Production and characterization of monoclonal antibodies against the DNA binding domain of the RE1-silencing transcription factor

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.

Production and characterization of a monoclonal antibody against the sialidase of Gardnerella vaginalis using a synthetic peptide in a MAP8 format

“…Eight-week-old female BALB/c mice were immunized weekly with 30 μg of the SLD-MAP8 peptide using Freund's complete and incomplete adjuvants (Sigma-Aldrich Cat # F5881 and Cat # F5506). Hybrid generation was performed as previously described by our workgroup (Cortés-Sarabia et al 2019). Culture supernatant samples were monitored by indirect enzyme-linked immunosorbent assay (ELISA) using SLD-MAP8 peptide as antigen, and hybridomas secreting monoclonal antibodies (mAb) were subcloned twice by limiting dilution and maintained in DMEM cell culture medium (Sigma-Aldrich Cat # D5796) with 10% fetal bovine serum (FBS, Gibco Cat # 16000044) and antibiotics.…”

Aβ-Induced Repressor Element 1-Silencing Transcription Factor (REST) Gene Delivery Suppresses Activation of Microglia-Like BV-2 Cells

“…Repressor element 1-silencing transcription factor (REST), also named neuron-restricted silencing factor (NRSF), is a zinc finger protein which binds to a 21 bp repressor element-1 (RE-1) to keep silence of hundreds of genes, many of which are neurally expressed genes [ 7 , 8 ]. REST is known to play a key role in neuronal differentiation, including neurogenesis, synaptogenesis, excitability, and synaptic transmission [ 9 , 10 ]. Importantly, REST dysregulation has been associated with neurodegenerative diseases, such as Alzheimer's disease [ 11 – 13 ].…”