TY - JOUR

T1 - Efficient purification of unique antibodies using peptide affinity-matrix columns

AU - Jensen, Liselotte Brix

AU - Riise, Erik

AU - Nielsen, Leif Kofoed

AU - Dziegiel, Morten Hanefeld

AU - Fugger, Lars

AU - Engberg, Jan

PY - 2004/1

Y1 - 2004/1

N2 - Phage display technology was used to identify peptide ligands with unique specificity for a monoclonal model antibody, MK16, that recognises the human multiple sclerosis associated MHC class II molecule DR2 in complex with a myelin basic protein (MBP)-derived peptide corresponding to residue 85-99. Several peptide epitopes were identified and all of them recognised specifically MK16. One peptide, ER6.1, was selected and linked to beaded agarose and demonstrated excellent performance as a peptide affinity chromatography matrix. This epitope matrix was efficient in the purification of MK16 Fab fragments and had no affinity for other antibodies. Using this peptide matrix MK16 IgG could be purified from cell culture supernatants thereby separating MK16 IgG from bovine IgG normally present in the enriched growth media used for such cells. Investigations of the fine specificity of the ER6.1 peptide demonstrated that it recognised a unique epitope within the heavy chain CDR3 region of the MK16 antibody. Thus, variants of MK16 antibody, which had retained the specificity and affinity of the original antibody but had slightly different amino acid composition in the CDR3 region, were not recognised by the ER6.1 peptide.

AB - Phage display technology was used to identify peptide ligands with unique specificity for a monoclonal model antibody, MK16, that recognises the human multiple sclerosis associated MHC class II molecule DR2 in complex with a myelin basic protein (MBP)-derived peptide corresponding to residue 85-99. Several peptide epitopes were identified and all of them recognised specifically MK16. One peptide, ER6.1, was selected and linked to beaded agarose and demonstrated excellent performance as a peptide affinity chromatography matrix. This epitope matrix was efficient in the purification of MK16 Fab fragments and had no affinity for other antibodies. Using this peptide matrix MK16 IgG could be purified from cell culture supernatants thereby separating MK16 IgG from bovine IgG normally present in the enriched growth media used for such cells. Investigations of the fine specificity of the ER6.1 peptide demonstrated that it recognised a unique epitope within the heavy chain CDR3 region of the MK16 antibody. Thus, variants of MK16 antibody, which had retained the specificity and affinity of the original antibody but had slightly different amino acid composition in the CDR3 region, were not recognised by the ER6.1 peptide.

KW - Amino Acid Sequence

KW - Antibodies, Monoclonal

KW - Antibody Affinity

KW - Antibody Specificity

KW - Base Sequence

KW - Chromatography, Affinity

KW - Complementarity Determining Regions

KW - Epitopes

KW - Humans

KW - Molecular Sequence Data

KW - Peptide Fragments

KW - Peptide Library

KW - Sequence Alignment

M3 - Journal article

C2 - 14736416

VL - 284

SP - 45

EP - 54

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

IS - 1-2

ER -