Caffeine ELISA

The Caffeine ELISA is a competitive immunoassay using a capture antibody technique. Calibrators, controls, and samples containing Caffeine are incubated overnight with a highly specific monoclonal anti-Caffeine antibody. During this incubation the formed antibody-Caffeine complexes are captured onto the pre-coated wells of the microplate. Then, enzyme label (Caffeine conjugated to horseradish peroxidase) is added, which competes with Caffeine present in calibrators, controls and samples for the free binding sites captured on the coated wells during a 1-hour incubation step. Unbound enzyme label is then removed by a washing step, and TMB (tetramethylbenzidine) substrate is added to the wells. In a further 30-minutes incubation step a chromophore is formed (turns from colorless to blue) in inverse proportion to the amount of Caffeine originally present in the samples, controls and calibrators. The color turns from blue to yellow after the addition of an acidic stop solution and can be measured at 450 nm. The Caffeine ELISA is particularly suited and validated for saliva. However, serum, plasma, food extracts, drinks, and other biological fluids can be measured with this ELISA, with a dilution of at least 1 in 10 using Dilution Buffer recommended for these sample types. The NovoLytiX Caffeine ELISA shows virtually no cross-reaction with Paraxanthine (0.6%).