Immunoaffinity chromatography utilizing monoclonal antibodies. Factors which influence antigen-binding capacity.
Differences in antigen-binding capacity of a monoclonal antibody coupled to Sepharose under varying conditions were explored. The extent of cyanogen bromide activation, and the pH of the coupling reaction had a profound effect upon the rate of antibody coupling, but only small differences in antigen-binding capacity were observed if the antibody coupling reaction was terminated when 80-90% of the antibody was covalently coupled to Sepharose. However, if antibody was incubated with activated resin until 100% coupling was attained, the antigen-binding capacity of the resulting immunoadsorbent decreased significantly. Monoclonal antibody coupled to Sepharose via an N-hydroxysuccinimide ester linkage and approximately half the antigen-binding capacity of antibody coupled by CNBr activation. Concentrations of monoclonal antibodies as high as 13 mg/ml of packed resin could be used without noticeable steric hindrance.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001217:Aspartate-Ammonia Ligase / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003488:Cyanogen Bromide; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008025:Ligases / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D012685:Sepharose
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007202:Indicators and Reagents; D012685:Sepharose; D008025:Ligases; D001217:Aspartate-Ammonia Ligase; D003488:Cyanogen Bromide