Principle of Sandwich Enzyme-linked Immunosorbent Assay Kit

 For a sandwich ELISA test, the microtiter plate has been pre-coated with an antibody specific to analyte. Standards or samples are then added to the appropriate microtiter plate wells and the anaylte present is bound by the immobilized Ab. Next, Biotin conjugated to Detection Ab (Detection reagent A) is added and binds to the analyte absorbed on the plate. The binding of two antibodies and analytes in the wells act as a “sandwich” structure. After the unbound biotin conjugated to Detection Ab are washed away, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added and development is completed, the enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The deeper the color is, the higher the concentration of aimed protein is. The concentration of analyte in the samples can be calculated from the standard curve.

The principle scheme is listed below.