Label-free assessment of high-affinity antibody-antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry.
J Immunol Methods
; 365(1-2): 50-7, 2011 Feb 28.
Article
in En
| MEDLINE
| ID: mdl-21115013
ABSTRACT
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biological Assay
/
Immunoassay
/
Surface Plasmon Resonance
/
Antigen-Antibody Reactions
Type of study:
Evaluation_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
J Immunol Methods
Year:
2011
Document type:
Article
Affiliation country:
Netherlands