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

Abstract

Background and objective: Allergic diseases in horses have remained unknown since IgE in horses has not been precisely measured. CRE-DR is a unique anti-IgE antibody cross-reactive to human and dog IgE. Amino acids of CRE-DR epitope are well-conserved among animal species except for rodents. In addition, CRE-DR recognizes IgE losing FcRI binding. Therefore, this work aimed to identify pathogenic (heat-sensitive) and non-pathogenic (heat-resistant) IgE in horses, by CRE-DR and its caninized one, CRE-DR-B, in order to allow a better understanding of the sensitization/allergy. Horse 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/nonpathogenic allergen-specific IgE to D. farinae and D. pteronyssinus was estimated in horse sera by 87ELISA, using the anti-IgE chimeric antibodies CRE-DR and CRE-DR-B

Materials and methods Sera from a total of 22 allergic horses and 7 from non-allergic, were tested. Determinations were performed with and without previous thermal treatment of sera, with CRE-DR for the determination of total and non-pathogenic IgE, respectively, and with previous thermal treatment, with CRE-DR-B to determine pathogenic IgE. 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-DRB 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 for standard curve and serum samples. 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 horses when compared to non-allergic individuals. With regard to D. farinae and D. pteronyssinus, 14 out of 22 horses presented with higher levels of pathogenic IgE when compared to non-pathogenic IgE. However, some non-allergic individuals also showed high levels of allergen-specific IgE, suggesting the possibility of being just sensitized individuals without allergy manifestation. Moreover, allergen-specific IgE levels should be carefully monitored until the onset of allergy.

Conclusions Understanding the pathogenic/nonpathogenic relation may permit further efficacy of immunotherapy, by allowing to avoid including in vaccination extracts species to which patients are just sensitized, without allergy triggering. It will also allow choosing the real relevant pool of molecular allergens for each individual immunotherapy as a true component-resolved immunotherapy, in a highly precision medicine context.