Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody
AbstractClostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry. View Full-Text
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Rohrbeck, A.; Fühner, V.; Schröder, A.; Hagemann, S.; Vu, X.-K.; Berndt, S.; Hust, M.; Pich, A.; Just, I. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody. Toxins 2016, 8, 100.
Rohrbeck A, Fühner V, Schröder A, Hagemann S, Vu X-K, Berndt S, Hust M, Pich A, Just I. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody. Toxins. 2016; 8(4):100.Chicago/Turabian Style
Rohrbeck, Astrid; Fühner, Viola; Schröder, Anke; Hagemann, Sandra; Vu, Xuan-Khang; Berndt, Sarah; Hust, Michael; Pich, Andreas; Just, Ingo. 2016. "Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody." Toxins 8, no. 4: 100.
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