Development and optimization of an indirect enzyme-linked immunosorbent assay for 4-nitrophenol. Application to the analysis of certified water samples Original Research Article

A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the detection of 4-nitrophenol (4-NP) was developed, optimized and validated for the analysis of this polar compound in water samples. The concentrations of the immunoreagents were selected to provide the highest sensitivity on an indirect ELISA format. Moreover, the influence of several physico-chemical parameters, such as incubation time, ionic strength, detergent concentration and pH were also studied. The IC50 and limit of detection (LOD) of the optimized assay As34/3-BSA (antiserum 34 and hapten 3 conjugated to bovine serum albumin) were 9.32 and 0.61 μg l−1, respectively. The specificity of this immunoassay was also evaluated by using eleven phenolic compounds, four different structurally related organophosphorus pesticides and a surfactant. Two kinds of sample matrices (Milli-Q and river water) were evaluated for possible interferences. A good correlation between the analysis by conventional techniques and by immunoassay was obtained, when five certified water samples were analyzed.