Development of a monoclonal antibody for ELISA of CYP1A in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes

Abstract

Induction of cytochrome P4501A CYP1A in cultured cells can be used to determine the induction potencies of xenobiotics or complex environmental samples. This report describes the development of an enzyme linked immunosorbent assay ELISA for measurement of CYP1A expression in primary cultures of rainbow trout Oncorhynchus mykiss hepatocytes. Juvenile rainbow trout were injected with naphthoflavone BNF 25 mg kg-1 body weight to induce the synthesis of CYP1A. The CYP1A isoenzyme 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 ELISA methods, a direct and a competitive one, were evaluated, with both methods being of comparable sensitivity. Rainbow trout hepatocytes, maintained as monolayers in serum free, chemically defined medium, were exposed to naphthoflavone, and the induction response was measured both by 7 ethoxyresorufin O deethylase EROD activity and the direct ELISA method. Comparison between EROD activity and immunodetectable CYP1A protein can provide information on the catalytic efficiency of CYP1A.