Solid Phase Assays for Studying ECM Protein-Protein Interactions - Methods in Molecular Biology: Extracellular Matrix Protocols - page 281

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Solid Phase Assays
for Studying ECM Protein-Protein Interactions

A. Paul Mould

1. Introduction
Solid-phase assays provide a simple, rapid and robust method for the analy-
sis of protein-protein interactions; i.e., does protein A interact with protein B?
In this assay, protein A (here termed “receptor”) is adsorbed to the wells of an
enzyme-linked immunosorbent assay (ELISA) plate (solid phase). The plate is
then blocked using bovine serum albumin (BSA), and biotin-labeled protein B
(here termed 'ligand') is added. After washing the wells to remove unbound
ligand, bound ligand is detected by addition of an avidin-peroxidase conjugate
followed by a colorimetric detection step.
This type of assay is particularly well suited for studying the interaction of
ECM proteins with integrins. The first solid-phase integrin-ligand binding
assay was described by Charo et al. (1) for studying fibrinogen binding to
α 3. In our laboratory we have developed an extremely sensitive and highly

versatile assay for fibronectin binding to

IIb β

1 (2) . For example, we have
described how the assay can be used to investigate the effects of divalent cat-
ions, activating and inhibitory MAbs, peptide inhibitors and mutations on
ligand binding (2-5) . The pharmacological screening of inhibitors of integrin-
ligand interactions is an important area in which this particular type of assay is
finding use.
Our preferred method for labeling of ligands is biotinylation because of its
safety and simplicity. One potential drawback is that if one or more lysyl resi-
dues in the ligand are crucial for receptor binding, their modification may ren-
der the ligand inactive. In this case, a possible solution may be to reduce the
amount of biotinylation reagent so that some of the lysyl residues remain
unmodified. Other labeling methods such as radioiodination can also be used.

α 5 β