Avdieiev Stanislav, Savinska Liliia, Filonenko Valeriy, Kavsan Vadym
Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo str.Kyiv, Ukraine.
Hybridoma (Larchmt). 2012 Feb;31(1):32-9. doi: 10.1089/hyb.2011.0048.
Chitinase 3-like 2 (CHI3L2) is one of the most overexpressed genes in glioblastoma. Despite this, both the CHI3L2 gene and its protein product CHI3L2 are poorly characterized. Here we report the generation and characterization of monoclonal antibodies to CHI3L2 protein (CHI3L2 MAbs). Bacterially expressed 6 His-tagged full-length CHI3L2 was used as antigen. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells. Hybridoma clones 2D3 and 4D2 producing high titer CHI3L2 MAbs were identified by enzyme-linked immunosorbent assay (ELISA) and further examined for their activity with the CHI3L2 protein by Western blot analysis and immunoprecipitation. The 2D3 clone was chosen for mouse inoculation and ascites formation. Antibodies derived from the ascitic fluid specifically recognized the recombinant CHI3L2 protein and strongly interacted with CHI3L2 in glioblastoma tissue lysate, as determined by Western blot analysis. The antibodies generated may be useful as a tool in various aspects of CHI3L2 investigation.
几丁质酶3样蛋白2(CHI3L2)是胶质母细胞瘤中过度表达最为明显的基因之一。尽管如此,CHI3L2基因及其蛋白产物CHI3L2的特征仍鲜为人知。在此,我们报告了针对CHI3L2蛋白的单克隆抗体(CHI3L2单克隆抗体)的制备及其特性。以细菌表达的带6个组氨酸标签的全长CHI3L2作为抗原。收集免疫小鼠的脾细胞并与SP2/0骨髓瘤细胞融合。通过酶联免疫吸附测定(ELISA)鉴定出产生高滴度CHI3L2单克隆抗体的杂交瘤克隆2D3和4D2,并通过蛋白质印迹分析和免疫沉淀进一步检测它们与CHI3L2蛋白的活性。选择2D3克隆用于小鼠接种和腹水形成。通过蛋白质印迹分析确定,腹水中获得的抗体能够特异性识别重组CHI3L2蛋白,并与胶质母细胞瘤组织裂解物中的CHI3L2发生强烈相互作用。所产生的抗体可能作为CHI3L2研究各个方面的一种工具。