Label-free assessment of high-affinity antibody–antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry

• Published in: Journal of immunological methods Vol. 365; no. 1-2; pp. 50 - 57
• Main Authors: Rispens, Theo, te Velthuis, Henk, Hemker, Piet, Speijer, Han, Hermens, Wim, Aarden, Lucien
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 28-02-2011; Elsevier
• Subjects: adsorption; Animals; antibodies; Antibodies, Monoclonal, Murine-Derived - metabolism; Antigen-Antibody Complex - metabolism; Antigen-Antibody Reactions; Biacore; binding capacity; bioassays; Biological and medical sciences; Biological Assay - methods; biosensors; Cattle; cell growth; desorption; dissociation; Fundamental and applied biological sciences. Psychology; Fundamental immunology; High-affinity antibodies; hormones; Humans; Immunoassay - methods; immunoassays; In Vitro Techniques; Interleukin-6 - immunology; Interleukin-6 - metabolism; Kinetics; Mice; Molecular immunology; monitoring; PEIA-ellipsometry; Surface Plasmon Resonance - methods; Techniques; Biacore; High-affinity antibodies; PEIA-ellipsometry; Antigen; Bioassay; Antibody; Association constant; Immunological method
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2010.11.010

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 PEIA11PEIA = Precipitate-Enhanced ImmunoAssay.-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.22SPR = Surface Plasmon Resonance.
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.