Mass Spectrometry Detects Api m 10 in Venom Immunotherapy Products With Suspected Absence of Api m 10

Materials and MethodsNatural HBV and VIT-products 31 different batches of four purified aqueous HBV VIT products (A, B, C, D) by three different manufacturers were analyzed for the presence of Api m 10.The batches of each particular product differed in terms of the time of their manufacture and submission for state batch testing.They included 18 out of 19 batches that had been previously analyzed with IgG-immunoblotting and were reported with partly contradicting results [Frick M et al 2016; Blank S et al. 2016].The lyophilized VIT products were stored until use at 4 C and freshly reconstituted with ultrapure water to a concentration of 1 mg/mL immediately prior to the respective analyses.Crude HBV (Latoxan, Portes ls Valence, France) and rApi m 10 (expressed in E. coli with an N-terminal His tag) was used as control for IgG Immunoblotting experiments. SDS-PAGETo determine the presence of Api m 10 in the individual VIT-batches, 5 or 25 g, respectively, of reconstituted HBV was incubated for 5 min at 95 C in Lmmli buffer and analyzed on a 13% SDS-PAGE (VWR Perfect Blue TwinS, VWR International GmbH, Darmstadt Germany)), followed by Semi-Dry blotting onto a 0.45 m nitrocellulose membranes (GE Healthcare, Freiburg, Germany).For transfer control, Western blot membranes were stained for 5 minutes with 0.1% Ponceau S solution in 5% acetic acid.After documentation, membranes were destained with ultrapure water.