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.