Interleukin-6 (IL-6) expression and secretion, induced by inflammatory processes, stimulate the acute phase response cascade. The overexpression of IL-6 contributes to a variety of inflammatory diseases, e.g. rheumatoid arthritis, Castleman’s disease, multiple myeloma, and prostate cancer. Screening for high amounts of IL-6 in the patients’ blood serum can be crucial for an adequate treatment. In this study, five novel murine monoclonal antibodies (mAbs) reactive to human IL-6 were generated. The mAbs were characterized for potential diagnostic purposes and recombinant antibodies were derived thereof. Initial epitope mapping using a combination of blocking experiments and Hyper-IL-6, a fusion protein consisting of IL-6 and the soluble IL-6 receptor revealed distinct but overlapping binding sites. At least one of the mAbs was found to interact with the region of IL-6/ IL-R complex formation. Three mAbs were applied successfully in intracellular staining by flow cytometry, whereas one of the mAbs showed comparable binding as a reference reagent. Furthermore, the mAbs were tested for applications in various immunological assays such as ELISA, Western blot and surface plasmon resonance spectroscopy (SPR), using IL-6 from commercial sources as well as in-house produced protein (IL-6_IME). The limit of detection was determined by sandwich ELISA (0.5 ng/mL, SD ±0.005). Our results also demonstrated that the recombinant IL- 6 produced was functional and correctly folded. These findings support the use of the generated mAb clones as promising candidates for application in various immunological assays for diagnostic and scientific purposes.

中文翻译：

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新的抗IL-6抗体的表征揭示了用于细胞内细胞因子检测和IL-6受体结合位点特异性靶向的高效候选物。

炎症过程诱导的白细胞介素6（IL-6）表达和分泌刺激急性期反应级联反应。IL-6的过度表达导致多种炎症性疾病，例如类风湿性关节炎，Castleman病，多发性骨髓瘤和前列腺癌。筛查患者血清中大量的IL-6对于适当的治疗至关重要。在这项研究中，产生了五种对人IL-6有反应性的新型鼠单克隆抗体（mAb）。表征mAb用于潜在的诊断目的，并衍生出重组抗体。通过结合阻断实验和Hyper-IL-6（一种由IL-6和可溶性IL-6受体组成的融合蛋白）的组合进行的初始表位作图显示出独特但重叠的结合位点。发现至少一种mAb与IL-6 / IL-R复合物形成区域相互作用。通过流式细胞术成功地将三种单克隆抗体应用于细胞内染色，而其中一种单克隆抗体显示出与参考试剂相当的结合力。此外，使用商业来源的IL-6以及内部生产的蛋白质（IL-6_IME），测试了单克隆抗体在各种免疫学测定中的应用，如ELISA，Western印迹和表面等离子体共振光谱（SPR）。通过夹心ELISA（0.5 ng / mL，SD±0.005）确定检测限。我们的结果还证明，产生的重组IL-6是功能性的并且正确折叠。这些发现支持将产生的mAb克隆用作有希望的候选物，以用于诊断和科学目的的各种免疫测定中。