DEVELOPMENT OF A MONOCLONAL-ANTIBODY FOR ELISA OF CYP1A IN PRIMARY CULTURES OF RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) HEPATOCYTES

Induction of cytochrome P4501A (CYP1A) in cultured cells can be used t o determine the induction potencies of xenobiotics or complex environm ental samples, This report describes the development of an enzyme-link ed immunosorbent assay (ELISA) for measurement of CYP1A expression in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. J uvenile rainbow trout were injected with beta-naphthoflavone (BNF) (25 mg kg(-1) body weight) to induce the synthesis of CYP1A. The CYP1A is oenzyme was purified, characterized by immunological cross-reactivity and N-terminal sequencing and used to prepare a monoclonal antibody in Balb-C mice. The specificity of the antibody for CYP1A was proved by Western blotting of samples from control and BNF-injected fish. Two EL ISA methods, a direct and a competitive one, were evaluated, with both methods being of comparable sensitivity. Rainbow trout hepatocytes, m aintained as monolayers in serum-free, chemically defined medium, were exposed to beta-naphthoflavone, and the induction response was measur ed both by 7-ethoxyresorufin-O-deethylase (EROD) activity and the dire ct ELISA method. Comparison between EROD activity and immunodetectable CYP1A protein can provide information on the catalytic efficiency of CYP1A.