This IL-16 enzyme-linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for IL-16. Standards or samples are then added to the appropriate microtiter plate wells and incubated. IL-16, 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 IL-16 and other components of sample. In order to quantitatively determine the amount of IL-16 present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated monoclonal antibody specific for IL-16 is added to each well to "sandwich" the IL-16 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. The enzyme (HRP) and substrate solution are allowed to react over a short incubation period. Only those wells that contain IL-16 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 IL-16 in the samples, this kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing). According to the testing system, the provided standard is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D) versus IL-16 (pg/mL). The concentration of IL-16 in the samples is then determined by comparing the O.D. of the samples to the standard curve.