Identification Of Antigens Recognized By The Monoclonal Antibody, Ibmr3 On Lymphocytes And Established Cell Lines
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Date
2016-09
Authors
Aris, Farizan
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Abstract
Monoclonal antibodies have demonstrated significant utilities in both research and clinical applications. IBMR3 monoclonal antibody (IBMR3 MAb) has been shown to recognize a molecule that may be associated with the human interleukin-4 receptor (IL-4R) complex. IL-4 has been shown to be extensively associated with cancer. The aim of this study is to identify the IBMR3 antigen (IBMR3 Ag) expressed in normal peripheral blood mononuclear cells (PBMC) and in a panel of established cell lines. Immunostaining revealed that IBMR3 MAb can recognize antigen in a variety of cell types, including epithelial, fibroblast, haemopoietic, cancer, and normal cells. Interestingly, IBMR3 Ag was found to be expressed in the cytoplasm but was absent on the cell surface except for normal lymphocytes. The analysis of IBMR3 Ag expression on lymphocytes showed that IBMR3 Ag expression was higher on activated lymphocytes compared to resting lymphocytes regardless of the type of mitogenic agents used. The lymphocytes activated by CD3 MAb demonstrated the highest expression (60.00 ± 1.94%) followed by those activated only by phytohaematoglutinin (PHA) stimulation (41.23 ± 0.63%). However, the combination of PHA with interleukin 4 (IL-4) stimulation (38.78 ± 4.82%) exhibited almost the same expression as PHA alone (41.23 ± 0.63%). Stimulation by IL-4 alone (22.51 ± 1.00%) and pokeweed (PWM) alone (22.00 ± 1.00%) produced almost the same level of expression as unstimulated control (20.61 ± 0.88%). MTS assay was used to investigate the effect of IBMR3
MAb incubation in PBMC and Jurkat cultures. According to the results, the PBMC proliferation was slightly inhibited by 10.00 ± 5.30% and 6.60 ± 5.10% at 48 and 72 hours of incubation time, respectively. Conversely, when IBMR3 MAb was incubated with Jurkat cells, significant proliferation stimulation was observed and reached up to 57.90 ± 14.40% after 48 hours of incubation. However, longer culture incubation at 72 hours decreased culture proliferation by 16.42 ± 12.70% compared with control. Mass spectrometry on Jurkat cells revealed eight candidate proteins for IBMR3 Ag , seven of which are common molecules associated with cancer. One of the proteins with high significance score was YWHAZ. Overexpressed YWHAZ has been correlated with malignant outcomes of several cancers. Moreover, the substrate for YWHAZ binding is known to be a protein involved in the IL-4 proliferation signaling pathway. Therefore, IBMR3 Ag could be a molecule that participates in tumorigenesis through involvement in cell proliferation.
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Keywords
Identify the IBMR3 antigen (IBMR3 Ag) expressed , in normal peripheral blood mononuclear cells.