Enzyme-linked immunosorbent assays for the sensitive analysis of 2,4-dinitroaniline and 2,6-dinitroaniline in water and soil |
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Authors: | Petra M Krämer Stephan Forster Elisabeth Kremmer |
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Institution: | 1. Helmholtz Center Munich, German Research Center for Environmental Health (GmbH), Institute of Ecological Chemistry, Ingolst?dter Landstrasse 1, 85764, Neuherberg, Germany 3. Wehrwissenschaftliches Institut für Werk-, Explosiv- und Betriebsstoffe (WIWEB), Gesch?ftsfeld 520 Chemie, Analytik, Institutsweg 1, 85435, Erding, Germany 2. Helmholtz Center Munich, German Research Center for Environmental Health (GmbH), Institute of Molecular Immunology, Marchioninistr. 25, 81377, Munich, Germany
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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. |
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Keywords: | 2-Bromo-4 6-dinitroaniline 2-Chloro-4 6-dinitroaniline 4-Chloro-2 6-dinitroaniline Disperse Blue 79 Rat monoclonal antibodies Water Soil |
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