Purification of anti-epithelial mucin monoclonal antibodies by epitope affinity chromatography.
Monoclonal antibodies against the protein core of epithelial mucins have been found to react with the immunodominant sequence P D T R P A P (Burchell et al., 1989; Price et al., 1990a). Two immunoadsorbent matrices were prepared by linking the peptide A P D T R P A P G to CNBr-activated Sepharose and by linking the peptide C A P D T R P A P G to activated thiol-Sepharose, so that each immunoadsorbent contained the immunodominant motif. Anti-epithelial mucin antibodies (anti-breast carcinoma antibodies, anti-purified mucin antibodies and anti-human milk fat globule antibodies) were examined for reactivity with these preparations. The initial tests indicated that the substituted CNBr-activated Sepharose displayed lower non-specific antibody binding and this matrix was selected for further investigation. The anti-mucin antibodies were shown to react specifically with this affinity matrix and irrelevant antibodies failed to bind. A Sepharose-peptide immunoadsorbent column was examined for its capacity to purify several of these anti-mucin antibodies and it was determined that this procedure was highly efficient--purified IgG and IgM antibodies could be isolated from either hybridoma tissue culture supernatants or ascitic fluids. The capacity of the column was in excess of 40 mg antibody protein per ml of gel for the IgG3 antibody, C595 (anti-urinary mucin) and at least 10 mg antibody protein per ml of gel for the IgM antibody, NCRC-11 (anti-breast carcinoma). The procedure described permits the efficient purification of anti-mucin antibodies and provides a product which would be suitable for further investigations requiring highly immunoreactive antibodies (e.g., for radioimmunotherapy or immunoscintigraphy in patients with malignant disease).
Price
M R
MR
Cancer Research Campaign Laboratories, University of Nottingham, U.K.
Sekowski
M
M
Tendler
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004848:Epithelium / Q000276:immunology; D000939:Epitopes; D007164:Immunosorbents; D051379:Mice; D008969:Molecular Sequence Data; D009077:Mucins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007164:Immunosorbents; D009077:Mucins