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

Abstract

To clarify the question as to why different solid-phase assays yield different results in terms of interaction strength, we used fihrinogen binding to immobilized aIIbfl3 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.

Binding of fibulin-1 to nidogen depends on its C-terminal globular domain and a specific array of calcium-binding epidermal growth factor-like (EG) modules

“…Most of these surface plasmon resonance data could be con®rmed by a second solid phase assay which did not measure kinetics, except for ®bulin-1D and mutant D498G, which were less active by at least one order of magnitude. This lack of correlation with independently measured af®-nities has also been observed in similar integrin binding assays (Tangemann & Engel, 1995), indicating that the higher af®nities measured by surface plasmon resonance are more reliable.…”

Microfibrils at Basement Membrane Zones Interact with Perlecan via Fibrillin-1

“…Although it is not possible to calculate binding affinities based on such assays (80), rough estimates suggest lower affinities as compared with affinities observed by Biacore using full-length tissue perlecan. These observations suggest that individual interaction epitopes located in the adjacent perlecan domains I and II contribute to a cooperative binding mechanism with the central region of fibrillin-1, resulting in an overall high affinity interaction between full-length fibrillin-1 and perlecan.…”

Electron microscopic evidence for a mucin‐like region in chick muscle α‐dystroglycan

“…ELISA experiments have been performed as described by [11] using a polyclonal anti-serum against mouse (EHS) tumor laminin. For laminin overlay experiments iodinated-laminin was prepared by the chloramine-T procedure, based on the protocol of Hunter and Greenwood All rights reserved.…”