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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Authors: | Karinna?Chouman,Birgit?Korioth-Schmitz,Markus?Sack,J?rn?Engelbert?Schmitz,Anh?Tuan?Pham,Rainer?Fischer,Stefan?Barth,Torsten?Klockenbring author-information" > author-information__contact u-icon-before" > mailto:torsten.klockenbring@ime.fraunhofer.de" title=" torsten.klockenbring@ime.fraunhofer.de" itemprop=" email" data-track=" click" data-track-action=" Email author" data-track-label=" " >Email author,Rolf?Fendel |
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Affiliation: | 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),Indianapolis,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,Tübingen,Germany |
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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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