Studies on the development of antibodies for the highly hydrophobic plasticizers DINCH and DEHT.

Diisononylcyclohexane-1,2-dicarboxylate (DINCH) and di-2-ethylhexyl terephthalate (DEHT), two of the most important substitutes for phthalate plasticizers, are used for a wide range of applications. Consequently, an increasing occurrence in urine and environmental samples is reported. Reliable and fast analytical methods for the quantification of these plasticizers are needed. So far, mainly GC-MS or LC-MS methods are used. We aimed to develop the first antibodies and immunoassays allowing for high-throughput analysis of samples. We designed two DINCH hapten structures and one DEHT hapten structure and employed hapten-protein conjugates for the immunization of rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISAs) against each hapten using the produced polyclonal antibodies were established. Yet, binding of DINCH to the respective antibodies was not observed in neither direct nor indirect assay formats, even when using protein conjugates with the heterologous haptens and different carrier proteins in the indirect format. The use of surfactants and solvents in the sample buffer did not result in recognition of the plasticizers. Also, no binding of DEHT in ELISA employing the respective antibodies was detected. We speculate that the production of antibodies against these highly hydrophobic molecules is not possible via our route, however a different hapten design could overcome this obstacle.

Enzyme-linked immunosorbent assay (ELISA) for the anthropogenic marker isolithocholic acid in water.

Bile acids are promising chemical markers to assess the pollution of water samples with fecal material. This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized assay was between 0.09 and 15 µg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 µg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement. However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters.

Structural considerations on the selectivity of an immunoassay for sulfamethoxazole.

Sulfamethoxazole (SMX), a sulfonamide, is a widely used bacteriostatic antibiotic and therefore a promising marker for the entry of anthropogenic pollution in the environment. SMX is frequently found in wastewater and surface water. This study presents the production of high affinity and selective polyclonal antibodies for SMX and the development and evaluation of a direct competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of SMX in environmental water samples. The crystal structures of the cross-reacting compounds sulfamethizole, N(4)-acetyl-SMX and succinimidyl-SMX were determined by x-ray diffraction aiming to explain their high cross-reactivity. These crystal structures are described for the first time. The quantification range of the ELISA is 0.82-63µg/L. To verify our results, the SMX concentration in 20 environmental samples, including wastewater and surface water, was determined by ELISA and tandem mass spectrometry (MS/MS). A good agreement of the measured SMX concentrations was found with average recoveries of 97-113% for the results of ELISA compared to LC-MS/MS.