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

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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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Authors and Affiliations

  1. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany

    Karinna Chouman, Birgit Korioth-Schmitz, Anh Tuan Pham, Torsten Klockenbring & Rolf Fendel

  2. Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany

    Markus Sack & Rainer Fischer

  3. Federela Office of Bundeswehr Personnel Management, Cologne, Germany

    Jörn Engelbert Schmitz

  4. Indiana Biosciences Research Institute (IBRI), 1345 W. 16th Street Suite 300, Indianapolis, IN, 46202, USA

    Rainer Fischer

  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

    Stefan Barth

  6. Institute of Tropical Medicine, University of Tübingen, Wilhelmstraβe 27, Tübingen, Germany

    Rolf Fendel

Authors
  1. Karinna Chouman
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  2. Birgit Korioth-Schmitz
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  3. Markus Sack
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  4. Jörn Engelbert Schmitz
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  5. Anh Tuan Pham
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  6. Rainer Fischer
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  7. Stefan Barth
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  8. Torsten Klockenbring
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  9. Rolf Fendel
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Corresponding author

Correspondence to Torsten Klockenbring.

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Chouman, K., Korioth-Schmitz, B., Sack, M. et al. Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites. Eur Cytokine Netw 29, 59–72 (2018). https://doi.org/10.1684/ecn.2018.0409

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  • DOI: https://doi.org/10.1684/ecn.2018.0409

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