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

摘要

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 I 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 mu g L-1 (n = 20). Assay 2 (mAb DNT4 4G4) has an IC50 of 0.35 +/- 0.07 mu g L-1 (n = 18), assay 3 (mAb DNT4 1A3) has an IC50 of 0.73 +/- 0.14 mu g L-1 (n = 15), and assay 4 (mAb DNT4 1A7) has an IC50 Of 2.32 +/- 0.70 pg L-1 (n = 15). Assay 5 (mAb DNT2 4B4) is very selective for 2-ADNT and has an IC50 Of 8.5 +/- 1.7 mu g 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-dini-trotoluene (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 4134, 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.