Assessment of affinity constants by rapid solid phase detection of equilibrium binding in a flow system.
We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilised analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference spectroscopy (RIfS) using flow injection analysis. The capabilities of the model were investigated using different analyte-antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10(7) M(-1) and higher. For lower affinity constants significant deviations due to dissociation of the analyte-antibody complex during detection were observed.
Piehler
J
J
Institut fur Physikalische und Theortische Chemie, Tubingen, Germany. jacob.piehler@uni-tuebingen.de
Brecht
A
A
Giersch
T
T
Hock
B
B
Gauglitz
G
G
D000818:Animals; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D004058:Diffusion; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D012212:Rheology; D014227:Triazines
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008024:Ligands; D014227:Triazines