Suppression of sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody

Abstract

Human IgG4 subtype antibodies have often been reported to have a significant portion (5–50%) of a heavy chain–light chain dimer (“half-antibody”) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), in which the heavy chain is not covalently linked through the hinge disulfides to another heavy chain. We demonstrate here that there can be artifactual sources of half-antibody. One occurred during SDS–PAGE sample preparation where rapid disulfide scrambling was initiated by preexisting free sulfhydryls in the monoclonal antibody (mAb) and by free sulfhydryl produced by destruction of disulfide bonds during heating. Inclusion of N-ethylmaleimide in the sample buffer prevented the disulfide scrambling. Presumably, cyclization of the flexible IgG4 hinge during this disulfide scrambling leads to the preferential separation of heavy chains. A second condition producing half-antibody was reoxidation after exposure to reductant, where 46% of the antibody was trapped in the intrachain disulfide form. The amount of half-antibody was reduced to 4% by reoxidation in the presence of a mixture of oxidized and reduced glutathione. When the improved sample preparation conditions were used, IgG4 mAb freshly isolated from cells contained 4.5–15% half-antibody, indicating that equilibration of the interchain and intrachain hinge disulfide pairing was not always attained in cells.