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      A direct binding assay for the vascular cell adhesion molecule-1 (VCAM1) interaction with alpha 4 integrins.

      Cell adhesion and communication

      Integrins, Animals, Antibodies, Monoclonal, metabolism, Antigens, CD, Binding, Competitive, drug effects, physiology, Cell Adhesion, Fluorescent Antibody Technique, Humans, Integrin alpha4, Leukemia, Magnesium, pharmacology, Mice, Protein Binding, Rats, Tumor Cells, Cultured, chemistry, cytology, Vascular Cell Adhesion Molecule-1, immunology

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          Abstract

          Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the alpha 4 integrins alpha 4 beta 1 (very late antigen 4: VLA4) and alpha 4 beta 7, 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 alpha 4 beta 1 we directly demonstrated by fluorescence analysis that the alpha 4 beta 1 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-beta 1 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 alpha 4 integrins in a microtiter plate format. This assay has allowed us to evaluate directly the effects of metal ions, anti-beta 1 mAbs, and different cell types and species on the VCAM1/alpha 4 integrin interaction. Most importantly, the assay system provides a means to rapidly evaluate alpha 4 integrin-directed inhibitors without the complication of post-ligand binding events inherent in adhesion assays.

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          8640376

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