Label-free assessment of high-affinity antibody-antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry.
Assessment of high-affinity antibody-antigen binding parameters is important in such diverse areas as selection of therapeutic antibodies, detection of unwanted hormones in cattle and sensitive immunoassays in clinical chemistry. Label-free assessment of binding affinities is often carried out by immobilization of one of the binding partners on a biosensor chip, followed by monitoring the binding equilibrium of the other partner. However, for the measurement of high-affinity binding, with dissociation constants in the picomolar range or lower, equilibration times exceed practical limits and one has to resort to the measurement of sorption kinetics. Here we evaluate a new technique, using PEIA(1)-ellipsometry and establishment of equilibrium in solution. Binding parameters are determined for two high-affinity anti-interleukin 6 antibodies, anti-IL6.16 and anti-IL6.8, and compared with values obtained by a bioassay, based on IL6-dependent cell growth, and with values obtained by a standard technique based on SPR.(2) The high affinities of both antibodies as found with the bioassay (5 and 50pM for anti-IL6.8 and anti-IL6.16, respectively), could be conveniently measured by PEIA-ellipsometry. Using SPR, equilibrium measurements indeed proved too time-consuming and analysis of adsorption/desorption kinetics revealed that the binding of the antibodies on the chip caused the appearance of different populations of antibodies with different affinities.
Department of Immunopathology, Sanquin Research, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. t.rispens@sanquin.nl
Theo
T
Rispens
Henk
H
Te Velthuis
Piet
P
Hemker
Han
H
Speijer
Wim
W
Hermens
Lucien
L
Aarden
D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000378:metabolism; D000936:Antigen-Antibody Complex / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D020349:Surface Plasmon Resonance / Q000379:methods*
D058846:Antibodies, Monoclonal, Murine-Derived; D000936:Antigen-Antibody Complex; C508600:IL6 protein, human; D015850:Interleukin-6