Clinical Chemistry, Vol 33, 1741-1746, Copyright © 1987 by American Association for Clinical Chemistry

Stability of model immune complexes during zone electrophoresis and isoelectric focusing in agarose gels

FT Vertosick Jr and RH Kelly
Department of Neurological Surgery, University of Pittsburg School of Medicine, PA.

We evaluated the stability of model immune complexes, consisting of radioiodinated follitropin (125I-FSH), bound either to a mouse monoclonal antibody to FSH or to sheep polyvalent anti-FSH antiserum, during zone electrophoresis and isoelectric focusing in agarose gels. The complexes were relatively stable during zone electrophoresis at conventional voltages (less than 40 V/cm), about 80% of the initially bound antigen migrating in the gamma region. Highter voltages resulted in a linear increase in the antigen stripped from the gamma region. By contrast, isoelectric focusing very effectively dissociated the acidic 125I-FSH from the basic sheep immunoglobulins, even if the complexes were subjected to electromotive forces alone, i.e., by loading them onto the neutral region of the gel. The monoclonal complexes were not dissociated by high voltage alone. The monoclonal complexes could be completely dissociated by loading them at the anode, where gel acidification enhances complex dissociation. These observations can form the basis of a simple technique for isolating and dissociating human immune complexes before Western immunoblot analysis. Furthermore, the ease and rapidity with which bound and free antigen can be separated during zone electrophoresis in agarose suggests that this technique may have some application in clinical immunoassays.