Development of a comprehensive protein microarray for immunoglobulin E profiling in horses with severe asthma

Abstract

Background Severe asthma in horses, known as severe equine asthma (SEA), is a prevalent, performance‐limiting disease associated with increased allergen‐specific immunoglobulin E (IgE) against a range of environmental aeroallergens. Objective To develop a protein microarray platform to profile IgE against a range of proven and novel environmental proteins in SEA‐affected horses. Animals Six SEA‐affected and 6 clinically healthy Warmblood performance horses. Methods Developed a protein microarray (n = 384) using protein extracts and purified proteins from a large number of families including pollen, bacteria, fungi, and arthropods associated with the horses, environment. Conditions were optimized and assessed for printing, incubation, immunolabeling, biological fluid source, concentration techniques, reproducibility, and specificity. Results This method identified a number of novel allergens, while also identifying an association between SEA and pollen sensitization. Immunolabeling methods confirmed the accuracy of a commercially available mouse anti‐horse IgE 3H10 source (R2 = 0.91). Biological fluid source evaluation indicated that sera and bronchoalveolar lavage fluid (BALF) yielded the same specific IgE profile (average R2 = 0.75). Amicon centrifugal filters were found to be the most efficient technique for concentrating BALF for IgE analysis at 40‐fold. Overnight incubation maintained the same sensitization profile while increasing sensitivity. Reproducibility was demonstrated (R2 = 0.97), as was specificity using protein inhibition assays. Arthropods, fungi, and pollens showed the greatest discrimination for SEA. Conclusions and Clinical Importance We have established that protein microarrays can be used for large‐scale IgE mapping of allergens associated with the environment of horses. This technology provides a sound platform for specific diagnosis, management, and treatment of SEA.

Science‐in‐brief: Latex in riding arenas and racetracks identified as a risk factor for equine respiratory health

“…• The development of accurate diagnostic tests has been hampered because of a lack of statistical approaches for clear disease classification, and the limited range of allergens tested to date. • Collaborative studies between the Royal Agricultural University and the University of Nottingham in the UK and clinical researchers in Switzerland, France, USA and Canada [3,4] have overcome many of these problems and provided insights into the allergens responsible for this condition. Equine Veterinary Journal 52 (2020) 11-12 © 2019 EVJ Ltd BALF and sera of 'true' sEA affected horses (defined by applying strict inclusion and exclusion criteria based on clinical workup, physiological tests and cytology) in mixed/matched groups.…”

“…Two papers by Sam White and colleagues have overcome many of these problems and provided insights into the allergens responsible for this condition by examining ‘true’ sEA affected horses (defined by applying strict inclusion and exclusion criteria based on clinical workup, physiological tests and cytology) in mixed/matched groups. The technology is based on the development of a comprehensive protein microarray comprising extracts and pure proteins from a wide range of protein families derived predominantly from fungi, bacteria, pollen, and arthropods (384 putative allergens tested).…”

“…Points of clinical relevance: Clinical diagnosis of sEA (RAO, COPD) is often compounded by complex histories of previous exposures to respiratory and other allergens. Allergen‐specific IgE has been shown to relate to the pathogenesis of sEA. The development of accurate diagnostic tests has been hampered because of a lack of statistical approaches for clear disease classification, and the limited range of allergens tested to date. Collaborative studies between the Royal Agricultural University and the University of Nottingham in the UK and clinical researchers in Switzerland, France, USA and Canada have overcome many of these problems and provided insights into the allergens responsible for this condition. This was achieved by developing a comprehensive protein microarray system (comprising 384 putative allergens) to examine IgE antibodies in BALF and sera of ‘true’ sEA affected horses (defined by applying strict inclusion and exclusion criteria based on clinical workup, physiological tests and cytology) in mixed/matched groups. Strong correlation was found between allergen‐specific IgE measured both in BALF and serum. Thus, the majority of mathematical modelling and other studies were conducted with serum alone because it is far easier to obtain, less invasive, more economical and stable to transport. A plethora of novel environmental derived allergenic proteins was identified which cause production of sEA symptoms in horses and a rise IgE antibody in sensitised horses.…”

Characterization of fungal exposure and dectin-1 expression in healthy horses and horses with severe asthma

“…In its more common form, susceptible horses become symptomatic when exposed to hay and straw, which contain high levels of fungi, mites, and endotoxins. 1,2 Similarly, asthma in humans is associated with exposure to antigens, which are usually proteins, and more specifically to aeroallergens. 3 Visibly moldy hay, compared to well-cured hay, is associated with more rapid asthma exacerbations, and horses with fungal particles in their tracheal aspirates are more likely to be diagnosed with equine asthma than those without fungi in their trachea.…”

Untitled

“…Determination of IgE in serum was performed by protein microarray, as previously described [10,22,23], using the same protein microarray as in Novotny et al [10] which included a total of 27 Culicoides r-allergen, Culicoides and Simulium vittatum extracts, as well as proteins irrelevant for IBH as controls (Table 2).…”