TY - JOUR

T1 - Multifunctional fractionation of polyclonal antibodies by immunoaffinity high-performance monolithic disk chromatography

AU - Ostryanina, Natalia D.

AU - Vlasov, Guennady P.

AU - Tennikova, Tatiana B.

N1 - Funding Information: The authors are grateful to Dr G.A. Platonova, Dr V.I. Korol’kov, G.A. Pankova and O.V. Lojkina, all from the Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg, Russia, for valuable and fruitful discussions and methodological assistance. The preparation of serum from immunized rabbits by S.B. Ivanova, Department of Molecular Genetics of the Institute of Experimental Medicine of the Russian Academy of Medical Sciences, St. Petersburg, is gratefully acknowledged. This project was partly funded by grant “Scientific Schools” (00-24678950) of the Russian Basic Research Foundation. BIA Separations, Slovenia is greatly acknowledged for providing CIM ® epoxy disks.

PY - 2002/3/8

Y1 - 2002/3/8

N2 - 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.

AB - 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.

KW - Affinity sorbents

KW - Albumin

KW - Antibodies

KW - Immunoaffinity chromatography

KW - Monolithic disks

KW - Peptides

KW - Proteins

UR - http://www.scopus.com/inward/record.url?scp=0037040587&partnerID=8YFLogxK

U2 - 10.1016/S0021-9673(02)00007-9

DO - 10.1016/S0021-9673(02)00007-9

M3 - Article

C2 - 11999732

AN - SCOPUS:0037040587

VL - 949

SP - 163

EP - 171

JO - Journal of Chromatography

JF - Journal of Chromatography

SN - 0021-9673

IS - 1-2

ER -