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(22-49) Antibodies against fish CYP1A proteins used for purification and development of a quantitative CYP1A-ELISA. Langvik, Magnus1, Nilsen, Bente2, Goksøyr, Anders*,1,2, 1 Department of Molecular Biology, Bergen, Norway2 Biosense Laboratories AS, Bergen, Norway ABSTRACT- Cytochrome P450 1A (CYP1A) is a well-known biomarker for organic xenobiotics (PCBs, dioxins, PAHs, oil etc.) in fish. The CYP1A induction is routinely being measured in catalytic assays (EROD), but this assay is time consuming and sensitive to inactivation of the enzyme by inhibitors and during sampling and storage. As an alternative to the EROD assay, the aim of this study is to develop a sensitive, robust and quantitative ELISA method for detection of CYP1A-induction in fish. Several monoclonal and polyclonal antibodies against CYP1A from fish are available. As a first step towards a quantitative ELISA assay we have investigated the epitope specificity of 5 monoclonal and 4 polyclonal antibodies using overlapping synthetic peptides covering a conserved region in the fish CYP1A-sequence (aa 271-310 in rainbow trout). Several of the antibodies bind to epitopes within this conserved region, indicating that they may be useful for the development of an ELISA for CYP1A from a variety of fish species. One of these monoclonal antibodies (designated C10-7) was used for immunoaffinity purification of liver microsomal CYP1A from Key words: cytochrome P450, CYP1A, fish, biomarker |
-naphthoflavone-treated rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio), and plaice (Pleuronectes platessa). Since CYP1A is a membrane-bound microsomal protein, interference of the membrane in the immunoassay is a major issue, requiring optimalization of detergent type and concentration. In a sandwich ELISA set-up with purified rainbow trout CYP1A protein and CYP1A-specific antibodies, the conditions for obtaining a quantitative and sensitive fish CYP1A assay are currently being evaluated.