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

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.     

Selection of hapten structures for indirect immunosensor arrays

A multianalyte immunosensor array can be implemented by immobilization of different haptens in distinct areas of a single cavity or flow cell. In this case a mixture of different antibodies for different analytes is used in an indirect ELISA-format. The selection of the right hapten structures is very important to build up an array successfully. A system of independent hapten/antibody combinations is needed, with one immobilized hapten (coating antigen) reacting only with one antibody. If more than one antibody binds to a coating antigen no ideal calibration curves are obtained. This phenomenon is known as shared-reactivity and can lead to double-sigmoidal curves. To use monoclonal antibodies to 2,4,6-trinitrotoluene (TNT) and 2,4-dichlorophenoxyacetic acid (2,4-D), two different haptens had to be found, one only reacting with the TNT-antibody, the other only binding to the 2,4-D-antibody. 2,4-Dichlorophenoxybutyric acid was used for the 2,4-D antibody and 2,4,6-trinitrophenyl-8-aminooctanoic acid for the TNT antibody. Although 4-nitrotoluene, 2,4-dinitrotoluene and 4-amino-2,6-dinitrotoluene showed only very low cross-reactivities to the 2,4-D antibody the corresponding haptens 4-nitrophenylacetic acid, 2,4-dinitrophenyl-6-aminohexanonic acid, and 4-amino-2,6-dinitrotoluyl-(N)-glutarate are useful coating antigens for this antibody. The structure of the coating antigens had no significant influence on the midpoints (IC50) of the test for 2,4-D and even haptens with very low cross-reactivities could be used. With all haptens a test midpoint of about 0.2 μg/L for 2,4-D was achieved. For the direct assay format with immobilized antibodies the same test midpoint of 0.2 μg/L for 2,4-D was obtained. As a conclusion, the selectivity of a monoclonal antibody should not be influenced by the used tracer or coating antigen as well. It could be shown that the affinity constants of an antibody to the analytes are the main sensitivity and selectivity determining parameters for competitive immunoassays. A two-dimensional microtiter plate array was used to determine the analytes 2,4-D and TNT in parallel with a mixture of antibodies. Received: 29 July 1998 / Revised: 21 October 1998 / Accepted: 10 November 1998     

Rapid voltammetric determination of nitroaromatic explosives at electrochemically activated carbon-fibre electrodes

The electrochemical behaviour of some nitroaromatic explosives (2,4,6-trinitrotoluene, TNT; 2,6-dinitrotoluene, 2,6-DNT; 2-nitrotoluene, 2-NT; 2-amino-4,6-dinitrotoluene, 2-A-4,6-DNT; 3,5-dinitroaniline, 3,5-DNA; and nitrobenzene, NB) at electrochemically activated carbon-fibre microelectrodes is reported. Electrochemical activation of such electrode material by repeated square-wave (SW) voltammetric scans between 0.0 and +2.6 V versus Ag/AgCl, produced a dramatic increase in the cathodic response from these compounds. This is attributed to the increase of the carbon-fibre surface area, because of its fracture, and the appearance of deep fissures along the main fibre axis into which the nitroaromatic compounds penetrate. Based on the important contribution of adsorption and/or thin layer electrolysis to the total voltammetric response, a SW voltammetric method for rapid detection of nitroaromatic explosives was developed. No interference was found from compounds such as hydrazine, phenolic compounds, carbamates, triazines or surfactants. The limits of detection obtained are approximately 0.03 g mL^–1 for all the nitroaromatic compounds tested. The method was applied for the determination of TNT in water and soil spiked samples; recoveries were higher than 95% in all cases.     

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

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 g L^–1 (n=20). Assay 2 (mAb DNT4 4G4) has an IC₅₀ of 0.35±0.07 g L^–1 (n=18), assay 3 (mAb DNT4 1A3) has an IC₅₀ of 0.73±0.14 g L^–1 (n=15), and assay 4 (mAb DNT4 1A7) has an IC₅₀ of 2.32±0.70 g L^–1 (n=15). Assay 5 (mAb DNT2 4B4) is very selective for 2-ADNT and has an IC₅₀ of 8.5±1.7 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-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.Dedicated to the memory of Wilhelm Fresenius     

Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor

A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-β-alanine-ovalbumin (TNP-β-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-β-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3 × 10⁻¹¹ to 3 × 10⁻⁷ g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1 × 10⁻¹¹ g/ml.     

