Effect of competitor design on immunoassay specificity: Development and evaluation of an enzyme-linked immunosorbent assay for 2,4-dinitrophenol Original Research Article

The specificity of an enzyme-linked immunosorbent assay (ELISA) has been modified by changing the chemical structure of the haptenized coating antigen. A competitive indirect immunoassay has been developed, evaluated and validated to measure 2,4-dinitrophenol (2,4-DNP) in wastewater samples. The assay uses polyclonal antisera originally produced to analyze 4-nitrophenol (4-NP), but changing the chemical structure of the competitor, the resulting immunoassay is able to detect specifically 2,4-DNP in the presence of 4-NP. The IC50 of the optimized immunoassay As36/4-OVA (antiserum 36 and hapten 4 attached to ovalbumin as coating antigen) is 2.03 μg l−1 and the limit of detection (LOD) 0.26 μg l−1. The working range of the assay is placed between 0.55 and 7.32 μg l−1. Moreover, the influence of several physico-chemical parameters such as incubation time, ionic strength, detergent concentration and pH were studied. The specificity of this immunoassay was evaluated using 35 structurally related compounds. Two kinds of water matrices (Milli-Q and river water) have been used to study possible interferences and to establish the accuracy and precision of the assay. Finally, a good correlation was observed between the chromatographic and immunoassay techniques while analyzing five certified water samples.