Dog saliva – an important source of dog allergens

Background Allergy to dog (Canis familiaris) is a worldwide common cause of asthma and allergic rhinitis. However, dander extract in routine diagnostics is not an optimal predictor of IgE-mediated dog allergy. Our objective was to evaluate saliva as an allergen source for improved diagnostics of allergy to dog. Methods IgE-binding proteins in dog saliva and dander extract were analysed by immunoblot and mass spectrometry (LC-MS/MS) using pooled or individual sera from dog-allergic patients (n = 13). Sera from 59 patients IgE positive to dander and 55 patients IgE negative to dander but with symptoms to dog were analysed for IgE against saliva and dander by ELISA. Basophil stimulation with dog saliva and dander extract was measured by flow cytometry among three dog-allergic patients. Additionally, IgE-binding protein profiles of saliva from different breeds were investigated by immunoblot. Results Greater number and diversity of IgE-binding proteins was found in saliva compared to dander extract and varied among dog breeds. In saliva, Can f 1, 2, 3 and 6 were identified but also four new saliva allergen candidates. The majority of the 59 dog dander–positive sera (n = 44) were IgE positive to dog saliva. Among patients IgE negative to dander, but with symptoms to dog, 20% were IgE positive to saliva. The biological activity of saliva was confirmed by basophil degranulation. Conclusions Dog saliva is an allergen source for improved diagnostics of dog allergy. The IgE-binding protein profile of saliva from different dogs varies.


SDS-PAGE and immunoblotting
Protein concentrations were determined by the BCA™ protein assay (Pierce, Rockford, IL, USA). SDS-PAGE of individual dog saliva samples, dog saliva pool and dog dander extracts were analysed by immunoblot under reducing conditions. Twelve µg of proteins per lane were resolved by 12% PAGE and electroblotted to Immobilon TM polyvinylidene difluoride (PVDF) transfer membranes (Millipore, Billerica, MA, USA) following manufacturer's instruction.

2D PAGE
Desalted dog saliva pool or dog dander proteins (100 µg) were loaded on 7.7 cm ZOOM strips (pH 3-10) (Invitrogen, Carlsbad, CA, USA) for the first dimension separation, followed by separation in the second dimension using homogeneous 12% SDS-PAGE and Coomassie Brilliant Blue (CBB) staining or electroblotting. After electroblotting, the membranes were blocked as above and incubated overnight with 9 ml of the pool containing serum from 13 dog allergic patients (2.5 kU A /l). Bound IgE was detected as described above.

Protein identification by mass spectrometry
For protein identification by tandem mass spectrometry (MS/MS) sequencing of tryptic peptides, protein spots were cut from the 2D gel after staining with CBB and subjected to trypsin in-gel digestion. The CBB-stained protein spots were digested using a MassPREP robotic protein handling system (Waters, Milford, MA, USA). Briefly, washing was carried out in 50 mM ammonium bicarbonate containing 50% acetonitrile. The protein was reduced (dithiothreitol) and alkylated (iodoacetamide) followed by in-gel digestion with 0.3 µg Modified Trypsin (Promega, Madison, WI, USA) in 50 mM ammonium bicarbonate for 5 h at 40 o C. The tryptic peptides were extracted with 1% formic acid/ 2% acetonitrile, followed by extraction with 50% acetonitrile twice. The acetonitrile was evaporated and the peptide extract concentrated to 5-10 µl under a stream of nitrogen.
MS/MS data was acquired with a QTOF Premier API instrument (Waters) equipped with the standard Z-spray source. Sample was introduced via a nanoAcquity liquid chromatography system (Waters) and electrosprayed using a PicoTip emitter (SilicaTip, New Objective, Woburn, MA, USA). Samples were desalted for 1 min using a Waters Symmetry C 18 column (5 µm particles; 180 µm x 2 cm) using 0.1% formic acid at 15 µl/min, followed by separation using a Waters BEH C 18 column (1.7 µm particles; 75 µm x 15 cm) employing a solvent system of 0.1% formic acid (solvent 1) and acetonitrile / 0.1% formic acid (solvent 2). The peptides were eluted with a linear gradient: 3-60% solvent 2 for 30 min at 300 nl/min. The

Basophil activation test
Allergen-specific basophil degranulation was analysed by monitoring the basophil activation markers CD203c and CD63 (19). Briefly, 10-fold serial dilutions of dog saliva or dog dander