Metabolic studies of explosives. 5. Detection and analysis of 2,4,6-trinitrotoluene and its metabolites in urine of munition workers by micro liquid chromatography/mass spectrometry

Metabolites of 2,4,6-trinitrotoluene (TNT) were found in the urine of a group of TNT munition workers. The urine extracts were analysed by micro liquid chromatography/mass spectrometry. The metabolites found included 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene, 2,6-diamino-4-nitrotoluene and untransformed TNT. The detection limit of the metabolites in urine was 0.1 ng/ml for 20 ml urine samples.     

Homogeneous immunoassay for the detection of trinitrotoluene (TNT) based on the reactivation of apoglucose oxidase using a novel FAD-trinitrotoluene conjugate

A flavin adenine dinucleotide-trinitrotoluene derivative (FAD-TNT) was synthesized by coupling N ⁶-(2-aminoethyl)-FAD covalently to the N-hydroxysuccinimidyl ester of trinitrophenyl-γ-aminobutyric acid and characterized by negative-ion electrospray-ionization mass spectrometry (ESI-MS) after purification by reversed-phase HPLC. Free FAD-TNT can be detected at very low levels by recombination with apoglucose oxidase, since the FAD-TNT-glucose oxidase complex is enzymatically active. On the contrary, if FAD-TNT has been bound by an anti-TNT antibody, the conjugate cannot recombine with apoglucose oxidase any more. Based on these two phenomena, a homogeneous apoenzyme reactivation immunoassay system (ARIS) was developed for the detection of TNT. No separation step is needed in this assay. Proportionality between the TNT concentration and enzyme activity was demonstrated with a detection limit of 5 μg/L TNT. Received: 5 September 1997 / Revised: 3 December 1997 / Accepted: 9 December 1997     

Applications of liquid chromatography-mass spectrometry in metabolic studies of explosives

Liquid chromatography-mass spectrometry was used for the detection and identification of metabolites of 2,4,6-trinitrotoluene (TNT) in urine and blood. The metabolites were found in the urine of rats and in the blood of rabbits fed with TNT, in the urine of rats exposed to TNT by skin absorption and in the urine of TNT munition workers. The detected metabolites, formed by reduction processes, included 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2,6-diamino-4-nitrotoluene, in addition to untransformed TNT.     

A new simple approach to predict entropy of fusion of nitroaromatic compounds

In this paper, a new model is introduced to predict entropy of fusion of nitroaromatic compounds, which can be used for some well-known high explosives such as 2,4,6-trinitrotoluene (TNT) and N-methyl-N,2,4,6-tetranitroaniline (TETRYL). Novel correlation contains two different functions for which elemental composition and symmetry of nitroaromatic compounds are used to present additive and non-additive contributions, respectively. The presence of some molecular fragments may influence the value of non-additive function. The root-mean-square (rms) deviation of predictions of entropy of fusion from 66 measured values (corresponding to 61 molecules) is 7.88 J/K mol. The predicted results are compared with one of the best available semi-empirical methods, which show the reliability of the new method is relatively good.     

Synthesis in aqueous solution and characterisation of a new cobalt-doped ZnS quantum dot as a hybrid ratiometric chemosensor

