Affinity electrophoresis and its applications to studies of immune response

Affinity electrophoresis (AEP) is a useful technique for separation of biomolecules such as plasma proteins, enzymes, nucleic acids, lectins, receptors, and extracellular matrix proteins by specific interactions with their ligands in electric fields and for the determination of dissociation constants for those interactions. Two-dimensional affinity electrophoresis (2-D AEP), which was newly developed by a combination of isoelectric focusing with AEP, has been used for studies on immune response to haptens. Antihapten antibodies, which were induced by immunization of a mouse with the hapten-conjugated bovine serum albumin, were separated by 2-D AEP into a large number of groups of IgG spots with a few microliters of antiserum. Each group of spots showed an identical affinity for the hapten but different isoelectric points as in the case of monoclonal antibodies specific to the hapten. This enabled us to study the diversification and affinity maturation of antihapten antibodies in the course of immunization of a single mouse. Furthermore, effects of a carrier and a hapten array on the production of antihapten antibodies and the cause of charge heterogeneity of monoclonal antibodies were also examined to understand the molecular basis of the immune response in vivo.