Description
Principle of the Assay: SEMA4D ELISA kit applies the quantitative sandwich enzyme immunoassay technique. The microtiter plate has been pre-coated with a monoclonal antibody specific for SEMA4D. Standards or samples are then added to the microtiter plate wells and SEMA4D if present, will bind to the antibody pre-coated wells. In order to quantitatively determine the amount of SEMA4D present in the sample, a standardized preparation of horseradish peroxidase (HRP) -conjugated polyclonal antibody, specific for SEMA4D are added to each well to "sandwich" the SEMA4D immobilized on the plate. The microtiter plate undergoes incubation, and then the wells are thoroughly washed to remove all unbound components. Next, substrate solutions are added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain SEMA4D and enzyme-conjugated antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. A standard curve is plotted relating the intensity of the color (O.D.) to the concentration of standards. The SEMA4D concentration in each sample is interpolated from this standard curve