In this paper, cobalt (Co(2+))-doped (CoD) ZnS quantum dots (QDs) are synthesised in aqueous solution and characterised for the first time. L-Cysteine (L-Cys) ligands on the surface of CoD ZnS QDs can bind 2,4,6-trinitrotoluene (TNT) to form Meisenheimer complexes (MHCs) mainly through acid-base pairing interactions between TNT and L-Cys and the assistance of hydrogen bonding and electrostatic co-interactions among L-Cys intermolecules. The aggregation of inter-dots induced by MHCs greatly influenced the light scattering property of the QDs in aqueous solution, and Rayleigh scattering (RS) enhancement at the defect-related emission wavelengths as well as its left side was observed with the excitation of CoD ZnS QDs by violet light. RS enhancement, combining with the quenching of the orange transition emission induced by TNT anions, resulted in a change in the ratiometric visualisation of the system being investigated. A novel CoD ZnS QD-based hybrid ratiometric chemosensor has therefore been developed for simple and sensitive analysis of TNT in water. This ratiometric probe can assay down to 25 nM TNT in solution without interference from a matrix of real water sample and other nitroaromatic compounds. Because of the excellent electron-accepting ability and strong affinity of TNT to L-Cys on the surface of CoD ZnS QDs, the CoD photoluminescent nanomaterials reported here are well suited for detecting ultra-trace TNT and for distinguishing different nitro-compounds in aqueous solution.     

Use of reversed-phase high-performance liquid chromatography-diode array detection for complete separation of 2,4,6-trinitrotoluene metabolites and EPA Method 8330 explosives: influence of temperature and an ion-pair reagent

Explosives such as 2,4,6-trinitrotoluene (TNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) are widely distributed environmental contaminants. Complete chromatographic separation is necessary in order to accurately determine and quantify explosives and their degradation products in environmental samples and in (bio)transformation studies. The present study describes a RP-HPLC method with diode array detection using a LC-8 guard column, a Supelcosil LC-8 chromatographic column, and a gradient elution system. This gradient method is capable of baseline separating the most commonly observed explosives and TNT transformation metabolites including 2,4,6-triaminotoluene (TAT) in a single run. The TNT metabolites separated were 2-hydroxylamino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, 2,4-dihydroxylamino-6-nitrotoluene, 4,4',6,6'-tetranitro-2,2'-azoxytoluene, 2,2',6,6'-tetranitro-4,4'-azoxytoluene, 4,4',6,6'-tetranitro-2,2'-azotoluene, 2,2',6,6'-tetranitro-4,4'-azotoluene, 2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,6-diamino-4-nitrotoluene, 2,4-diamino-6-nitrotoluene, and TAT. The same gradient method at a different column temperature can also be used to baseline separate the explosives targeted in the Environmental Protection Agency (EPA) Method 8330 with approximately 22% reduction in total run time and 48% decrease in solvent consumption compared to previously published methods. Good separation was also obtained when all TNT metabolites and EPA Method 8330 compounds (a total of 23 compounds) were analyzed together; only 2,6-DANT and HMX co-eluted in this case. The influence of temperature (35-55 degrees C) and the use of an ion-pair reagent on the chromatographic resolution and retention were investigated. Temperature was identified as the key parameter for optimal baseline separation. Increased temperature resulted in shorter retention times and better peak resolution especially for the aminoaromatics investigated. The use of an ion-pair reagent (octanesulfonic acid) generally resulted in longer retention times for compounds containing amine functional groups, more baseline noise, and decreased peak resolution.     

Analysis of aqueous 2,4,6-trinitrotoluene (TNT) using a fluorescent displacement immunoassay

We report a rapid, simple, and sensitive assay that is potentially amenable to high throughput screening for analysis of 2,4,6-trinitrotoluene (TNT) present in aqueous solutions. The assay is based on the change in fluorescence emission intensity of a fluorescently labeled TNT analogue pre-bound to an anti-TNT antibody that occurs upon its competitive displacement by TNT. The assay can be performed in both cuvette- and 96-well plate-based formats. TNT at a level of 0.5 micro g L(-1) (0.5 ppb) was detected in phosphate buffered saline; detection improved to 0.05 micro g L(-1) (0.05 ppb) for TNT dissolved in artificial seawater.     

Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay

Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl–keyhole limpet hemocyanin (TNPh–KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh–KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.      

