Immunochemical detection methods for bioactive pollutants

Abstract

Recently, environmental contaminants were found to include pharmaceuticals. Regarding the convenient applicability of the ELISA, especially in water samples, we have raised antibodies against two target compounds that have been found at rather high levels in effluents, groundwater, surface water and even drinking water, i.e. ibuprofen and naproxen. Several polyclonal antibody preparations were obtained from rabbits and used to design ELISAs. In addition, one of the antibodies against ibuprofen was also used in a preliminary fashion for the construction of an immunosensor. Ibuprofen has been found in the highest concentrations in waste water and influents of up to 16 nM, whereas in surface water it can reach levels of 7.8 nM. In our indirect competitive ELISA a detection limit of 0.1 nM was achieved, with a working range of 0.1–146 nM, which is well in the range required for monitoring purposes. The ELISA was validated in the usual way by determining several parameters, e.g. cross-reactivity (<1% for all compounds tested (related and unrelated)), with the exception of naproxen (2.2%); matrix effects, linearity (R ² = 0.9580), precision and recoveries (105–162% depending on the matrix and the concentration added). Naproxen has been detected in concentrations up to 0.126 μM in effluents and waste water. Our ELISA displayed a detection limit of 15 μM and a working range of 15–150 μM, which is not yet suitable to monitor this compound. One of the antibodies appeared to be rather specific for naproxen, showing less than 10% cross-reactivity with various related and unrelated substances. Similar to ibuprofen, linearity of the assay (R ² = 0.9850), matrix effects, precision (intra-assay variation 11–24%, inter-assay variation 9–19% at levels of 62.5 to 250 μM) and recoveries were determined. From the results it appeared, for example, that reliable values were obtained by adding methanol to standards and samples before analysis. In addition, using the polyclonal antibodies against ibuprofen preliminary measurements have been performed on the Biacore™ 3000 in various spiked real water samples containing 200, 1000 and 8000 nM of ibuprofen. The results were compared with those determined in the ELISA; the correlation between both methods was highly significant showing an R ² = 0.9998. Attempts are being made to transfer the Biacore immunoassay on a Spreeta™ format for field use. Herein, a standard curve with a detection limit of 3.5 μM was achieved. In conclusion, the pharmaceutical contaminant ibuprofen may be measured specifically both by ELISA and an immunosensor assay (Biacore). An ELISA for naproxen, a compound with comparable bioactivity, has been developed, but should either be optimized with respect to the required sensitivity or combined with a preconcentration step. This assay, however, is very robust and seems promising for further research.

Keywords:

• Environment
• ELISA
• Immunosensor
• Pharmaceuticals