Multifunctional fractionation of polyclonal antibodies by immunoaffinity high-performance monolithic disk chromatography

High-performance monolithic disk chromatography (HPMDC), including its affinity mode, is a very efficient method for fast separations of biological molecules of different sizes and shapes. In this paper, protein and peptide ligands, immobilized on the inner surface of thin, monolithic supports (Convective Interaction Media or CIM® disks), have been used to develop methods for fast, quantitative affinity fractionation of pools of polyclonal antibodies from blood sera of rabbits, immunized with complex protein–peptide conjugates. The combination of several disks with different affinity functionalities in the same cartridge enables the separation of different antibodies to be achieved within a few minutes. The apparent dissociation constants of affinity complexes were determined by frontal analysis. Variation of elution flow rate over a broad range does not affect the affinity separation characteristics. Indifferent synthetic peptides used as biocompatible spacers do not change the affinity properties of the ligands. The highly reproducible results of immunoaffinity HPMDC are compared with data obtained by widely used enzyme-linked immunosorbent assay.