Enzyme-linked immunosorbent assays for the sensitive analysis of 2,4-dinitroaniline and 2,6-dinitroaniline in water and soil

Abstract

The development and characterization of one rat monoclonal antibody (mAb) for 2,4-dinitroaniline and of two rat mAbs for 2,6-dinitroaniline are described. With the immunization of rats with 2,4,6-trinitrophenyl-glycylglycine–keyhole limpet hemocyanine (KLH) conjugate one mAb (PK 5H6) has been developed and formatted into a competitive enzyme-linked immunosorbent assay (ELISA). This assay no. 1 is very sensitive for 2,4-dinitroaniline with a test midpoint of 0.24 ± 0.06 μg L⁻¹ (n = 19) in 40 mM phosphate-buffered saline (PBS). A second hapten, 3-(4-amino-2,6-dinitrophenyl)propionic acid, which was also conjugated to KLH and used for the immunization of rats, led to two sensitive ELISAs for 2,6-dinitroaniline in 40 mM PBS with test midpoints of 0.61 ± 0.08 μg L⁻¹ (n = 15; mAb DNT4 3C6; assay no. 2) and 0.94 ± 0.29 μg L⁻¹ (n = 17; mAb DNT4 1A7, assay no. 3). Selectivities of all mAbs were checked with more than 20 compounds, including nitroaromatic compounds, 2,6-dinitroaniline pesticides, and other substituted derivatives of aniline. As very noticeable cross-reactivities, all mAbs recognize 2-chloro-4,6-dinitroaniline, 4-chloro-2,6-dinitroaniline and 2-bromo-4,6-dinitroaniline, the last of these being a major metabolite of the azo dye Disperse Blue 79. As first demonstrations of applications, two ELISAs (assays no. 1 and 2) were used for the analysis of 2,4- or 2,6-dinitroaniline in spiked water and soil samples. Recovery data were determined and the majority of these data were in the range of 90–120%. These assays can contribute to a very cost-effective and environmentally friendly immunochemical surveillance monitoring of environmental samples for contaminations with these compounds. To the best of the authors’ knowledge, these are the first antibodies described for 2,4-dinitroaniline and for 2,6-dinitroaniline.