EP-4735882-A1 - BAT-BASED METHOD FOR DETERMINING THE PREVALENCE AND/OR SEVERITY OF AN ALLERGY IN AN INDIVIDUAL AND KITS THEREFORE

Abstract

The present invention relates in a first aspect to a method for determining the prevalence and/or severity of an allergy in an individual with or suspected to be afflicted with allergy against an allergy-triggering source whereby the method is a basophil activation test (BAT)-based method substituting the current, potentially risky method of allergy challenge considered as the gold standard of allergy diagnostic measures. In addition, the present invention relates to the method which can be conducted automatically and preferably in a computer-implemented method for fast and reliable determination of the prevalence and/or severity of allergy in said individual. In particular, the present method allows the calculation of a combined symptom and tolerance class (CSTC), which allows the individual to be categorized into different classes of allergy severity, e.g., to guide therapeutic decisions and/or behavioural recommendations for said individual. Moreover, the present invention provides a computer-implemented method for doing so and in a further aspect, a test kit or a kit of parts for determining the prevalence and/or severity of an allergy according to the present invention. The use of the test kit includes the application of a basophil activation test following the method according to the present invention.

Inventors

• Walsemann, Theresa
• BEHRENDS, Jochen
• JAPPE, Uta

Assignees

• Forschungszentrum Borstel, Leibniz Lungenzentrum

Dates

Publication Date

20260506

Application Date

20240624

Claims (1)

