Demonstration of non‐linear detection in ELISA resulting in up to 1000‐fold too high affinities of fibronogen binding to integrin αIIbβ3

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Abstract
To clarify the question as to why different solid-phase assays yield different results in terms of interaction strength, we used fibrinogen binding to immobilized alpha IIb beta 3 integrin as a test system. A classical 'three step' enzyme-linked (ELISA), a 'two step' biotin enzyme-linked streptavidin and a 'one step' radioligand assay were compared under otherwise identical conditions. Only the last assay yielded binding constants comparable to earlier data by total internal reflection fluorescence microscopy while the other assays yielded apparent binding constants 5- to 1000-fold too high. These effects are explained by non-linearity of detection signals.