Quantification of 2,4-dichlorophenoxyacetic acid in oranges and mandarins by chemiluminescent ELISA

Direct competitive enzyme-linked immunosorbent assay (ELISA) for 2,4-dichlorophenoxyacetic acid (2,4-D) was developed. Varying the concentrations of monoclonal anti-2,4-D-antibody and the conjugate of soybean peroxidase and 2,4-D the conditions of ELISA performance were optimised. The chemiluminescent method based on peroxidase-catalysed oxidation of luminol was applied to measure the enzyme activity of the conjugate. A mixture of 3-(10′-phenothiazinyl)propane-1-sulfonate and 4-morpholinopyridine was used as potent enhancer of chemiluminescence signal. It was shown that the values of the lower detection limit, IC50 and the working range were 1.5, 64.0, and 6.5–545 ng/mL, respectively. The recovery values of CL-ELISA from 10 spiked samples of oranges (n = 5) and mandarins (n = 5) cultivated in green house without use of 2,4-D and containing different 2,4-D concentrations (10–300 ng/mL) were ranged from 92% to 104% that indicated on the absence of matrix effect for the fruit extracts of interest. Determination of 2,4-D in peel of five oranges and five mandarins purchased from stores in Vietnam showed that 2,4-D content in oranges fruits (79–104 μg/kg) was significantly higher than that in mandarins (1.66–2.82 μg/kg).