This AFP enzyme-linked immunosorbent assay (ELISA) applies a technique called quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for AFP. Standards or samples are then added to the appropriate microtiter plate wells and incubated. AFP if present, will bind and become immobilized by the antibody pre-coated on the wells. The microtiter plate wells are thoroughly washed to remove any unbound AFP and other components of sample. In order to quantitate the amount of AFP present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated monoclonal antibody specific for AFP is added to each well to "sandwich" the AFP immobilized during the first incubation. The microtiter plate then undergoes a second incubation. The wells are thoroughly washed to remove all unbound HRP-conjugated antibodies and a TMB (3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain AFP and enzyme-substrate reaction will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the colour change measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.

In order to measure the concentration of AFP in the samples, this kit includes a set standard. This allows the operator to produce a standard curve of Optical Density (O.D) versus AFP (ng/mL). The concentration of AFP in the samples is then determined by comparing the O.D. of the samples to the standard curve.