Polyclonal Antibody Selection of Partially Disulfide-Bonded Protein States.

Polyclonal antibodies are used extensively to characterize their protein antigens in many experimental settings, including measuring protein concentration and function in vitro, ex vivo and in fixed and living tissues. Disulfide bonds constrain the polypeptide backbone of proteins and reduce the conformational variations of the structure. Several proteins are constitutively produced as multiple partially disulfide-bonded states that result in populations of protein states with a variety of conformational forms, which can influence polyclonal antibody binding to the protein. For two protein antigens described herein, polyclonal antibodies preferentially recognize states with more unformed disulfides and the avidity of the antibodies correlates with extent of unformed disulfide bonds in one of the protein antigens. This selectivity of polyclonal antibodies for their target antigens should be considered when interpreting the results of antibody-based experiments.