Rapid field screening test for determination of 2,4,6-trinitrotoluene in water and soil with immunofiltration

A rapid field screening test for 2,4,6-trinitrotoluene (TNT) in water and soil is described. The immunofiltration assay is based on a simplified enzymelinked immunosorbent assay (ELISA) performed in a pre-packed portable device. Performance of the test was assayed in spiked water, methanolic soil extracts (dilution 1: 10) and natural water samples. A quantitative colour response to concentrations of TNT in the range of 1 to 30 g/l in water and 50 to 1000 g/kg in soil is demonstrated. The relative average standard deviations are 11.9% and 14.1%, respectively. High correlations between immunofiltration, ELISA and gas chromatography have been demonstrated. The cross reactivities of the antibody are comparable in both techniques.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday     

Monitoring carbamazepine in surface and wastewaters by an immunoassay based on a monoclonal antibody

The pharmaceutical compound carbamazepine (CBZ) is an emerging pollutant in the aquatic environment and may potentially be used as a wastewater marker. In this work, an enzyme-linked immunosorbent assay (ELISA) for the detection of carbamazepine in surface and sewage waters has been developed. The heterogeneous immunoassay is based on a commercially available monoclonal antibody and a novel enzyme conjugate (tracer) that links the hapten via a hydrophilic peptide (triglycine) spacer to horseradish peroxidase. The assay achieves a limit of detection of 24 ng/L and a quantitation range of 0.05–50 μg/L. The analytical performance and figure of merits were compared to liquid chromatography–tandem mass spectrometry after solid-phase extraction. For nine Berlin surface water samples and one wastewater sample, a close correlation of results was observed. A constant overestimation relative to the CBZ concentration of approximately 30% by ELISA is probably caused by the presence of 10,11-epoxy-CBZ and 2-hydroxy-CBZ in the samples. The ELISA displayed cross-reactivities for these compounds of 83% and 14%, respectively. In a first screening of 27 surface water samples, CBZ was detected in every sample with concentrations between 0.05 and 3.2 μg/L. Since no sample cleanup is required, the assay allowed for the determination of carbamazepine with high sensitivity at low costs and with much higher throughput than with conventional methods.      

Highly Stable Voltammetric Detection of Nitroaromatic Explosives in the Presence of Organic Surfactants at a Polyphenol‐Coated Carbon Electrode

A polyphenol‐coated screen‐printed carbon electrode is used for highly sensitive voltammetric measurements of the 2,4,6‐trinitrotoluene (TNT) explosive in the presence of surface‐active substances. The permselective/protective polyphenol coating offers excellent resistance to surfactant fouling, while allowing facile transport of the target TNT. High levels of gelatin, humic acid and sodium dodecyl sulfate (SDS) (up to 50 mg/L) have negligible effects upon the square‐wave voltammetric TNT response. The TNT peak current and potential remain nearly the same in the presence of these organic macromolecules, as compared to substantial peak suppressions and shifts at the bare electrode. Control of the electropolymerization time was used for achieving the desired exclusion of interfering surface‐active macromolecules while allowing transport of the target TNT. The response for ppm levels of TNT is highly linear and stable for prolonged operations in the presence of surface‐active substances. By meeting the high sensitivity, selectivity, stability, portability and low‐cost demands, such voltammetric sensing holds great promise for field‐based voltammetric monitoring of nitroaromatic explosive compounds.     

Simultaneous determination of nitroaromatic compounds in water using capillary electrophoresis with amperometric detection on an electrode modified with a mesoporous nano‐structured carbon material

In this article, a carbon disk electrode modified with mesoporous carbon material (CMK‐3) was used in CE with amperometric detection system for the simultaneous determination of four types of important nitroaromatic compounds, including 2,4,6‐trinitrotoluene (TNT), 1,3,5‐trinitrobenzene (TNB), 2,4‐dinitrotoluene (DNT) and 1,3‐dinitrobenzene (DNB). Compared with the bare carbon electrode, the CMK‐3 modified electrode greatly improved the sensitivity at a relatively positive detection potential due to its excellent electrocatalytic activities, high conductivity and large effective surface area. The four analytes could be well separated and detected within 480 s. A good linear response was obtained for TNB, DNB, TNT and DNT from 8.4 to 5.0×10³ μg/L, with correlation coefficients higher than 0.9992. And the detection limits were established between 3.0 and 4.7 μg/L for the four investigated nitroaromatic compounds (S/N=3). The CMK‐3‐modified electrode was successfully employed to analyze coking wastewater, tap water and river samples with recoveries in the range of 94.8–109.0%, and RSDs less than 5.0%. The presented results demonstrated that the CMK‐3‐modified carbon electrode used in CE with amperometric detection was of convenient preparation, high sensitivity and good repeatability, which could be employed in the rapid determination of practical samples.     

