Production and characterization of monoclonal antibodies against the DNA binding domain of the RE1-silencing transcription factor.
J Biochem
; 166(5): 393-402, 2019 Nov 01.
Article
em En
| MEDLINE
| ID: mdl-31198928
ABSTRACT
The use of monoclonal antibodies for the detection of cellular biomarkers during carcinogenesis provides new strategies for cancer diagnosis or prognosis in patients. Loss of the Restrictive Element 1-Silencing Transcription (REST) factor has been observed in previous molecular and immunological approaches in aggressive breast cancer, small cell lung cancer, liver carcinoma, and colo-rectal cancer; however, for clinic diagnosis, monoclonal antibodies for REST recognition are unavailable. The goal of this work was to design, produce and characterize monoclonal antibodies against the REST DNA binding damain (DBD) that would be suitable for immunoassays. We searched for conserved domains, and immunogenic and antigenic sites in the REST structure via in silico analysis. For mice immunization, we used a recombinant REST DBD purified by affinity chromatography, and then Hybridomas were generated by mouse spleen fusion with myeloma cells. Finally, for monoclonal antibody characterization, we performed enzyme-linked immunosorbent (ELISA), western blot, dot blot, immunocytochemistry (ICC) and immunoprecipitation assays. Results showed that the DBD is conserved in REST isoforms and contains immunogenic and antigenic sites. We generated three clones producing monoclonal antibodies against REST DBD, one of them specifically recognized native REST and was suitable for ICC in samples from patients.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Repressoras
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DNA
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Anticorpos Monoclonais
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Neoplasias
Tipo de estudo:
Diagnostic_studies
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Prognostic_studies
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article