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

• Published in: Journal of Chromatography A 1999-08, Vol.852 (1), p.129-140
• Main Authors: Platonova, Galina A., Pankova, Galina A., Il’ina, Irma Ye, Vlasov, Guennady P., Tennikova, Tatiana B.
• Format: Article
• Language: English
• Subjects: Antibodies; Antibodies - isolation & purification; Chromatography, Affinity - methods; Peptides - immunology; Spectrophotometry, Ultraviolet
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(99)00672-X

A new affinity method for the direct quantitative analysis of monospecific anti-peptide immunoglobulins (antibodies) and, simultaneously, their semi-preparative isolation from blood serum of the immunized animals has been developed. Immunoaffinity discs based on macroporous poly(glycidyl methacrylate–co-ethylene dimethacrylate) were used as the supporting stationary phase. The specifically prepared synthetic peptides with biological activity imitating that of the immunoglobulin binding sites of various proteins were used as the selective ligands instead of native proteins. These ligands were immobilized by a single-step reaction that involves epoxy groups located on the pore surface of the porous polymer disc with amine groups of the peptide molecules. A spacer between biospecific ligands and the linking site was not required to achieve good separation. These novel immunosorbents characterized by large binding capacity are well suited for high throughput screening. Dissociation constants of the peptide–antibody complexes calculated from the experimental adsorption isotherms confirm the excellent selectivity of the proposed separation method. The discs were used in a single step enrichment of antibodies both from precipitated blood fraction and crude blood serum of immunized animals. The quantitative data of the immunoaffinity disc chromatography were compared to those obtained by an enzyme-linked immunosorbent assay. Gel electrophoresis was also used to demonstrate the high degree of purity of the final product. In contrast to typical techniques that involve proteins, this immunoaffinity approach allows for the first time direct determination of concentration of specific antibodies using the immunosorbent prepared from the short peptide molecules immobilized on the internal surface of reactive porous polymer discs.