Antibodies to a synthetic peptide that react specifically with rainbow trout hepatic cytochrome P450 1A1

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Abstract

The purpose of this investigation was to develop a specific immunological probe to rainbow trout cytochrome P450 1A1 (CYP1A1). Three oligopeptides corresponding to different regions of trout CYP1A1 (amino acids 162-181, 250-267, 277-294) were coupled to keyhole limpet hemo-cyanin (KLH) using two different methods. All three peptides were coupled to KLH through side-chain amine and carboxyl groups of the peptide; peptides 162-181 and 277-294 were also coupled to KLH through the sulfhydryl 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 peptides used to generate the antisera. Four of these five antisera, however, did not react with the trout CYP1A1 protein. In contrast, the antiserum directed against peptide 277-294 (which was coupled to KLH through a sulfhydryl linkage) reacted strongly and specifically with the trout CYP1A1 protein. These antipeptide antibodies had a high affinity for CYP1A1 in liver microsomes from rainbow trout that had been exposed to β-naphthoflavone G3-NF), a known CYP1A1 inducer in trout. Microsomal proteins from control trout were not recognized by the antipeptide antibodies. Preimmune serum from the rabbits did not recognize any proteins in control or β-NF-treated trout. These findings demonstrate that antipeptide antibodies directed against peptide 277-294 can be easily produced in large quantities and used in research or biomonitoring studies for the detection of CYP1A1 in rainbow trout.

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