Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity

Developing a preventive immunization approach against insect bite hypersensitivity using recombinant allergens: A pilot study

“…1-12 were vaccinated with four recombinant C. nubeculosus allergens (rCul n 1, rCul n 2, rCul n 5 and rCul n 9) produced in Escherichia coli and purified (Schaffartzik et al, 2011). These allergens have previously been shown to be relevant allergens for IBH, as allergic horses have significantly more often serum IgE specific for these recombinant allergens than healthy control horses and they induce immediate type reaction in intradermal tests in IBH-affected horses (Schaffartzik et al, 2010(Schaffartzik et al, , 2011. The horses were vaccinated three times (week 0, 5 and 9) with 10 g of each r-allergen in a mixture of 136 L. Six horses were immunized with IC31 ® adjuvant (250 L), provided by Valneva Austria GmbH, Vienna and six without (250 L PBS, instead of IC31 ® ).…”

“…The strips were rewashed in TBS-T and bound antibodies detected with BCIP/NBT (Roche) diluted 1:50 in alkaline phosphatase buffer (0.1 M Tris-HCl, 0.1 M NaCl, 0.05 M MgCl 2 , pH 9.5). As controls, strips were incubated with TBS-T alone or with protein specific antibodies (Schaffartzik et al, 2011).…”

“…Strips all derived from the same blot were incubated with the pre-immune sera and sera taken 2 weeks after the last immunization (week 11), diluted 1:30. Furthermore, for identification of protein bands in SGE corresponding to the recombinant proteins, mouse antibodies specific for the recombinant proteins were also incubated on SGE strips (Schaffartzik et al, 2011).…”

“…In all steps 100 L were added to each well except for the blocking step and for the phosphatase substrate, where 200 L/well were added. ELISA plates (Thermo Scientific) were coated with recombinant allergens (rCul n 1, rCul n 2, rCul n 5 or rCul n 9) (Schaffartzik et al, 2010(Schaffartzik et al, , 2011, 2 g/mL, diluted in 0.2 M Carbonate-Bicarbonate buffer, pH 9.4 (Thermo scientific) for 2 h at 37 • C. Plates were then washed with 150 mM NaCl (Merck) and 0.05% Tween 20 (Sigma). Non-specific binding sites were blocked with 5% dried milk powder and 5% Tween 20 in PBS (Calbiochem ® ), for 1 h at 37 • C. The sera (weeks 0, 2, 7, 11, 19, 30) were diluted in blocking buffer, 1:5 for IgE and 1:200 for IgG subclass detection and incubated overnight (o.n.)…”

“…The first salivary gland proteins that bind IgE from IBH affected horses to be identified and produced as recombinant proteins were derived from laboratory-produced species. These comprised one protein from Culicoides sonorensis (Langner et al, 2009) and eleven from Culicoides nubeculosus (Schaffartzik et al, 2010(Schaffartzik et al, , 2011. These two species are not very common in Europe, but the salivary proteins from them bind IgE from IBH horses with variable frequency.…”