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

Abstract

Background and objective: In Europe approximately 23% of the population is affected by allergic respiratory diseases. The most common clinical signs are sneezing, itchy nose, rhinorrhea, and/or nasal congestion. In humans, several respiratory disorders have also been associated with allergen-specific IgE. Various clinical manifestations such as asthma or allergic rhinitis are among the main reasons for allergy consultation in people. This work aimed to identify heat-sensitive (pathogenic) and heat-resistant (non-pathogenic) IgE in human patients by using an anti-dog x human IgE mouse monoclonal antibody, CRE-DR, and its caninized one (CRE-DR-B) to allow a better understanding of the sensitization/allergy duality. Prevalence of pathogenic/nonpathogenic allergen-specific IgE to Dermatophagoides. farinae and D. pteronyssinus was estimated in human sera by ELISA, using the two anti-IgE antibodies

Materials and methods Sera from a total of 12 dust-mite allergic human patients (6 of those with sera from before and following allergen immunotherapy) 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. CRE-DR-B ELISAs were used to determine pathogenic IgE with non-heated sera. 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, respectively, streptavidin-β-Gal and 4-methylumbelliferyl β-D-galactopyranoside substrates for 1h at RT. After the enzymatic reaction was stopped by adding stop solution, fluorescence was measured at 460 nm. The average of fluorescence intensity units (FU) for data points was calculated for the standard curve. The graphic plot FU (y-axis) towards the equivalent IgE concentrations (x-axis) was obtained.

Results Regarding D. farinae, 8 out of 12 human patients presented with higher levels of pathogenic IgE, when compared to nonpathogenic IgE. Following immunotherapy, all patients showed a decrease in total, pathogenic and nonpathogenic IgE levels, although 2 of them kept with higher levels of pathogenic IgE levels than nonpathogenic. Moreover, serum IgE levels usually do not correlate with the severity of disease.

Conclusions Several patients did not show a decrease in allergen-specific IgE levels following immunotherapy, despite an improvement in clinical condition. Understanding the pathogenic/nonpathogenic relation may allow further efficacy of immunotherapy, by avoiding including in immunotherapy extracts species to which patients are just sensitized. It will 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.