CD106 (Vascular Cell Adhesion Molecule-1, VCAM-1, INCAM-110) BioAssay(TM) ELISA Kit

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).\n\nVCAM-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).\n\nVCAM-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).\n\nAn 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. \n\nThe 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.\n\nAnalytical Sensitivity and Detection Limits:\nSensitivity: 0.59ng/ml \nRange of Detection: 3.2-100ng/ml \nIntra-Assay Variation: 3.1% \nInter-Assay Variation: 5.2% \nRecovery: 89% average \nAssay Time: 135 minutes \n\nCrossreactivity: \nNo interference from other members of the immunoglobulin superfamily. \n\nKit Components:\nC2446-86A1: 1 aluminum pouch with a Microwell Plate coated with Mab (murine) to human VCAM-1\nC2446-86A2: 1 vial (0.1ml) Conjugate Mixture containing Biotin-Conjugated Anti-VCAM-1 Mab (murine) mixed with Streptavidin-HRP*\nC2446-86A3: 2 vials (200ng/ml as reconstituted) soluble VCAM-1 Standard, lyophilized*\nC2446-86A4: 1 vial lyophilized control\nC2446-86A5: 1 bottle (50ml) Wash Buffer Concentrate 20X (PBS, 1% Tween 20)* \nC2446-86A6: 1 vial (5ml) Assay Buffer Concentrate 20X (PBS, 1% Tween 20, 10% BSA)*\nC2446-86A7: 1 vial (15ml) Substrate Solution \nC2446-86A8: 1 vial (12ml) Stop Solution (1M Phosphoric Acid, H3PO4), ready to use \nC2446-86A9: 1 Microwell Strip Holder\nC2446-86A10: 2 Adhesive Plate Covers

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SPECIFICATIONS

Catalog Number

C2446-86

Size

1Kit

Applications

ELISA

References

1. Cybulsky, M.I., et al., Am. J. Pathol. 138: 815 (1991). 2. Hession, C., et al., J. Biol. Chem. 266: 6682 (1991). 3. Carlos, T.M., et al.,Blood 76: 965 (1990) . 4. Rice, G.E., et al., J. Exp. Med. 171: 1369 (1990). 5. Hession, C., et al., Biochem. Biophys. Res. Commun. 183: 163 (1992). 6. Sharar, S.R., et al., Immunopathol. 16: 359 (1995). 7. Ryan, D.H., et al., J. Clin. Invest. 88: 995 (1991). 8. Freedman, A.S., et al., Blood 79: 206 (1992). 9. Carlos, T.M. & Harlan, J.M., Immunol. Rev. 114: 5 (1990). 10. Osborn, L., et al., Cell 59: 1203 (1989). 11. Masinovsky, B., et al., J. Immunol. 145: 2886 (1990). 12. De Maeyer, E. & De Maeyer-Guignard, J., Interferons. In The Cytokine Handbook. (A. Thomson, ed.) Academic Press Harcourt Brace & Company, London, p. 265 (1995). 13. Lobb, R.R., et al., J. Immunol. 147: 124 (1991). 14. Gearing, A.J.H., et al., Ann. N. Y. Acad. Sci. 324 (1995). 15. Banks, R.E., et al., Br. J. Cancer 68: 122 (1993). 16. Fortis, C., et al., Clin. Immunol. Immunopathol. 76: 142 (1995). 17. Gearing, A.J.H. & Newman, W., Immunol. Today 14: 506 (1993). 18. Lai, L.C., et al., Br. J. Surg. 82: 83 (1995). 19. Doreduffy, P., et al., Ann. Neurol. 37: 55 (1995). 20. Gruschwitz, M.S., et al., Arthritis Rheum. 38: 184 (1995). 21. Mason, J.C., et al., Arthritis Rheum. 36: 519 (1993). 22. Mrowka, C. & Sieberth, H.G., Clin. Nephrol. 43: 288 (1995). 23. Spronk, P.E., et al., Clin. Exp. Immunol. 97: 439 (1994). 24. Wellicome, S.M., et al., Clin. Exp. Immunol. 92: 412 (1993). 25. Jakobsen, P.H., et al., Immunology 83: 665 (1994). 26. Adams, D.H., et al., Hepatology 19: 588 (1995). 27. Lim, A.G., et al., J. Hepatol. 22: 416 (1995).