Vascular cell adhesion molecule-1 (VCAM-1, CD106) is a member of the immunoglobulin gene superfamily. Two forms of VCAM-1 with six or seven extracellular Ig-like domains arise due to alternative splicing from a seven-domain VCAM-1 (7D VCAM-1). 7D VCAM-1 is the dominant form expressed by cultured human endothelial cells (1,2). Homology of domains 1 through 3 with domains 4 through 6 suggests a gene duplication event (1,2). The cDNA of 7D VCAM-1 predicts a core protein of approximately 81kD with seven sites for N-linked glycosylation. Upon complete glycosylation the mature protein has a molecular weight of approximately 102kD. This observation is in general agreement with the 110kD protein immunoprecipitated from cytokine-activated endothelium (3,4).
VCAM-1 appears to have been highly conserved through evolution. Both rat and mouse VCAM-1 are highly homologous at the protein level to the human VCAM-1 (77% and 76%, respectively) (5). VCAM-1 supports the adhesion of lymphocytes, monocytes, natural killer cells, eosinophils and basophils, through its interaction with integrin a4b1 (VLA-4). VCAM-1/VLA-4 interaction mediates firm adherence of circulating nonneutrophilic leukocytes to endothelium (6). VCAM-1 also participates in leukocyte adhesion outside of the vasculature, mediating precursor lymphocyte adhesion to bone marrow stromal cells (7) and B cell binding to lymph node follicular dendritic cells (8).
VCAM-1 is not constitutively expressed on endothelium, but can be up-regulated in vitro in response to LPS, TNF-a, and IL- 1 (9,10), as well as to interferon-g and IL-4 (11). VCAM-1 is also present on tissue macrophages, dendrite cells, bone marrow fibroblasts, myoblasts and myotubes. A soluble form of VCAM-1 has been described (12,13), and soluble VCAM-1 has been found in serum (14). Increased levels of soluble VCAM-1 can be detected in several disease states, including cancer (15-18), autoimmune disorders (17,19-24), infections (25) and inflammations (17,22,26,27).
An anti-VCAM-1 monoclonal antibody is adsorbed onto microwells. Soluble VCAM-1 present in a sample or standard then binds to antibodies adsorbed to the microwells. A mixture of biotin-conjugated monoclonal anti-VCAM-1 antibody and Streptavidin-HRP conjugate is added. Biotinylated anti-VCAM-1 antibody binds to VCAM-1 captured by the first antibody. Streptavidin-HRP binds to the biotinylated anti-VCAM-1. Unbound biotinylated anti-VCAM-1 and Streptavidin-HRP conjugate is removed with a wash step and HRP substrate solution is added to the wells. An amount of colored product is formed, proportional to the amount of soluble VCAM-1 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve is prepared from six soluble VCAM-1 standard dilutions and the VCAM-1 sample concentration is determined.
The VCAM-1 ELISA is an enzyme-linked immunosorbent assay for quantitative detection of soluble Vascular Cell Adhesion Molecule-1 in cell culture supernatants, serum, plasma, or other biological fluids.
Analytical Sensitivity and Detection Limits:Sensitivity: 0.59ng/ml Range of Detection: 3.2-100ng/ml Intra-Assay Variation: 3.1% Inter-Assay Variation: 5.2% Recovery: 89% average Assay Time: 135 minutes
Crossreactivity:No interference from other members of the immunoglobulin superfamily.
Kit Components:C2446-86A1: 1 aluminum pouch with a Microwell Plate coated with Mab (murine) to human VCAM-1C2446-86A2: 1 vial (0.1ml) Conjugate Mixture containing Biotin-Conjugated Anti-VCAM-1 Mab (murine) mixed with Streptavidin-HRP*C2446-86A3: 2 vials (200ng/ml as reconstituted) soluble VCAM-1 Standard, lyophilized*C2446-86A4: 1 vial lyophilized controlC2446-86A5: 1 bottle (50ml) Wash Buffer Concentrate 20X (PBS, 1% Tween 20)* C2446-86A6: 1 vial (5ml) Assay Buffer Concentrate 20X (PBS, 1% Tween 20, 10% BSA)*C2446-86A7: 1 vial (15ml) Substrate Solution C2446-86A8: 1 vial (12ml) Stop Solution (1M Phosphoric Acid, H3PO4), ready to use C2446-86A9: 1 Microwell Strip HolderC2446-86A10: 2 Adhesive Plate Covers
