Direct Competitive Enzyme-Linked Immunosorbent Assay for the Determination of the Highly Polar Short-Chain Sulfophenyl Carboxylates

Abstract

A direct enzyme-linked immunosorbent assay for the detection of the short-chain sulfophenylcarboxylic acids (SPCs), the main metabolites of the linear alkylbenzenesulfonates, is reported. Six SPCs (2C3, 2C4, 3C4, 2C5, 3C5, 3C6), differing in the length of the alkyl chain (between C3 and C6) and in the position of the phenylsulfonic group versus the carboxylic group, have been synthesized. Antibodies have been raised against a mixture of the corresponding horseshoe crab hemocyanin conjugates prepared by coupling the carboxylic acid to the lysine amino acid residues. The immunoassay As115/3C4-HRP achieves an IC50 value of 23 nM (6.67 microg L(-1)) and a detection limit of 0.85 nM (0.24 microg L(-1)), using as standard analyte an equimolar mixture of the six SPCs. The immunoassay has found to work better in media with low or moderate ionic strength (4-30 mS cm(-1)). The decrease in the detectability produced by the potential formation of SPC salts with divalent cations such as Ca2+ can be prevented by lowering the pH of the assay medium below the pKa value of the SPC carboxylic group and using a buffer chelating with properties such as citrate buffer. The assay can be considered specific for short-chain SPCs since congeners with longer alkyl chains and other pollutants containing sulfonic groups in their structure do not interfere significantly in the assay. Preliminary experiments addressed to evaluate the potential application of this assay to environmental water samples demonstrate the usefulness of the assay.