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Cross-reactivity of monoclonal antibodies against peptide 277-294 of rainbow trout CY
By C. D. Rice, D. Schlenk, J. Ainsworth, A. Goksoyr
Marine Environmental Research, Volume 46, Issues 1-5, Pollutant Responses in Marine Organisms, July-December 1998, Pages 87-91, ISSN 0141-1136, DOI: 10.1016/S0141-1136(97)00122-0.
Exposure to a variety of xenobiotics, including polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), results in the induction of CYP1A and related biological activity. Historically, antibodies against purified CYP1A have been raised for ELISA and western immunoblotting techniques to quantitate this protein. However, recent advances in molecular cloning and sequencing of CYP1A genes, followed by Hopp-Woods hydrophilicity plotting, have allowed investigators to raise polyclonal antibodies to specific peptide sequences of interest [Myers et al. (1993) Environmental Toxicology and Chemistry 12, 1619-1626]. We sensitized RBF/dnj mice with KLH-conjugated peptide 277-294 of rainbow trout CYP1A1. Splenic lymphocytes were then fused with FOX myeloma cells, resulting in hybridoma mixtures secreting antibodies that reacted strongly with BSA-conjugated peptide and PCB-126 induced channel catfish hepatic microsomes. Using ELISA, dot immunoblots, and SDS-PAGE/immunoblotting as screening techniques, hybridomas were cloned that secrete monoclonal antibodies exhibiting cross-reactivity with CYP1A from several species of fish. To date, these include rainbow trout (Onchorhynchus mykiss), Atlantic cod (Gadus morhua), Atlantic salmon (Salmo salar), pinfish (Lagodon rhomboides), killifish (Fundulus sp.), carp (Cyprinus carpio), channel catfish (Ictalurus punctatus) and others. Studies characterizing inhibitory effects of these antibodies on CYP1A-related enzyme activity (EROD) are underway.