Purification of monoclonal antibodies by epitope and mimotope affinity chromatography.
Murine monoclonal antibodies raised against the carcinoma-associated MUC1 mucin have applications in the diagnosis and therapy of human cancer. Many of these antibodies define linear epitopes of three, four or five amino acids within an immunodominant region of the MUC1 protein core. Various synthetic peptides which incorporated this region were prepared and covalently linked to agarose beads for use as affinity matrices. An unrelated peptide was identified as a mimotope for one of the anti-MUC1 antibodies using phage display technologies and this was also evaluated as a potential ligand in an affinity matrix. Epitope affinity chromatographic purification of an anti-MUC1 antibody was performed using hybridoma tissue culture supernatants as sample. Following sample application and column washing, antibody was desorbed from the matrix by gradient elution with increasing concentrations of NaSCN. The procedure has proved efficient for the purification of anti-MUC1 antibodies and the concentration of NaSCN required for antibody desorption gives a measure of the relative binding affinity of the antibody for the peptide epitope matrix so that separation strategies may be optimised.
Murray
A
A
Department of Pharmaceutical Sciences, University of Nottingham, UK.
Sekowski
M
M
Spencer
D I
DI
Denton
G
G
Price
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018396:Mucin-1