Quantitative fast fractionation of a pool of polyclonal antibodies by immunoaffinity membrane chromatography

A new affinity method for the direct quantitative analysis of monospecific anti-peptide immunoglobulins (antibodies) and, simultaneously, their semi-p reparative isolation from blood serum of the immunized animals has been dev eloped. Immunoaffinity discs based on macroporous poly(glycidyl methacrylat e-co-ethylene dimethacrylate) were used as the supporting stationary phase. The specifically prepared synthetic peptides with biological activity imit ating that of the immunoglobulin binding sites of various proteins were use d as the selective ligands instead of native proteins. These ligands were i mmobilized by a single-step reaction that involves epoxy groups located on the pore surface of the porous polymer disc with amine groups of the peptid e molecules. A spacer between biospecific ligands and the linking site was not required to achieve good separation. These novel immunosorbents charact erized by large binding capacity are well suited for high throughput screen ing. Dissociation constants of the peptide-antibody complexes calculated fr om the experimental adsorption isotherms confirm the excellent selectivity of the proposed separation method. The discs were used in a single step enr ichment of antibodies both from precipitated blood fraction and crude blood serum of immunized animals. The quantitative data of the immunoaffinity di sc chromatography were compared to those obtained by an enzyme-linked immun osorbent assay. Gel electrophoresis was also used to demonstrate the high d egree of purity of the final product. In contrast to typical techniques tha t involve proteins, this immunoaffinity approach allows for the first time direct determination of concentration of specific antibodies using the immu nosorbent prepared from the short peptide molecules immobilized on the inte rnal surface of reactive porous polymer discs. (C) 1999 Published by Elsevi er Science B.V. All rights reserved.