A sensitive competitive ELISA for 2,4-dinitrophenol using 3,6-fluorescein diphosphate as a fluorogenic substrate.

A competitive ELISA for sensitive detection of 2,4-dinitrophenol (DNP) was established. Certain amounts of bovine serum albumin conjugate of DNP were readily coated on a polystyrene microplate. Free DNP was then quantitated by its competition with the coated DNP for binding to anti-DNP (antibody)-alkaline phosphatase conjugate. The enzyme conjugate remaining on the plate surface as a result of the competition was detected by an enzymatic reaction with a fluorogenic substrate, 3,6-fluorescein diphosphate (FDP), or comparatively with a conventional chromogenic substrate, p-nitrophenyl phosphate (PNPP). The results showed that the ELISA with FDP at the optimal conditions of enzymatic reaction can detect as little as 10 fmol DNP, a detection limit of 50 times less than that with PNPP. The easy and sensitive DNP assay procedures in this work can be generalized to ELISAs for other antigens, particularly small antigens, haptens or drugs.