Alkaline phosphatase (AP) is a widely used marker enzyme in ELISA as the enzyme is easily conjugated with antibodies, is extremely stable, has high catalytic activity and a broad substrate spectrum. Highly sensitive sensing of alkaline phosphatase activity is therefore of great significance for the development of AP-based ELISA platforms. Our AquaSpark™ phosphatase substrate is designed for the most sensitive ELISA and Western blot detection methods. AquaSpark™ -based substrates work as single reagent without the need for enhancers, co-substrates or any additional reagent as opposite to traditional dioxetanes. In ELISA procedures the AquaSpark™ phosphatase substrate solution is applied to the test at the last step of the procedure. Green light is emitted immediately if AP-conjugated (secondary) antibody is bound to its corresponding detecting (first) antibody.