Denaturing action of adjuvant affects specificity of polyclonal antibodies

Influence of the immunization procedure on the specificity of the produced antibodies towards different conformations of the antigen was investigated. It was demonstrated that intravenous immunization of a rabbit with an adjuvant-free solution of recombinant sperm-specific glyceraldehyde-3-phosphate dehydrogenase (dN-GAPDS) resulted in production of antibodies recognizing only native conformation of dN-GAPDS and exhibiting no cross-reaction with somatic isoenzyme of glyceraldehyde-3-phosphate dehydrogenase. A subcutaneous immunization with human dN-GAPDS mixed with Freund's complete adjuvant yielded antibodies recognizing both native and denatured conformation of dN-GAPDS. The oil component of the adjuvant was shown to cause inactivation and partial denaturation of dN-GAPDS, leading to exposure of the epitopes that are masked in the native protein, which resulted in production of the antibodies to the denatured antigen. These results may be of importance for biochemical research that often require polyclonal antibodies recognizing different conformations of antigens.