Development of a highly sensitive enzyme-immunoassay for the determination of triazine herbicides

A monoclonal antibody (Mab) with extraordinary sensitivity and high class selectivity to triazine herbicides is described. With an enzyme-linked immunosorbent assay (ELISA) using Mab 4A54 IC₅₀ values for terbuthylazine, atrazine, propazine and simazine below 0.1 μg/L (the EU maximum admissible concentration for individual pesticides) have been obtained. Detection limits of 0.004 μg/L for terbuthylazine, 0.006 μg/L for atrazine, 0.003 μg/L for propazine, 0.01 μg/L for simazine and 0.05 μg/L for deethylterbuthylazine could be achieved. Therefore, Mab 4A54 allows a sum screening of these five triazines in a relevant concentration range. To our knowledge, this is the most sensitive antibody to terbuthylazine at all and also the most sensitive Mab to all these four triazines. Another monoclonal antibody resulting from the same immunization, clone 4A118, exhibits best sensitivity for propazine (detection limit: 0.02 μg/L) at lower cross-reactivity to terbuthylazine and atrazine compared to clone 4A54. Affinity constants of both Mabs towards several triazines have been calculated. The application of both Mabs for the analysis of triazines in water samples of different origin has been tested and their resistance towards humic acid influence could be shown. A good correlation of the analysis of water samples with GC and ELISA was observed. Received: 17 February 1997 / Revised: 1 April 1997 / Accepted: 3 April 1997     

Evaluation of suspected interferents for TNT detection by ion mobility spectrometry

The use of ion mobility spectrometry systems to detect explosives in high security situations creates a need to determine compounds that interfere and may compromise accurate detection. This is the first study to identify possible interfering air contaminants common in airport settings by IMS. Seventeen suspected contaminants from four major sources were investigated. Due to the ionization selectivity gained by employing chloride reactant ion chemistry, only 7 of the 17 compounds showed an IMS response. Of those seven compounds, only 4,6-dinitro-o-cresol (4,6DNOC) was found to have a similar mobility to 2,4,6-trinitrotoluene (TNT) with K(o) values of 1.55 and 1.50 cm(2) V(-1) s(-1), respectively. Although baseline resolution between TNT and 4,6DNOC was not achieved, the drift time for TNT was still easily identified. Alkyl-nitrated phenols, due to acidic fog, responded the strongest in the IMS. The effect of contamination on TNT sensitivity was investigated. Charge competition between TNT and 2,4-dinitrophenol (2,4DNP) was found to occur and to effect TNT sensitivity.     

Immunological method for the detection of nitroaromatic residues covalently bound to humic acids

The potential of the earlier reported sandwich-immunoassay principle for the detection of nitroaromatic residues bound to humic acids was examined. A synthetic conjugate derived by coupling a 2,4,6-trinitrotoluene derivative to humic acid was used as a model substance for bound nitroaromatic residues. Spectrophotometric determination gave a content of 14 ± 1.6 μmol/g TNT-derivative in the synthetic conjugate, which was used as a calibration standard. Extensive blocking optimization was necessary to establish the sandwich-immunoassay. Experiments were carried out to check the selectivity of the developed test system. Trace amounts of 2,4,6-trinitrotoluene (25 μg/L) suppressed the assay signal completely by blocking the antibody binding sites. This was a proof for the excellent selectivity of the assay. The potential of quantitative determinations was examined with dilution experiments. During assay optimization strong non-immunological interactions between various proteins and humic acids were observed. This led to a significant improvement of the original sandwich-immunoassay, where the humic acid antibody was substituted by basic proteins. More sensitive calibration curves with higher signal intensities were achieved. This new immunoassay seems to be the method of choice for further immunological investigations of bound residues.