Mapping epitopes and antigenicity by site-directed masking

Abstract

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 (␤-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 ␤-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 ␤-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.backbone flexibility ͉ Freund's adjuvant ͉ conformational epitope ͉ antisera

Stiff-person syndrome (SPS) and anti-GAD-related CNS degenerations: Protean additions to the autoimmune central neuropathies

“…Moreover, the binding site of GAD65 for antibodies displays greater polarity and more negatively charged amino acid residues on its surface whereas for GAD67 the C-terminal exhibits a subdued, neutral electrostatic field and hence possesses less polarity [50]. Finally, the C-terminal and catalytic loop residues of GAD65 display more flexibility and mobility than their GAD67 counterparts [30], recalling here that Paus and Winter [51] suggested that increases in protein flexibility and charge were associated with augmented protein antigenicity. Thus differing structural properties of the two GAD isoforms would explain at least to some degree why, for T1D sera, only the GAD65 isoform is independently autoantigenic.…”

Antibody-independent localization of the electroneutral Na+-HCO3− cotransporter NBCn1 (slc4a7) in mice

“…The molecular identification in the remaining studies hinges on RT-PCR analyses and immunohistochemical localization, using antibodies directed against the transporter (14,15,18,21,31,32,37). The use of antibodies depends on immunoreactivity, which can be influenced by several factors, including the conformation of the antigen, phosphorylation, or masking from other structures (14,23,25). Furthermore, eventual splice variants without the targeted epitope will not be immunoreactive.…”

Constraining Enzyme Conformational Change by an Antibody Leads to Hyperbolic Inhibition

“…33 In summary, we have shown how an antibody can inhibit enzyme activity hyperbolically by constraining conformation change. In view of the apparent correlation between antigenicity and epitope flexibility, 34 we anticipate that this mechanism will recur in allosteric effector antibodies that modulate the activities of other enzymes and receptors.…”