Dermatophagoides farinae‐specific IgG subclass responses in atopic dogs undergoing allergen‐specific immunotherapy

Abstract

Canine atopic dermatitis (CAD) is known to involve IgE‐mediated mechanisms, but the role of IgG in the pathogenesis is less well understood. Previous studies have shown that total serum IgG concentrations are increased in CAD and suggested that a subclass of IgG, IgGd, is involved in the pathogenesis. Furthermore, induction of IgG blocking antibodies has been suggested as a possible mode of action of allergen‐specific immunotherapy (ASIT). The aim of this study was to characterize the changes in Dermatophagoides farinae (DF)‐specific total IgG and IgG subclasses during treatment of atopic dogs with ASIT. Twenty‐one dogs with confirmed atopic dermatitis and showing positive reactions to DF on intradermal testing were treated with alum‐precipitated vaccines for 9 months. Blood samples were collected before and after 3, 6, and 9 months of ASIT. Lyophilized whole‐body DF allergens were reconstituted to 1 mg/mL in sterile phosphate‐buffered saline (PBS), and SDS–PAGE was used to separate mite proteins. Separated proteins were transferred onto blotting membranes, cut into strips, and incubated with canine sera. For DF‐specific total IgG responses, strips were probed with horseradish peroxidase‐conjugated goat anti‐dog IgG and subsequently incubated with the colorimetric substrate DAB (3,3’‐diaminobenzidine). For DF‐specific IgG subclass responses, strips were probed sequentially with one of a panel of four monoclonal antibodies with specificity for each subclass, bovine anti‐mouse IgG conjugated to horseradish peroxidase, and finally, the chemiluminescent substrate ECL that was used for its higher sensitivity. Images of the positive bands were captured using Kodak Image Station, and the number of bands and their intensity was analysed by Kodak 1D Image Analysis Software. Prior to ASIT, a number of proteins of different molecular weights were recognized by antisera specific for total IgG and the subclasses IgG1 and IgG4, the most common being the high‐molecular‐weight chitinase, Df 15. There was virtually no IgG2 or IgG3 response to DF allergens. During ASIT, the total IgG response to DF allergens varied widely between dogs and could increase, decrease, fluctuate or remain the same. For each dog, the changes in total DF‐specific IgG were paralleled by equivalent changes in IgG1 and IgG4, but there was no induction of IgG2 or IgG3 antibodies. There were no significant increases in total IgG or IgG subclass responses in dogs showing a good response to ASIT. These findings suggest that atopic dogs mount an IgG1 and IgG4 response against multiple antigens of Dermatophagoides farinae, but these responses are not consistently augmented by ASIT. This suggests that successful ASIT in dogs is not associated with the production of blocking antibodies. Funding: Self‐funded.