How do you develop and validate an enzyme activity assay?

Enzyme assay development involves several steps. Assay design: select detection method (spectrophotometric, fluorometric, radiometric, or coupled assay), establish signal relationship to product formation, and ensure linear range. Optimization: determine optimal pH, temperature, ionic strength, and cofactor concentrations; establish substrate concentrations spanning Km; identify and minimize interfering substances. Validation: assess precision (intra-/inter-assay CV typically <15%), accuracy (recovery, comparison with reference methods), linearity with enzyme concentration, specificity (inhibitor studies, blank substrates), stability (reagent and sample stability), and robustness (small parameter variations). For kinetic studies, ensure initial velocity conditions (product < 10% substrate). Documentation should enable reproducibility by independent operators.