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Published on: April 6, 2019
Preparation and application of anti-peptide antibodies for detection of orphan cytochromes P450
Petr Hodek1, Johana Hrdinova1, Iva Macova1
1Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
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.
Insights
Researchers developed specific antibodies to detect orphan Cytochromes P450 (CYP) 2S1 and 2W1. These antibodies successfully identified these CYPs in cancer cell lines and tissue samples, highlighting their potential as malignancy markers.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Cytochromes P450 (CYP) are crucial enzymes for metabolizing compounds.
- Orphan CYPs, lacking clear metabolic roles, are notably over-expressed in tumor tissues.
- This overexpression suggests orphan CYPs may serve as potential malignancy biomarkers.
Purpose of the Study:
- To develop specific antibodies for the immunodetection of orphan CYPs, specifically CYP2S1 and CYP2W1.
- To evaluate the utility of these antibodies as potential markers for cancer detection.
Main Methods:
- Synthesis of unique peptides corresponding to CYP2S1 and CYP2W1 sequences.
- Immunization of chickens with synthesized peptides to generate antibodies.
- Isolation, purification, and validation of antibodies using ELISA and Western blot analysis.
- Detection of CYP2S1 and CYP2W1 expression in cancer cell lines and human tissue samples.
Main Results:
- Specific antibodies against CYP2S1 and CYP2W1 peptides were successfully developed.
- Antibodies demonstrated reactivity with their respective CYP targets in Western blot assays.
- CYP2S1 and CYP2W1 were detected in various cancer cell lines and human tissue samples, often co-expressed.
- Differential expression patterns were observed, with CYP2S1 in BxPC3 and CYP2W1 in MCF7 and HeLa cell lines.
Conclusions:
- Anti-peptide antibodies are effective tools for detecting homologous CYPs.
- The developed antibodies successfully recognized CYP2S1 and CYP2W1 in cancer contexts.
- These findings support the potential of orphan CYPs as diagnostic markers for malignancy.
