Endolysosomal protease susceptibility of Amb a 1 as a determinant of allergenicity

[...]because of low sequence identity, cross-reactivity is not expected. Because Dickeya species are known to cause soft root disease on many crops (eg, bell pepper, tomatoes, potatoes, and onions)4 and ornamental indoor plants, contact with this phytopathogen has been recently demonstrated by metagenomic sequencing of the microbiota of human buccal mucosa.5,6 These studies unambiguously identified Dickeya dadantii as an oral commensal bacterium. [...]we performed ELISAs to evaluate immune responses to DC-pelC. [...]derivatives were calculated by applying the Savitzky-Golay algorithm with 25 smoothing points. Experiments using anonymized serum samples from allergic patients were approved by the ethics committee of the Medical University of Vienna (712/2010).ELISA For ELISA experiments, Maxisorp plates (Nunc, Thermo Fisher, Waltham, Mass) were coated with 100 ng protein/well in 50 μL PBS overnight at 4°C. After washing and blocking, plates were incubated either with diluted human sera (1:50 for IgG and 1:250 for IgE detection) (buffer: TBS, pH 7.4, 0.05% (v/v) Tween, 1% (w/v) BSA) or with serial dilutions of murine sera (buffer: TBS, pH 7.4, 0.05% (v/v) Tween, 1% (w/v) BSA) overnight at 4°C. Bound human IgE was detected using an AP-conjugated mouse anti-human IgE antibody (Clone G7-26) (BD Biosciences, Franklin Lakes, NJ), human IgG1 was detected by an AP-conjugated mouse anti-human IgG1-hinge antibody (Clone 4E6), and human IgG4 was detected using an AP-conjugated mouse anti-human IgG4 pFc' antibody (Clone HP6023).