Comparison of the results obtained by ELISA and surface plasmon resonance for the determination of antibody affinity.
The aim of this study was to compare the affinity values obtained for a monoclonal antibody/antigen complex using two different techniques, surface plasmon resonance (SPR) and an enzyme linked immunosorbent assay (ELISA) approach recently described by Bobrovnik S.A. and by Stevens F.J. These two techniques can be used in particular to determine the equilibrium dissociation constant, K(D), of the complex in solution or on a surface. Bobrovnik's method gives two K(D) values that differ by a factor of 100, demonstrating that two populations of complexes are present in solution. In an initial step, one protein binds relatively weakly to the other (high K(D)) and this is followed by a conformational change in the most flexible portion of the antigen, which increases the affinity (low K(D)). Only the higher of the two K(D) values can be detected when complex formation in solution is investigated using SPR, because the interaction measured concerns the fibronectin/antibody complexes of lowest affinity. In contrast, when measuring association at the sensor surface, SPR gives an average result between the two K(D) values because complexes corresponding to both affinities can form in this situation. The constants that characterise the kinetics of the fibronectin-antibody interaction obtained by SPR and ELISA are therefore different, because the methods do not allow the same phenomena to be observed. However they are consistent and complementary.
Heinrich
Laurence
L
Université de Lyon, Université Lyon 1, ISPB, UPSP 2007-03-135 Réparation Tissulaire Interactions Biologiques et Biomatériaux, 8 Avenue Rockefeller F-69373 Lyon Cedex 08, France. laurence.heinrich@recherche.univ-lyon1.fr
Tissot
Nathalie
N
Hartmann
Daniel Jean
DJ
Cohen
Richard
R
D000136:Acid-Base Equilibrium; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D055592:Biophysical Phenomena / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005353:Fibronectins / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000379:methods*
D000911:Antibodies, Monoclonal; D005353:Fibronectins