Mapping epitopes and antigenicity by site-directed masking.
Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (beta-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to beta-lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with beta-lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops.
Paus
Didrik
D
Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.
Winter
Greg
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000737:chemistry / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D005620:Freund's Adjuvant / Q000378:metabolism; D007114:Immunization / Q000379:methods; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D001618:beta-Lactamases / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D005620:Freund's Adjuvant; D001618:beta-Lactamases