A Direct Binding Assay for the Vascular Cell Adhesion Molecule-1 (VCAM1) Interaction with α4 Integrins

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Abstract
Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the α4 integrins α4β1 (very late antigen 4: VLA4) and α4β7, which are constitutively expressed on many leukocyte subsets and play a key role in cell trafficking and activation. Using a recombinant VCAM-IgG fusion protein (VCAM-Ig) as a soluble ligand for α4β1 we directly demonstrated by fluorescence analysis that the α4β1 receptor can exist in different affinity states on the cell surface, and that a high affinity state is induced by manganese ions or certain activating anti-β1 monoclonal antibodies (Jakubowski et al., 1995b). Here we have extended these observations by developing a rapid and reproducible assay using alkaline phosphatase (AP)-coupled VCAM-Ig (VCAM-Ig-AP) which measures the interaction between VCAM1 and α4 integrins in a microtiter plate format. This assay has allowed us to evaluate directly the effects of metal ions, anti-β1 mAbs, and different cell types and species on the VCAM1/α4 integrin interaction. Most importantly, the assay system provides a means to rapidly evaluate α4 integrin-directed inhibitors without the complication of post-ligand binding events inherent in adhesion assays.