Production of a monoclonal antibody and development of enzyme-linked immunosorbent assay for alkyl ethoxylates

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) has been developed for the determination of alkyl ethoxylates (AEs) that are the most widely used nonionic surfactants in the world. Three types of hapten, hemi-succinated AEs (C12EO7suc, C16EO23suc, and C18EO10suc), were synthesized and conjugated to bovine serum albumin (BSA) for mouse immunization. The mice immunized with the C12EO7suc-BSA that showed the high immune responses were used for cell fusion. The obtained monoclonal antibody (TFG2-76) was specific to AEs, which had alkyl (C) and ethoxy (EO) chain lengths of C10-12 and EO5-15, respectively. Two types of solid support, namely, a polystyrene tube and a 96-well microplate, were used for antibody immobilization. The working ranges of the tube-type and plate-type ELISAs were 2-100 and 20-1000 μg/L with IC₅₀ values of 12 and 71 μg/L AE (C12EO7), respectively. Moreover, the lowest quantification limit of plate-type ELISA could be lowered to 5 μg/L by decreasing the coated antibody concentration. Cross-reactivities with non-AE surfactants were determined, and the assay proved highly selective for AEs. The application of plate-type ELISA to determine spiked AEs in distilled water, tap water and river water provided good recoveries without matrix effects.