Principle of the Assay

This assay measures membrane-bound E-Selectin expressed on stimulated HUVEC using enzyme-linked immunosorbent assay (ELISA). After stimulation of HUVEC (with E.coli LPS, for example), the cells are fixed and incubated with a specific mouse monoclonal primary antibody to human membrane-bound E- Selectin. The cells are then incubated with an anti-mouse IgG antibody conjugated with peroxidase to detect the primary Ab. A chromogen substrate solution is added to the wells and color develops in proportion to the amount of membrane-bound E-Selectin present on the cells. As the E-Selectin is membrane-bound, a protein concentration standard cannot be performed. Rather, this assay measures test sample stimulation HUVEC E-Selectin expression relative to positive and negative controls.

Reagents Included

1. Stimulation medium (1X) - 20ml. Contains 5% human serum as a source of soluable CD14.
2. E.coli LPS - 20µl (1mg/ml). Positive control.
3. Cell Fixative (16X) – 0.65ml (8%). Dilute 1:16 in 1XPBS.
4. Diluent/Wash Buffer (10X) – 50ml. Dilute 1:10 with dH₂O.
5. Antibody Dilution Buffer (1X) – 25ml. Dilution buffer for anti-E-Selectin and secondary Abs.
6. Anti-E-Selectin Ab. Lyophylized. Dilute the contents of the vial in 10.0 ml 1X Antibody Dilution Buffer (E).
7. Secondary Ab. Lyophylized. Anti-mouse IgG, peroxidase conjugated. Dilute the contents of the vial in 10.0 ml 1X Antibody Dilution Buffer (E).
8. TMB (1X) - 10ml. Chromogen substrate for peroxidase detection.
9. Stop Solution (1X) – 5ml. Diluted sulfuric acid to stop the color reaction.

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