Generation of Broad Specificity Antibodies for Sulfonamide Antibiotics and Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Analysis of Milk Samples

Immunoreagents appropriately produced to detect a wide range of sulfonamide antibiotic congeners have been used to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA). The selectivity has been achieved by combining antibodies raised against 5-[6-(4-aminobenzenesulfonylamino)pyridin-3-yl]-2-methylpentanoic acid (SA1), covalently coupled to horseshoe crab hemocyanin (HCH), and 5-[4-(amino)phenylsulfonamide]-5-oxopentanoic acid (SA2), coupled to ovalbumin (OVA), on an indirect ELISA format. The immunizing hapten has been designed to address selectivity against the common aminobenzenesulfonylamino moieties, using theoretical calculations and molecular modeling tools. Hapten SA1 has been synthesized in four steps from methyl 5-(4-amino-3-pyridinyl)-2-methyl-4-pentenoate through a Heck reaction, under Jeffery conditions, to avoid introduction of additional epitopes in the linker. The microplate immunoassay developed is able to reach the necessary delectability for the determination of the sulfonamide antibiotics most frequently used in the veterinary field, in compliance with the EC Regulation 2377/90. As an example, the IC50 and LOD values accomplished for sulfapyridine are 2.86 +/- 0.24 and 0.13 +/- 0.03 mu g L-1, respectively. Studies performed with different types of milk samples demonstrate that direct and accurate measurements can be performed in this type of matrix without any previous sample cleanup method.