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

Area of Science:

• Immunochemistry
• Analytical Chemistry

Background:

• Enzyme-linked immunosorbent assays (ELISAs) are crucial for detecting various analytes.
• Quantifying small molecules like 2,4-dinitrophenol (DNP) often requires highly sensitive methods.

Purpose of the Study:

• To establish a sensitive competitive ELISA for the detection of 2,4-dinitrophenol (DNP).
• To compare the sensitivity of a fluorogenic substrate with a chromogenic substrate for DNP detection.

Main Methods:

• Coating polystyrene microplates with a DNP-bovine serum albumin conjugate.
• Utilizing a competitive binding assay with anti-DNP-alkaline phosphatase conjugate.
• Detecting bound enzyme conjugate using either 3,6-fluorescein diphosphate (FDP) or p-nitrophenyl phosphate (PNPP) substrates.

Main Results:

• The competitive ELISA successfully detected 2,4-dinitrophenol (DNP).
• The assay using the fluorogenic substrate FDP achieved a detection limit of 10 fmol DNP.
• This detection limit was 50 times lower compared to using the chromogenic substrate PNPP.

Conclusions:

• A highly sensitive competitive ELISA for DNP detection was developed.
• The use of a fluorogenic substrate (FDP) significantly enhances assay sensitivity.
• The developed method is adaptable for sensitive ELISAs of other small antigens, haptens, or drugs.