Characterization of interleukin-6 monoclonal antibodies for future applications

Interleukin-6 (IL-6) is a cytokine not only involved in inflammation and infection responses but also in the regulation of metabolic, regenerative, and neural processes. Although its expression is strictly controlled by transcriptional and post-transcriptional mechanisms, deregulated continual synthesis of IL-6 plays a pathological role in chronic inflammation and autoimmunity. Strategies targeting IL-6 and IL-6 signalling lead to effective prevention and treatment of models of rheumatoid arthritis and other chronic inflammatory diseases. Screening for high amounts of IL-6 in the patients’ blood serum can be crucial for an adequate treatment. Considering the important role of IL-6, this study started with the production of the human IL-6 cytokine. The characterization of this cytokine was done with eight murine monoclonal antibodies (mAbs), raised against human IL-6 by the Mobiguide group. The recombinant formats were generated from the binding monoclonal antibodies and characterized in parallel. The antibodies were tested for applications in various immunological assays such as ELISA, Western blot, surface plasmon resonance (SPR), flow cytometry analysis and finally cellbased assays for blockage of signal transducer and activator of transcription 3 (STAT3) activation. The antibodies derived from hybridoma clones #3 and #4 were also implemented into an amperometric biochip platform, where some tests for the detection of IL-6 in peripherical blood were made. The results of the SPR analysis, ELISA and Western blot demonstrate that the produced IL-6 is functional and correctly folded. The limit of detection was determined by sandwich ELISA (0.5 ng/mL IL-6, SD ±0.013). For epitope analysis by SPR, hIL-6-IME, IL-6 from Conaris and hyper-IL-6 were included, demonstrating clear differences between the antibody clones. IL-6 spiked in full blood samples could be detected by the amperometric platform. Intracellular IL-6 was verified by flow cytometry, showing that the antibody #3 is a suitable antibody for this purpose. The antibodies #3 and #6 could block activation of STAT3 in two different cell lines assay. These findings emphasize the use of the generated mAbs as promising candidates for application in various immunological assays