ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE SPECIFIC DETECTION OF THE MERCAPTURIC ACID METABOLITIES OF NAPHTHALENE

The measurement of metabolites constitutes a useful tool for detection of exposure and in pharmacokinetic studies. Epoxidation with subsequent glutathione conjugation and mercapturic acid formation is an important deactivation pathway for naphthalene, a toxin which presumably causes lung disease. The mercapturic acid conjugates of naphthalene [NaphMA (1), N-acetyl-S-(1,2-dihydro-1-hydroxy-2-naphthyl)cysteine (1a), and N-acetyl-S-(1,2-dihydro-2-hydroxy-1-naphthyl)cysteine (1b)], its most important urinary metabolites, and other structurally related derivatives, such as N-acetyl-S-(1,2,3,4-tetrahydro-2-hydroxy-1-naphthyl)cysteine (2), N-acetyl-S-(3-hydroxy-1,2,3,4-tetrahydro-2-naphthyl)cysteine (3), and N-acetyl-S-(2-hydroxy-1-phenyl-ethyl)cysteine (4a) and N-acetyl-S-(2-hydroxy-2-phenylethyl)cysteine (4b) as an isomeric mixture, were synthesized to develop an ELISA (enzyme-linked immunosorbent assay) for the specific detection of NaphMA (1). Compound 1, as an isomeric mixture, was used to raise antibodies by immunizing six rabbits with the corresponding KLH (keyhole limpet hemocyanin) and BSA (bovine serum albumin) derivatives (1KLH and 1BSA). The remaining compounds were covalently attached to BSA, conalbumin, and ovalbumin to be used as coating antigens. The best assay was obtained in a homologous system combining serum Ab2357 (1KLH) and 1BSA as coating antigen. The immunoassay has an I50 of 4-6 ng/mL and a detection limit of 1-2 ng/mL. Because of the known instability of the mercapturic acid conjugate of naphthalene 1, leading to the fully aromatic compound 20, a system involving HPLC is described to check the stability of the NaphMA stock solutions used in the assay. Cross-reactivity studies show high specificity toward the NaphMA. Other related compounds as well as the dehydrated derivative 20 are not recognized by the antibody in this ELISA system.