Browsing by Subject "Antibodies--genetics."

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Open Access
Characterization of novel monoclonal antibodies that target proteins differentially expressed in hepatocellular carcinoma : a proteomics approach
(2011) Öztaş, Emin
Hepatocellular carcinoma (HCC) is the sixth common cancer in the world. Because of the late diagnosis of the disease, survival rates are still poor in the HCC patients. Surveillance strategies have to be developed in populations with high risk groups having premalignant diseases for HCC, such as liver cirrhosis. The usage of serum and histology-based biomarkers assists health professionals to evaluate the patients. Despite of the advances in diagnostic methods, there is still a need to develop novel biomarkers for early detection of HCC. Therefore, we aimed to develop new biomarkers with higher sensitivity and specificity for HCC to improve the surveillance of the patients. Using an apoptotic HCC cell line, HUH7, and SIP1 proteins, we generated novel monoclonal antibodies (mAbs). 6D5, 1C6 and 6E5 hybridoma clones were chosen for characterization studies because of their strong reactivity in cell-ELISA assays. We found differential reactivity pattern for those novel mAbs in a panel of human sections consisting of tumors, benign liver diseases, normal tissues and a variety of cell lines. Using proteomics methods, we identified candidate target proteins for the 6D5 mAb. Better characterization of these target proteins will provide a better understanding of the molecular pathways in the HCC and aid in the research for developing newer therapeutic agents. In conclusion, our candidate biomarker mAbs can be used in the early diagnosis of HCC as well as in drug development studies.
Open Access
Monoclonal antibody production for coiled-coil domain containing-124 (CCDC-124) and its molecular characterization
(2010) Gürbüz, İrem
Coiled-coil-domain-containing-124 (CCDC-124) is a novel protein of unknown function. The Ccdc-124 gene is highly conserved among eukaryotic species and is predicted to encode a 223 amino acids long protein. Yeast-two-hybrid assays had shown that CCDC-124 interacts with RasGEF1B guanine exchange factor, which was previously identified as a specific guanine exchange factor for Rap2, a member of the Rap subfamily of Ras-like G-proteins. In order to reveal the cellular function of CCDC-124 protein, different biochemical experiments, including Western Blotting and Sub-cellular localization studies were performed. In those experiments, a polyclonal antibody against an N-terminal peptide of CCDC-124 was used. Nevertheless, due to its polyclonal nature this antibody exhibited non-specific binding. Although it was determined that the gene encodes a 33 kDa protein and that the protein has diffused cytoplasmic localization, the results were not precise. In order to detect the protein more accurately, monoclonal antibodies were generated against CCDC-124. Throughout the project, mice were injected with pure HisTagged CCDC-124 protein. Via hybridoma technology antibodies were generated and selected for their recognition capacities. At the end, 3 positive hybridoma clones were produced: 7F7, 15C11 and 4B3. To characterize the produced monoclonal antibodies, Western Blot experiments were performed and their binding properties were compared to the polyclonal antibody. Among the three monoclones, 4B3 gave the most promising results at 33 kDa, in Western Blotting. The antibodies will be used in the determination of the protein's sub-cellular localization and in the analysis of its response to extracellular signals. These in turn will aid further analyses related to the protein's role within the cell.
Open Access
Novel monoclonal antibodies targeting conformational ERBB2 epitopes
(2012) Ceran, Ceyhan
ERBB2 is a tyrosine kinase receptor which can act as homodimers or heterodimers with other members of the ERBB family. Nearly 30% of breast cancers overexpress ERBB2, which can be effectively targeted by anti-ERBB2 monoclonal antibodies. Trastuzumab directed against an epitope on subdomain IV of the extracellular domain (ECD) of ERBB2 is a clinically used therapeutics but the response rate is poor and acquired resistance is frequent. Pertuzumab that binds to subdomain II and inhibits receptor dimerization is another promising therapeutics under clinical trials. Anti-ERBB2 antibodies directed to novel epitopes are potentially useful tools for replacement and combinatorial therapies. We produced five new anti-ERBB2 antibodies, all directed against epitope(s) present only on the native ECD. They performed selective growth inhibitory effects depending on the level of ERBB2 expression and cellular background. When used alone, novel anti-ERBB2 antibodies displayed modest but significant growth inhibition on SK-BR-3, BT-474 and MDA-MB-361 cells with ERBB2 overexpression; while no detectable inhibition was observed on MCF-7 and T47D cells lacking ERBB2 amplification. When the antibodies were tested in combination with TNF-α, they acted synergistically on SK-BR-3 cells, producing upto 80% growth inhibition; but performed antagonistically on BT-474 cells. Detailed investigation of a representative antibody indicated G1-arrest as the main mechanism of the anti-proliferative effects exerted on SK-BR-3 cells. Antibody treatment induced permanent inhibition of DNA synthesis, leading to accumulation of cells at G1-phase; an effect which was accelerated in the presence of TNF-α. In addition, treated SK-BR-3 cells displayed inhibition of Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest, independently from TNF-α. Novel antibodies against conformational epitopes present on the extracellular domain of ERBB2 receptor may serve as new analytical and diagnostic tools, in addition to being potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-ERBB2 antibodies and TNF-α provide evidence for a complex interplay between ERBB2 and TNF-α signaling pathways. Such complexity may drastically affect the outcome of ERBB2-directed therapeutic interventions.