Open Access

ARTICLE

Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites

Karinna Chouman¹, Birgit Korioth-Schmitz¹, Markus Sack², Jörn Engelbert Schmitz¹, Anh Tuan Pham¹, Rainer Fischer^2,4, Stefan Barth⁵, Torsten Klockenbring¹, Rolf Fendel^1,6

1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany
2 Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany
3 Federela Office of Bundeswehr Personnel Management, Cologne; Germany
4 Indiana Biosciences Research Institute (IBRI), 1345 W. 16th Street Suite 300, Indianapolis, IN 46202, USA
5 Department of Integrative Biomedical Sciences, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
6 Institute of Tropical Medicine, University of Tübingen, Wilhelmstraße 27, Tübingen, Germany

* Corresponding Author: Torsten Klockenbring,

European Cytokine Network 2018, 29(2), 59-72. https://doi.org/10.1684/ecn.2018.0409

Accepted 04 June 2018;

Abstract

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.

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