to form a patch. Although these patches sometimes overlapped, each was different indicating no two MAbs bound identically to hGH. The MAbs bound to determinants in loops and helices that were generally most accessible to a 9 A radius probe. Only a few side-chains dominated each functional epitope and these tended to be Arg greater than Pro greater than Glu approximately Asp approximately Phe approximately Ile (Ala, Cys and Trp were not tested). Our studies indicate that most of the accessible surface of hGH is potentially antigenic in the mouse and suggest that functional epitopes are dominated by fewer side-chains than may be in the contact epitope.
Jin
L
L
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080.
Fendly
B M
BM
Wells
J A
JA
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D000936:Antigen-Antibody Complex; D000937:Antigen-Antibody Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D013006:Growth Hormone; D000409:Alanine