Identifying relevant specific IgE in dogs – a one health precision model.

Abstract

Background and objective: In dogs, several skin disorders have been associated with allergen-specific IgE and clinical manifestations like atopic dermatitis are one main reason for veterinary care regarding allergy. However, sensitization may occur without clinical manifestations, pointing to the occurrence of specific IgE lacking allergy triggering. Defective skin barrier function, favoring an in-depth penetration of allergens, also concurs to sensitization, predisposing to allergy. A dual state of skin barrier deficiency and immunological IgE production coincide in the development of allergy. This work aimed to identify FcRI-binding (pathogenic) and non-FcRI-binding (non-pathogenic) IgE in dogs, by using an anti-human x canine IgE monoclonal antibody, CRE-DR, to allow a better understanding of the sensitization/allergy duality. Dog sera were tested for pathogenic and non-pathogenic IgE and levels of these two forms of specific IgE from both allergic and non-allergic individuals were compared. Prevalence of pathogenic/non-pathogenic allergen-specific IgE to D. farinae and D. pteronyssinus in dog sera by ELISA, was estimated using CRE-DR and its caninized CRE-DR-B during allergen-specific immunotherapy. (1036 characters)

Materials and methods Sera from 12 dust-mite allergic dogs (6 of those with sera from before and following allergen immunotherapy) and 26 from non-allergic dogs, were tested. ELISAs with CRE-DR were performed with and without previous thermal treatment of sera for the determination of total and non-pathogenic IgE, heat-sensitive (FcRI-binding) and heat-resistant IgEs, respectively. ELISAs with CRE-DR-B were carried out for heated sera only for pathogenic IgE, which is not reactive to serum IgE without the heating. A standard curve was determined using a dog serum. ELISA 96-well microplate (FLUOTRAC™ 600, Greiner Bio-One) was prepared by overnight coating with D. farinae/pteronyssimus extracts, incubated (1h) with blocking buffer at room temperature (RT) and washed 3 times. Serum samples were used at a 5-80-fold dilution for overnight incubation at 4 ⁰C. Negative and blank controls were used together; 100 μL of CRE-DR/CRE-DR-B was added to each well and incubated 2h at RT. Detection was done with streptavidin-β-Gal and 4-methylumbelliferyl β-D-galactopyranoside for 1h at RT. Enzymatic reaction was stopped by adding stop solution and fluorescence was measured at 460 nm. The average of fluorescence intensity units (FU) for data points was calculated using the standard curve. The graphic plot FU (y-axis) towards the equivalent IgE concentrations (x-axis) was obtained.

Results In this study, higher levels of allergen-specific IgE were found in sera from allergic dogs when compared to non-allergic individuals. Regarding D. farinae, 9 out of 12 allergic dogs presented with higher levels of pathogenic IgE, when compared to nonpathogenic IgE. Following immunotherapy, 3 dogs showed a decrease in total IgE, either pathogenic or non-pathogenic. Nevertheless, several non-allergic individuals showed high levels of allergen-specific pathogenic IgE, suggesting being just sensitized individuals without allergy manifestation. There is still some room for refinement of IgE measurement to detect true pathogenic IgE.

Conclusions Understanding the pathogenic/nonpathogenic relation may allow further efficacy of immunotherapy, by avoiding including in vaccination extract species to which patients are just sensitized to, without allergy manifestation. Hence, it may also allow choosing the relevant pool of molecular allergens for each individual immunotherapy as a true component-resolved immunotherapy, in a highly precision medicine context.