Abstract
Analysis of the functional activity in polyclonal serum following immunization of a complex protein or glycoprotein immunogen is a very important but tedious process. Fine mapping of epitope-specific antibodies is difficult when they are elicited at relatively low levels. In our recent study focused on developing an HIV-1 vaccine, we immunized rabbits with hyperglycosylated stable core immunogens, which were designed using high-resolution structural information to elicit antibodies against the primary receptor-binding, CD4-binding site on HIV-1 gp120. Using a solid phase adsorption assay, we could map the serum antibodies to the conserved CD4-binding site, a known broadly neutralizing determinant on exterior envelope glycoprotein, gp120.
Materials and Reagents
Equipment
Procedure
Dynabeads MyOne Tosylactivated beads are superparamagnetic, polystyrene beads with polyurethane layer designed for bio-magnetic separations. These beads were freshly tosyl-activated, coupled with selected core gp120 variants. We used the core-conjugated Dynal beads to fractionate site-specific antibodies from polyclonal anti-sera elicited by HIV-1 core immunization. Anti-sera were adsorbed over isogenic glycoprotein antigens possessing epitope-specific mutations that were covalently attached to the Dynabeads. Following this adsorption method we were able to enrich and detect antibodies elicited against the desired epitope.
Recipes
Acknowledgments
This protocol was previously used in Ingale et al. (2014).
References
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