Preparation and application of anti-peptide antibodies for detection of orphan cytochromes P450.
OBJECTIVES: Cytochromes P450 (CYP) are monooxygenases, which metabolize mostly hydrophobic endogenous and exogenous compounds. CYPs without any clear connection to metabolism are called "orphans". Interestingly, these "orphan" CYPs are over-expressed in tumor tissues. Thus, the main aim of the paper is the development of antibodies for immunodetection of these CYPs as potential malignancy markers.
METHODS: Unique sequences of CYP2S1 and 2W1 were selected and peptides synthesized. Chickens were immunized with peptides bound to hemocyanin (KLH). The antibodies were isolated from egg yolks and their reactivity was tested by ELISA. Antibodies were further affinity purified on immobilized peptides. Western blots containing CYP2S1 and 2W1 standards were developed with purified antibodies.
RESULTS: Using unique peptide immunogens of CYP2S1 and 2W1 the antibodies were developed. As judged from ELISA all chickens produced specific antibodies against the respective peptides. Both affinity purified antibodies against CYP2S1 peptide recognized the CYP2S1 standard on Western blots, but only one of four anti-peptide antibodies against CYP2W1 reacted with CYP2W1 standard. The antibodies were used for the detection of CYPs in cancer cell lines and human tissues samples. Although both CYPs were frequently co-expressed in cancer cells, CYP2S1 was solely induced in the cell line BxPC3, while CYP2W1 was predominantly present in cell lines MCF7 and HeLa. Our data show that anti-peptide antibodies are an indispensable tool for detection of homologous CYPs.
CONCLUSIONS: The anti-peptide antibodies successfully recognized CYP2S1 and 2W1 in the cancer cell lines and tissue samples....