This EC50 determination for antibody employs an indirect ELISA method. In this assay, standard 96-well plates (12 strips with 8 wells/strip) are coated with a specific protein, which combines with the corresponding antibody present in a sample, which is diluted in a series of dilution fold for EC50 determination. The wells are washed, and biotinylated detection antibody such as anti-human IgG antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells, and color develops in proportion to the amount of the specific protein antibody bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. The EC50 concentration can be calculated by plot the series dilution antibody or a sample as 4-PL (4 parametric logistic) standard curve, with standard concentration on the x-axis and absorbance on the y-axis.