H. te Velthuis
- Label-free assessment of high-affinity antibody-antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry
- Journal of immunological methods
- Volume | Issue number
- 365 | 1-2
- Pages (from-to)
- Document type
- Faculty of Science (FNWI)
- Korteweg-de Vries Institute for Mathematics (KdVI)
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 PEIA1-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 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.
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