Biointeraction analysis by high-performance affinity chromatography: Kinetic studies of immobilized antibodies.
A system based on high-performance affinity chromatography was developed for characterizing the binding, elution and regeneration kinetics of immobilized antibodies and immunoaffinity supports. This information was provided by using a combination of frontal analysis, split-peak analysis and peak decay analysis to determine the rate constants for antibody-antigen interactions under typical sample application and elution conditions. This technique was tested using immunoaffinity supports that contained monoclonal antibodies for 2,4-dichlorophenoxyacetic acid (2,4-D). Association equilibrium constants measured by frontal analysis for 2,4-D and related compounds with the immobilized antibodies were 1.7-12x10(6)M(-1) at pH 7.0 and 25 degrees C. Split-peak analysis gave association rate constants of 1.4-12x10(5)M(-1)s(-1) and calculated dissociation rate constants of 0.01-0.4s(-1) under the application conditions. Elution at pH 2.5 for the analytes from the antibodies was examined by peak decay analysis and gave dissociation rate constants of 0.056-0.17s(-1). A comparison of frontal analysis results after various periods of column regeneration allowed the rate of antibody regeneration to be examined, with the results giving a first-order regeneration rate constant of 2.4x10(-4)s(-1). This combined approach and the information it provides should be useful in the design and optimization of immunoaffinity chromatography and other analytical methods that employ immobilized antibodies. The methods described are not limited to the particular analytes and antibodies employed in this study but should be useful in characterizing other targets, ligands and supports.
Nelson
Mary Anne
MA
Chemistry Department, University of Nebraska, Lincoln, 68588-0304, United States.
Moser
Annette
A
Hage
David S
DS
D015084:2,4-Dichlorophenoxyacetic Acid / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007700:Kinetics; D011485:Protein Binding
D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D015084:2,4-Dichlorophenoxyacetic Acid