ANTIBODIES TO A SYNTHETIC PEPTIDE THAT REACT SPECIFICALLY WITH RAINBOW-TROUT HEPATIC CYTOCHROME-P450 1A1

The purpose of this investigation was to develop a specific immunologi cal probe to rainbow bow trout cytochrome P450 1A1 (CYP1A1). Three oli gopeptides corresponding to different regions of trout CYP1A1 (amino a cids 162-181, 250-267, 277-294) were coupled to keyhole limpet hemocya nin (KLH) using two different methods. All three peptides were coupled to KLH through side-chain amine and carboxyl groups of the peptide; p eptides 162-181 and 277-294 were also coupled to KLH through the sulfh ydryl group of a cysteine residue of each peptide. These five peptide- KLH conjugates were used to immunize rabbits. Antibody production and specificity were monitored by Western immunoblot analyses. All of the antipeptide antisera showed strong reactivity with the corresponding p eptides used to generate the antisera. Four of these five antisera, ho wever, did not react with the trout CYP1A1 protein. In contrast, the a ntiserum directed against peptide 277-294 (which was coupled to KLH th rough a sulfhydryl linkage) reacted strongly and specifically with the trout CYP1A1 protein. These antipeptide antibodies had a high affinit y for CYP1A1 in liver microsomes from rainbow trout that had been expo sed to beta-naphthoflavone (beta-NF), a known CYP1A1 inducer in trout. Microsomal proteins from control trout were not recognized by the ant ipeptide antibodies. Preimmune serum from the rabbits did not recogniz e any proteins in control or beta-NF-treated trout. These findings dem onstrate that antipeptide antibodies directed against peptide 277-294 can be easily produced in large quantities and used in research or bio monitoring studies for the detection of CYP1A1 in rainbow trout.