Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Author: Bc. Petra Porubová Supervisor: prof. RNDr. Ctirad Andrýs, Ph.D. Title of diploma thesis: Allergic phenotypes in patients with respiratory allergy evaluated by microchip ISAC technique Key words: allergic inflammation, allergen specific IgE, component resolved diagnostics, microchip, ISAC Background: The aim of this diploma thesis was to evaluate the sensitization profile in the group of pacients with allergic rhinitis and/or bronhial asthma and to identify possible differences between patients with different disease severity. Methods: ImmunoCAP ISAC was used to determine specific IgE antibodies against 112 allergen components. The evaluated group included 152 patients (57.9 % women) from the allergology outpatient office of the Department of Clinical Immunology and Allergology, University Hospital Hradec Králové. Results: The most common allergens in this group of patients were the components Phl p 1 (68,4 %) and Bet v 1 (65,1 %). Differences in the number of positive and negative reactions were demonstrated between patients with different severity of allergic rhinitis. Correlations were found between the number of positive/negative reactions and the severity of bronchial asthma. And the...

<p>Immunoglobulin E (IgE)-mediated allergy occurs when our immune system causes a reaction to otherwise harmless substances (allergens). Allergens are predominantly proteins present in biological materials such as pollens, mites, animal epithelia, moulds and foods. </p><p><i>In vitro</i> tests for specific IgE antibodies usually employ an allergen source extract as an antibody capturing reagent. The proportion of allergenic molecules in these biochemically complex extracts may vary.</p><p>Recombinant allergens may be obtained in large quantities with biotechnological techniques. These proteins can be characterized biochemically and immunologically, resulting in tests with minimal batch-to-batch variation. This thesis describes different uses of recombinant allergens in component-resolved diagnostics (CRD).</p><p>In CRD, single allergenic proteins are used to establish a sensitization profile of the patient. Two timothy grass (<i>Phleum pratense</i>) pollen allergens, Phl p 11 and Phl p 4, were cloned and expressed as recombinant proteins. They were subsequently characterized and can, for example, be used in a panel for grass pollen CRD.</p><p>Single allergens may be useful as diagnostic markers for allergic sensitization. This phenomenon was studied using tropomyosin, a major allergen from the shrimp <i>Penaeus aztecus</i> (Pen a 1). The characteristics of the recombinant and natural proteins were compared. The recombinant tropomyosin was then extensively tested using specific competition for IgE binding against extracts of other crustacean species, house dust mite and cockroach.</p><p>In cases when an important allergen is missing or underrepresented in a natural extract, the corresponding recombinant allergen may be added to the extract as a spiking reagent. Previous studies have shown that latex extracts for diagnostic testing may lack the allergen Hev b 5. Recombinant Hev b 5 was expressed from a synthetic gene construct, incorporating several adaptations to enable efficient large scale production of the recombinant protein, to be used as a spiking reagent.</p>

Immunoglobulin E (IgE)-mediated allergy occurs when our immune system causes a reaction to otherwise harmless substances (allergens). Allergens are predominantly proteins present in biological materials such as pollens, mites, animal epithelia, moulds and foods. In vitro tests for specific IgE antibodies usually employ an allergen source extract as an antibody capturing reagent. The proportion of allergenic molecules in these biochemically complex extracts may vary. Recombinant allergens may be obtained in large quantities with biotechnological techniques. These proteins can be characterized biochemically and immunologically, resulting in tests with minimal batch-to-batch variation. This thesis describes different uses of recombinant allergens in component-resolved diagnostics (CRD). In CRD, single allergenic proteins are used to establish a sensitization profile of the patient. Two timothy grass (Phleum pratense) pollen allergens, Phl p 11 and Phl p 4, were cloned and expressed as recombinant proteins. They were subsequently characterized and can, for example, be used in a panel for grass pollen CRD. Single allergens may be useful as diagnostic markers for allergic sensitization. This phenomenon was studied using tropomyosin, a major allergen from the shrimp Penaeus aztecus (Pen a 1). The characteristics of the recombinant and natural proteins were compared. The recombinant tropomyosin was then extensively tested using specific competition for IgE binding against extracts of other crustacean species, house dust mite and cockroach. In cases when an important allergen is missing or underrepresented in a natural extract, the corresponding recombinant allergen may be added to the extract as a spiking reagent. Previous studies have shown that latex extracts for diagnostic testing may lack the allergen Hev b 5. Recombinant Hev b 5 was expressed from a synthetic gene construct, incorporating several adaptations to enable efficient large scale production of the recombinant protein, to be used as a spiking reagent.