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

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

Fast Detection of 2,4,6-Trinitrotoluene (TNT) at ppt Level by a Laser-Induced Immunofluorometric Biosensor

“…They often showed promising performance parameters. Unfortunately, not many antibodies against explosives are available, although some had been developed and characterized [ 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ]. However, their broader development seems to have stalled for some time.…”

A new, versatile field immunosensor for environmental pollutantsDevelopment and proof of principle with TNT, diuron, and atrazine

“…Continuation in the field of nitroaromatics will be achieved in applying a battery of newly developed mAbs for TNT and its metabolites (Krämer et al, 2004b(Krämer et al, , 2004c to the system. With these mAbs TNT can also be analyzed with the same sensitivity, but in addition the new mAbs show more and higher CRs than mAb A1.1.1.…”

“…A battery of six enzyme-tracers for nitroaromatic compounds as haptens were produced by hapten conjugation to HRP via N-hydroxysuccinimide active esters (for details, see Krämer et al, 2004b). In total, six conjugates were produced by this method and tested as enzyme-tracers: TNP-glycine-HRP; TNP-glycylglycine-HRP; TNP-␣-aminobutyric acid-HRP; DNP-glycine-HRP; DNP-␥-aminobutyric acid-HRP; DNP--aminocaproic acid-HRP.…”

Extremely sensitive and selective antibodies against the explosive 2,4,6‐trinitrotoluene by rational design of a structurally optimized hapten

“…This approach might prevent the formation of antibodies of ultimate affinity to TNT and have unwanted effects on the selectivity of the antibodies when used in the field of explosive detection. Only in one study, Krämer et al () have described a series of TNT antibodies, which are based on a conjugate of 3‐(4‐amino‐2,6‐dinitrophenyl)‐propionic acid and keyhole limpet hemocyanin. All TNT antibodies described up to now are in a strict sense antibodies against 2,4,6‐trinitroaniline or the TNT metabolite 4‐amino‐2,6‐dinitrotoluene, and only CRs are used for TNT detection.…”

“…All TNT antibodies described up to now are in a strict sense antibodies against 2,4,6‐trinitroaniline or the TNT metabolite 4‐amino‐2,6‐dinitrotoluene, and only CRs are used for TNT detection. The monoclonal antibody A1.1.1 (Zeck et al , ), for example, shows a high CR of 184% to 2,4,6‐trinitroaniline or monoclonal antibody 1A7 (Krämer et al , ) of 360% to 2,6‐dinitroaniline. The antibodies 3F6 and 4G4 (Krämer et al , ), both also monoclonal, have a slightly higher affinity to 1,3,5‐trinitrobenzene than to TNT (120%).…”