UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad Universitaria, Ruta Nacional 168, Km 472.4, C.C. 242 (S3000ZAA), Santa Fe, Argentina.
UNL, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Biotechnological Development Laboratory, Ciudad Universitaria, Ruta Nacional 168, Km 472.4, C.C. 242 (S3000ZAA), Santa Fe, Argentina.
Appl Microbiol Biotechnol. 2022 Dec;106(24):8121-8137. doi: 10.1007/s00253-022-12282-6. Epub 2022 Nov 19.
Human stem cell factor (hSCF) is an early-acting growth factor that promotes proliferation, differentiation, migration, and survival in several tissues. It plays a crucial role in hematopoiesis, gametogenesis, melanogenesis, intestinal motility, and in development and recovery of nervous and cardiovascular systems. Potential therapeutic applications comprise anemia treatment, mobilization of hematopoietic stem/progenitor cells to peripheral blood, and increasing gene transduction efficiency for gene therapy. Developing new tools to characterize recombinant hSCF in most native-like form as possible is crucial to understand the complexity of its in vivo functions and for improving its biotechnological applications. The soluble domain of hSCF was expressed in HEK293 cells. Highly purified rhSCF showed great molecular mass variability due to the presence of N- and O-linked carbohydrates, and it presented a 2.5-fold increase on proliferative activity compared to bacteria-derived hSCF. Three hybridoma clones producing monoclonal antibodies (mAbs) with high specificity for the glycoprotein were obtained. 1C4 and 2D3 mAbs were able to detect bacteria-derived and glycosylated rhSCF and demonstrated to be excellent candidates to develop a sandwich ELISA assay for rhSCF quantification, with detection limits of 0.18 and 0.07 ng/ml, respectively. Interestingly, 1A10 mAb only recognized glycosylated rhSCF, suggesting that sugar moieties might be involved in epitope recognition. 1A10 mAb showed the highest binding affinity, and it constituted the best candidate for immunodetection of the entire set rhSCF glycoforms in western blot assays, and for intracellular cytokine staining. Our work shows that combining glycosylated rhSCF expression with hybridoma technology is a powerful strategy to obtain specific suitable immunochemical assays and thus improve glycoprotein-producing bioprocesses. KEY POINTS: • Soluble glycosylated human SCF exerted improved proliferative activity on UT-7 cells. • Three mAbs with high specificity targeting glycosylated human SCF were obtained. • mAbs applications comprise sandwich ELISA, western blot, and immunofluorescence assays.
人干细胞因子（hSCF）是一种早期作用的生长因子，可促进几种组织中的增殖、分化、迁移和存活。它在造血、配子发生、黑素生成、肠道动力以及神经系统和心血管系统的发育和恢复中发挥着关键作用。潜在的治疗应用包括贫血治疗、动员造血干细胞/祖细胞进入外周血，以及提高基因治疗的基因转导效率。开发新的工具来尽可能以最接近天然的形式表征重组 hSCF 对于理解其体内功能的复杂性以及改进其生物技术应用至关重要。hSCF 的可溶性结构域在 HEK293 细胞中表达。由于存在 N 和 O 连接的碳水化合物，高度纯化的 rhSCF 显示出很大的分子量变异性，并且与细菌衍生的 hSCF 相比，增殖活性增加了 2.5 倍。获得了 3 株产生针对糖蛋白的高特异性单克隆抗体（mAb）的杂交瘤克隆。1C4 和 2D3 mAb 能够检测细菌衍生的和糖基化的 rhSCF，并被证明是开发 rhSCF 定量夹心 ELISA 测定法的优秀候选物，检测限分别为 0.18 和 0.07ng/ml。有趣的是，1A10 mAb 仅识别糖基化的 rhSCF，表明糖基可能参与表位识别。1A10 mAb 显示出最高的结合亲和力，并且是在 Western blot 测定中免疫检测整个 rhSCF 糖型的最佳候选物，并且用于细胞内细胞因子染色。我们的工作表明，将糖基化 rhSCF 的表达与杂交瘤技术相结合是获得特异性合适免疫化学测定法的有力策略，从而可以改进糖蛋白产生的生物工艺。关键点：• 可溶性糖基化人 SCF 对 UT-7 细胞表现出改善的增殖活性。• 获得了 3 株针对糖基化人 SCF 的高特异性 mAb。• mAb 的应用包括夹心 ELISA、Western blot 和免疫荧光测定。
