5 - Resolution of Immunoglobulin Patterns by Analytical Isoelectric Focusing

Abstract

This chapter discusses the resolution of immunoglobulin patterns by analytical isoelectric focusing (IEF). IEF in polyacrylamide slab gels has become a frequently used method to study the heterogeneity of antibody responses and to screen genetically defined populations of laboratory animals for the pattern of specific antibodies. IEF has also been used to screen human sera for the occurrence of naturally acquired antibodies to the streptococcal group polysaccharide antigens. IEF is a high-resolution separation method. It separates ampholytes such as amino acids and oligo- and polypeptides as well as protein molecules according to their isoelectric points in ion-free media. For the fractionation of immunoglobulins, the column approach is used mainly for preparative work; polyacrylamide gels have the capacity to highly resolve and, therefore, identify the product of a single lymphocyte clone (clonotype) in a mixture of other proteins and antibodies. However, IEF became a practical method for the fractionation of macromolecular ampholytes only when natural pH gradients and suitable carrier ampholytes were introduced.