In the first step, samples are incubated in the antibody-coated microtiter plate. During the second step, the plate is washed and incubated with the POD-labeled GMP-140 antibody. A substrate is added, and the reaction between POD and the substrate (H2O2, TMBZ) results in a color development. The amount of sample GMP-140 is determined by measuring absorbance using an EIA plate reader. Accurate GMP-140 sample concentrations can be determined by comparing their specific absorbances with the absorbance obtained for the Standard plotted on a standard curve.
Granule Membrane Protein 140 (GMP-140, P-selectin, CD62 or PADGEM) is a 140 kDa integral membrane adhesion glycoprotein categorized functionally as a member of the LEC-CAM (lectin-epidermal growth factor-complement binding cell adhesion molecules) family or the selectin family. In non-activated conditions, GMP-140 is present in the alpha-granules of platelets or in endothelial cell Weibel Palade bodies. However, when activated by stimulators, such as thrombin or by inflammatory or hemostatic mediators during intravascular inflammation, GMP-140 is rapidly secreted to the plasma membrane to promote adhesion of cells to vascular endothelia or to neutrophils and monocytes at the site of tissue injury. Previous studies suggested that GMP-140 is indeed a useful marker of platelet and endothelium activation in experimental inflammation models, and potentially in clinical situations. Recent data also suggest that free GMP-140 molecules present in the circulation may serve to maintain the non-adhesiveness of neutrophils and prevent the development of inflammatory responses.