Affinity immunoblotting. High resolution isoelectric focusing analysis of antibody clonotype distribution.

A sensitive and specific method has been developed for analyzing specific antibody clonotype changes during an immune response or comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies were separated by flatbed acrylamide isoelectric focusing (IEF) and analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels after IEF bound the antigen on the nitrocellulose when the coated nitrocellulose was laid over the gels. Non-specific protein binding was inhibited with Tween 20. Bound IgG antibody clonotypes were detected using peroxidase-conjugated anti-IgG. This method has been used for the analysis of Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentration for coating the nitrocellulose membranes ranged from 10 to 100 micrograms/ml. The reactions could be inhibited by saturating the nitrocellulose with soluble protein antigen or free hapten prior to immunoblotting. Antibodies of alternative allotypic forms were detected by probing the immunoblot with biotinylated anti-allotype antibodies instead of anti-IgG. The simultaneous analysis of alternative allelic forms of antibodies of defined specificity is not possible with methods which use labelled antigen for clonotype detection.