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Development of new rat monoclonal antibodies with different selectivities and sensitivities for 2,4,6-trinitrotoluene (TNT) and other nitroaromatic compounds

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Abstract

Five new rat monoclonal antibodies (mAbs) for 2,4,6-trinitrotoluene (TNT) and other nitroaromatic compounds, including, especially, the metabolite 2-amino-4,6-dinitrotoluene (2-ADNT), are described. Five heterogeneous, competitive enzyme-linked immunosorbent assays (ELISAs) were developed. Assay 1 uses mAb DNT4 3F6 as recognition element and gives a standard curve for TNT in 40 mmol L−1 phosphate buffered saline (PBS) with a test midpoint (IC50) of 0.26±0.08 μg L−1 (n=20). Assay 2 (mAb DNT4 4G4) has an IC50 of 0.35±0.07 μg L−1 (n=18), assay 3 (mAb DNT4 1A3) has an IC50 of 0.73±0.14 μg L−1 (n=15), and assay 4 (mAb DNT4 1A7) has an IC50 of 2.32±0.70 μg L−1 (n=15). Assay 5 (mAb DNT2 4B4) is very selective for 2-ADNT and has an IC50 of 8.5±1.7 μg L−1 (n=15) in PBS. These antibodies for nitroaromatic compounds differ not only in their sensitivity but also in their selectivity. Major cross-reactants are 1,3,5-trinitrobenzene, 2-ADNT, 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-dinitroaniline, 3,5-dinitroaniline, and 2,6-dinitroaniline. Although assay 5 is not highly sensitive, the mAb DNT2 4B4 in this assay is highly selective for 2-ADNT. Of all the compounds tested, only 2,4-dinitroaniline and 3,5-dinitroaniline had relevant cross reactivities, 18% and about 26%, respectively. Two ELISAs, using mAbs DNT4 3F6 and DNT2 4B4, were used to analyze different concentrations of TNT and 2-ADNT, respectively, in three different surface water matrices (river and lake water). Both assays were affected by the matrix, but usually performed well (recovery within the range 70–120%). In addition, these ELISAs were used to analyze mixtures of TNT, 2-ADNT, and 4-ADNT, at three different concentrations, in the same water matrices. A different recognition pattern was clearly visible with both assays and depended on the cross reactivities of the corresponding mAb.

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Abbreviations

2-ADNT:

2-Amino-4,6-dinitrotoluene

4-ADNT:

4-Amino-2,6-dinitrotoluene

BSA:

Bovine serum albumin

CR:

Cross reactivity

Da:

Dalton

ELISA:

Enzyme-linked immunosorbent assay

GC:

Gas chromatography

HMX:

1,3,5,7-Tetranitro-1,3,5,7-tetraazacyclooctane

HRP:

Horseradish peroxidase

IC50:

Inhibitory concentration 50%, test midpoint of standard curve

IgG:

Immunoglobulin G

KLH:

Keyhole limpet hemocyanin

LC:

Liquid chromatography

mAb:

Monoclonal antibody

MW:

Molecular weight

PBS:

Phosphate-buffered saline

RDX:

1,3,5-Trinitro-1,3,5-triazacyclohexane

TMB:

3,3′,5,5′-Tetramethylbenzidine

TNP:

Trinitrophenyl

TNT:

2,4,6-Trinitrotoluene

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Acknowledgments

The authors thank Sabine Rottmüller (IEC, GSF) for her excellent technical assistance. Dr Norbert Hertkorn (IEC, GSF) is acknowledged for NMR confirmation of structural data of the immunogen haptens. We acknowledge Dr Robert O. Harrison, Cape Technologies, MA, USA, for the spreadsheet with the additive model. Parts of this work were presented as a poster at the “8th World Congress on Biosensors”, 24–26 May, 2004, Granada, Spain, and in an oral presentation at the “6th Workshop on Biosensors and BioAnalytical μ-Techniques in Environmental and Clinical Analysis”, 8–12 October, 2004, Rome, Italy, and at the “First SWIFT-WFD Workshop on Validation of Robustness of Sensors and Bioassays for Screening Pollutants”, 2–3 December, 2004, Menorca, Spain. This research was funded by the BMBF (Ministry for Science and Education, Germany), FKZ 02WU0102.

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Authors and Affiliations

  1. GSF—National Research Centre for Environment and Health, Institute of Ecological Chemistry, Ingolstädter Landstraße 1, 85764, Neuherberg (Oberschleißheim), Germany

    Petra M. Krämer, Cristina M. Weber & Stephan Forster

  2. Wissenschaftszentrum Weihenstephan, Lehrstuhl für Ökologische Chemie und Umweltanalytik, Technische Universität München, Weihenstephaner Steig 23, 85350, Freising, Germany

    Petra M. Krämer, Ioan M. Ciumasu & Antonius A. Kettrup

  3. GSF—National Research Centre for Environment and Health, Institute of Molecular Immunology, Marchioninistraße 25, 81377, Munich, Germany

    Elisabeth Kremmer

Authors
  1. Petra M. Krämer
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  2. Elisabeth Kremmer
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  3. Cristina M. Weber
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  4. Ioan M. Ciumasu
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  5. Stephan Forster
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  6. Antonius A. Kettrup
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Correspondence to Petra M. Krämer.

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Dedicated to the memory of Wilhelm Fresenius

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Krämer, P.M., Kremmer, E., Weber, C.M. et al. Development of new rat monoclonal antibodies with different selectivities and sensitivities for 2,4,6-trinitrotoluene (TNT) and other nitroaromatic compounds. Anal Bioanal Chem 382, 1919–1933 (2005). https://doi.org/10.1007/s00216-005-3294-1

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  • DOI: https://doi.org/10.1007/s00216-005-3294-1

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