Characterisation of residues in antibody binding sites by chemical modification of surface-adsorbed protein combined with enzyme immunoassay.
Specific functional group modification of an antibody adsorbed to microtitre plates has been used to probe the binding site residues that determine antigen specificity. Chemical modification of adsorbed protein in tandem with enzyme immunoassay (termed CMAP-EIA) consumes only modest amounts of antibody, while allowing a variety of reagents to be rapidly screened in situ. Modification of tyrosine and arginine residues with 1-fluoro-2,4-dinitrobenzene, and p-hydroxyphenylglyoxal resulted in reduced binding of polysaccharide antigen from Yersinia enterocolitica O-polysaccharide to its homologous monoclonal antibody, YsT9-1. Modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide under various conditions indicated that carboxylate groups may also be involved. Parallel experiments with diethylpyrocarbonate and acetic anhydride were used to rule out the involvement of histidine and lysine residues respectively. In all cases, binding of an anti-idiotypic antibody, AJ5, could only be reduced at concentrations of modifying reagent substantially higher than those required to reduce polysaccharide antigen binding to YsT9-1. The results are discussed with regard to the structure of the combining site of YsT9-1 as determined by X ray crystallography and by modelling, and the role of particular residues in complex formation with antigen and in the idiotope.
Gudmundsson
B M
BM
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Young
N M
NM
Oomen
R P
RP
D000089:Acetic Anhydrides; D000907:Antibodies, Bacterial / Q000276:immunology*; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D001666:Binding Sites, Antibody / Q000187:drug effects / Q000276:immunology*; D002234:Carbodiimides; D004047:Diethyl Pyrocarbonate; D004139:Dinitrofluorobenzene; D004306:Dose-Response Relationship, Immunologic; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D008958:Models, Molecular; D019081:O Antigens; D010658:Phenylglyoxal / Q000031:analogs & derivatives; D011135:Polysaccharides, Bacterial / Q000276:immunology*; D014961:X-Ray Diffraction; D015008:Yersinia enterocolitica / Q000276:immunology*
D000089:Acetic Anhydrides; D000907:Antibodies, Bacterial; D002234:Carbodiimides; D019081:O Antigens; D011135:Polysaccharides, Bacterial; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; C028465:4-hydroxyphenylglyoxal; C031800:acetic anhydride; D004139:Dinitrofluorobenzene; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal