Label-free assessment of high-affinity antibody-antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry
| Autor: | Theo Rispens, Wim Th. Hermens, Henk te Velthuis, Han Speijer, Piet Hemker, Lucien A. Aarden |
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| Přispěvatelé: | Scientific Computing, Analysis (KDV, FNWI), Landsteiner Laboratory |
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Immunoassay
Chromatography Chemistry Interleukin-6 Immunology Antigen-Antibody Complex In Vitro Techniques Surface Plasmon Resonance Affinities Dissociation constant Antigen-Antibody Reactions Antibodies Monoclonal Murine-Derived Kinetics Mice Adsorption Antigen Ellipsometry Immunology and Allergy Bioassay Animals Humans Biological Assay Cattle Biosensor Label free |
| Zdroj: | Journal of Immunological Methods, 365(1-2), 50-57 Journal of immunological methods, 365(1-2), 50-57. Elsevier |
| ISSN: | 0022-1759 |
| Popis: | 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)-ellipsometty 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 50 pM 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. (C) 2010 Elsevier B.V. All rights reserved |
| Databáze: | OpenAIRE |
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