Hamed, Aljali
(2016)
Development of IgE microarray assays for classification of allergic individuals.
PhD thesis, University of Nottingham.
Abstract
Allergic reactions are pathological immune reactions against common, harmless environmental proteins, termed “allergens”. Type I hypersensitivity reactions are mediated by IgE antibodies; these reactions are the most prevalent, affecting all ages and both genders to almost the same extent. This form of allergic reaction is distributed widely affecting more than 25% of the population in the industrial world. The increased incidence of IgE-mediated allergy is thought to be a combination of environmental and genetic factors, each account for about 50%. The effects associated with this disease may reduce quality of life, daily activities, and in some cases threaten life, hence affecting work and leisure time, increasing healthcare and medication costs.
IgE-mediated allergy is defined a complex innate and adoptive immune response to natural harmless environmental allergens in which allergen- specific IgE antibodies are predominantly produced. In addition, it is believed that other immunoglobulin isotypes might be involved in atopic diseases (mainly IgG4), although the roles of these antibody isotype are still debatable whether they are pathogenic or protective antibodies.
Generally, diagnostic procedures for IgE-mediated allergy begin with the patient’s history, clinical symptoms, physical examination, in vivo provocation tests, and are eventually confirmed with laboratory investigations.
In this thesis we develop a microarray technology for use in-house for comprehensive investigations of immunoglobulin reactivity profiles against numerous allergens in atopic disease.
Several printing and blocking buffers were developed in-house to preserve functionality of the arrayed proteins, minimise background noise, and increase signal intensity. The new diagnostic platform was validated by set of quality controls, then applied on large-scale applications (allergic patients, general populations, suspected asthmatics and healthy control individuals) to measure total and specific IgE antibodies. Application of the developed antibody microarray platform identified the difficulty of setting cutoffs using a “healthy control group”. We tested both the 75th and the 95th percentiles of the healthy control group as a cut-off point for total IgE levels that resulted in 39.3ng/ml and 70.68ng/ml respectively. When the 75th percentile was selected, the number of participants in the allergy patients classed as positive above the cutoff points was 92 out of 105 (87.6%). However, if the 95th percentile was selected, the number of positive patients was 63 out of 105 (60%) in the same group. On the other hand, the developed allergen microarray platform was used to immunoprofile allergen specific immunoglobulin subtypes in the four different cohorts against 70 SPT allergens. MeV software was used to present the reactivity profiles of the immunoglobulin subtypes semi-quantitatively that resulted in the hierarchical clustering according to allergen species. The reactivity profiles of allergen specific IgE in the allergic group recognized multiple allergens while specific IgE in the healthy control group was negative except for one serum sample. Specific IgE in the random population was lower than in the suspected asthmatics although it recognized allergens from different species (ingested, inhaled, venom).
Similarly, this multi-allergen microarray platform was adapted to investigate the roles of other antibody isotypes (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) in IgE- mediated allergy. The study revealed no roles for these isotype except IgG4 that showed multiple recognition patterns in serum samples of healthy individuals and allergic patients. A comparison between microarray results and provocation testing (SPT) was performed for the samples from suspected asthmatics. To enable direct comparison, IgE:IgG4 ratio was utilized as IgE levels alone would not be expected to correlate with wheal and flare reaction. The IgE:IgG4 ratio showed good correlation with the six allergens tested by SPT (r=0.9299, p<0.0001). In addition, the multi-allergen chip can detect additional responses outside the standard six SPT- allergens performed routinely, and moreover reports on IgG4 responses that are never assessed by SPT, but are potentially important to the likelihood of asthma and allergy.
Overall, the results indicated that the new developed microarray platform is highly specific, sensitive and reproducible. Subsequently, this high-throughput microarray platform can work as a diagnostic tool for monitoring the development of allergic diseases, predicting allergic reactions particularly for polysensitised patients, evaluating patient response to treatments, and eventually allowing the classification of allergic individuals. In conclusion, a new high-throughput multi-allergen platform has been developed that will certainly improve the future of allergy diagnosis.
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