Kinetics study on recombinant alkaline phosphatase and correlation with the generated fluorescent signal

Alkaline phosphatase (AP) (EC 3.1.3.1) is one of the most commonly used enzymes in immunoassays. In VIDAS® assays (bioMérieux, Marcy l’Etoile, France), AP catalyzes the hydrolysis of 4-methylumbelliferyl phosphate (4-MUP) in 4-methylumbelliferone (4-MU) producing a fluorescent signal. This work introduces an original method of characterization of the kinetic parameters K_m, V_max, and K_cat of AP embedded in VIDAS® assays. Assessment of such constants allows us to predict the fluorescent signal generated for given amounts of enzyme and its associated substrate; in the particular case of VIDAS®, it has been estimated that 0.06 nmol/L of AP produces 3144 Relative Fluorescent Values (RFV).

Abbreviations: 4-MUP, 4-Methylumbelliferyl phosphate; 4-MU, 4-Methylumbelliferone; RFV, Relative Fluorescent Values; RFU, Relative Fluorescent Units; QDs, Quantum Dots; LoD, Limit of Detection