A direct binding assay for the vascular cell adhesion molecule-1 (VCAM1) interaction with alpha 4 integrins.
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.
Lobb
R R
RR
Biogen, Inc., Cambridge Center, MA 02142, USA.
Antognetti
G
G
Pepinsky
R B
RB
Burkly
L C
LC
Leone
D R
DR
Whitty
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002448:Cell Adhesion / Q000502:physiology; D005455:Fluorescent Antibody Technique; D006801:Humans; D039441:Integrin alpha4; D016023:Integrins / Q000378:metabolism*; D007938:Leukemia; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D016023:Integrins; D019010:Vascular Cell Adhesion Molecule-1; D039441:Integrin alpha4; D008274:Magnesium