Immunoassays for alkylphenolic pollutants with endocrine disrupting activity

Abstract

Among the numerous pollutants with endocrine/estrogenic activity, several detergents and plasticizers are found both in the environment and in food. Particularly, phenolic compounds such as alkylphenols (AP) and bisphenol A (BPA) may enter the food chain as a result of the application as emulgators in pesticides and plasticizers in plastic materials, respectively. In order to be able to measure and/or monitor these residues we designed several ELISAs. Antibodies were raised in rabbits and used in direct and indirect competitive ELISA, in IAC columns as well as in an immunosensor. Alkylphenolic compounds included octylphenol and nonylphenol. Validation of the ELISA revealed a detection limit of 1 and 2 nM for these compounds, respectively. Two of the antibodies showed a relatively broad cross-reactivity spectrum for 4-n-octylphenol (100%), nonylphenol (90%) and BPA (10%), whereas one other antibody was highly selective for 4-n-octylphenol. Further validation parameters included linearity of the assays (R 2 ≥ 0.9998), recovery (110–130%) and matrix effects. The same antibodies were coupled onto wide porous activated silica and off-line columns were evaluated with HPLC. The capacity of the columns was 1.3 nmol of nonylphenol and 1.0 nmol of octylphenol (reproducibility 9 and 3% RSD, respectively). Polyclonal anti-BPA antibodies performed very well in the indirect as well as the direct competitive ELISA, showing a detection limit of 0.035 nM. Linearity (R 2 ≥ 0.9996), intra-assay and inter-assay variation (about 14 and 8%, respectively), cross-reactivity and matrix effects were determined. IAC columns containing antibodies coupled onto wide porous activated silica were designed in both off-line and on-line (Guard) format coupled to HPLC. They were validated in terms of capacity, detection limit (0.03 nM), re-usability, and recovery (95–100% for spiked samples). An immunosensor assay for BPA was developed on the Biacore 3000 wherein either antibody or BPA-derivative was directly coupled onto the chip surface. A detection limit of 2 nM could be achieved and the assay appeared specific for BPA showing no cross-reactivity for other related and unrelated compounds. Real water samples (surface water and effluent) were spiked with varying concentrations of BPA with or without potentially interfering substances. A recovery of around 100% was found comparable to the ELISA. An attempt was also made to transfer the Biacore assay to the Spreeta™ using the same chip and polyclonal antibody. Herein an IC50 for BPA of 5 ng mL−1 was found, whereas again the antibody cross-reacted mainly with BPA and BVA.