1. Claims 1) Method for determining the prevalence and/or severity of an allergy in an individual afflicted with or suspected to be afflicted with allergy against an allergy-triggering source, comprising the steps of: a) providing a sample of the said individual; b) aliquoting the sample and performing an allergen-resolved BAT (basophil activation assay) wherein a single aliquot is incubated with exactly one stimulus selected from i) to v): i) an extract of the allergy-triggering source comprising at least two different allergens; ii) a major allergen of the allergy-triggering source being essentially free of other allergens of the allergy-triggering source; iii) optionally, a minor allergen of the allergy-triggering source being essentially free of other allergens of the allergy-triggering source; iv) one or more lipid-associated allergen(s) of the allergy-triggering source wherein the one or more lipid-associated allergen(s) is different to the major allergen of ii) and/or the optional minor allergen of iii); and v) no allergen of the allergy-triggering source as control; c) determining the basophil activation for each of the individual aliquots i) to v) from step b), d) determining the change of CD63 expression and CD203c expression in the individual aliquots i)-iv) compared to the control v); and e) calculating a combined symptom and tolerance class (CSTC index) as a measure for the prevalence and severity of an allergy against the allergytriggering source for the individual based on the data determined in steps c) and d). 2) The method according to claim 1 for differentiating between allergic individuals and sensitized but non-allergic individuals. 3) The method according to any one of the preceding claims, wherein the allergytriggering source is selected from the group of one or more food, inhalation or environmental allergy triggering source, preferably, wherein the allergy-triggering source is selected from peanut, dust mite, soy, milk, egg, fish, mussels, meat, tree nuts, wheat, apple, bee, wasp, cat, dog, weeds, grasses and trees. 4) The method according to any one of the preceding claims, like wherein the allergytriggering source is peanut, the extract is an alkaline peanut extract; the major allergen is Ara h 2, the optional minor allergen is Ara h 8, and the lipid-associated allergens are oleosins containing at least Ara h 14 and Ara h 15 from roasted peanuts. 5) The method according to any one of the preceding claims wherein the sample is selected from fluid or enzymatically digested tissue for single cell suspensions of said subject, in particular, the sample is selected from sputum, blood including whole blood, saliva, nasal secretion and urine, preferably the sample is whole blood. 6) The method according to any one of the preceding claims wherein the method for determining activation of basophils is a cytometric-based in vitro method using the fluorescence-labelled markers anti-CD63, anti- FccRIa, and anti-CD203c, preferably, wherein the change of CD63 expression and/or CD203c expression is determined as a change of median fluorescence intensity in cytometric measurement. 7) The method according to claim 5 or 6 wherein the basophil activation is determined as proportion of activated basophils using the identification marker CD63, FccRIa, and CD203c along with the following gating strategy: i) expression of FCER1 a and CD203c; ii) forward scatter area (FSC-A) vs. sideward scatter area (SSC-A); iii) sideward scatter width (SSC-W) vs. CD203c; and the proportion of activated basophils is calculated based on CD63-, FccRIa- and CD203c-positive cells. 8) The method according to any one of the preceding claims further comprising a step of excluding individuals not accessible to CSTC index classification wherein said individuals are characterized by non-responsiveness to IgE mediated basophil activation, in particular, determined by incubation of a single aliquot with a single IgE- independent activation stimulus, like fMLP, and/or incubation of a single aliquot with a single IgE-dependent activation stimulus, like a mixture of two anti- Ig E antibodies. 9) The method according to any one of the preceding claims wherein at least part of the steps is conducted by a computer-implemented analysis method. 10) The method according to any of the preceding claims wherein different stages of CSTC are calculated based on the degree of basophil activation obtained for the individual stimuli defined under claim 1 , step b) i)-iv)), as well as predetermined thresholds for the change in CD63 expression and CD203c expression, preferably, where the CTSC index classifies in ascending order the severity of an individual's allergy from 0 to 3, and the stages being determined as follows: i) CSTC 0 index with reactivity only to extract i) with a proportion of activated basophils less than 25% indicating no allergy with high tolerance to the allergy-triggering source; ii) CSTC 1 index with reactivity to not more than three stimuli i) to iv) with individual proportions of activated basophils less than 25%, respectively, and the change in CD63 expression and/or CD203c expression, in particular, a delta MFI, for the stimuli is less than 2-fold, indicating a mild allergy with substantial tolerance; iii) CSTC 2 index with reactivity to not more than three stimuli i) to iv) with individual proportions of activated basophils only for one stimulus exceeding 30% and the change in CD63 expression and/or CD203c expression, in particular, a delta MFI, for most stimuli is less than 2.5-fold, indicating a moderate allergy with moderate tolerance; and iv) CSTC 3 index with reactivity to at least three stimuli i) to iv) with individual proportions of activated basophils of at least two allergens exceeding 30% and the change in CD63 expression and/or CD203c expression, e.g., a delta MFI for most stimuli above 2.5-fold, indicating a severe allergy with low or no tolerance. 11) A computer-implemented method for determining the prevalence and/or severity of an allergy of an individual suspected to be afflicted with allergy against an allergytriggering source comprising the step of a) obtaining data of measured parameters, preferably data obtained by flow cytometry, to determine the percentage or absolute number of activated basophils at least in the individual samples i) to v) i) an extract of the allergy-triggering source comprising at least two different allergens; ii) a major allergen of the allergy-triggering source being essentially free of other allergens of the allergy-triggering source; iii) optionally, a minor allergen of the allergy-triggering source being essentially free of other allergens of the allergy-triggering source; iv) one or more lipid-associated allergen(s) of the allergy-triggering source wherein the one or more lipid-associated allergen(s) is different to the major allergen of ii) and/or the optional minor allergen of iii); and v) no allergen of the allergy-triggering source as control; of the individual, wherein the basophil activation is determined using the identification marker CD63, FceRla, and CD203c; b) computing the data of step a); c) identifying the percentage or absolute number of activated CD63 expressing basophils, the change of CD63 expression, and the change of CD203c expression of said activated basophils compared to the control v); d) determining the allergy status and/or severity, optionally showing the data on an output unit. 12) The method according to any one of the preceding claims wherein the sample processing and measurement are conducted automatically using a flow cytometer with integrated robotic sample handling and manipulation, and wherein the basophil activation is optionally determined using a computer-implemental method according to any one of claims 9 to 11. 13) The method according to any one of claims 11 or 12 wherein the data of measured parameter are of measured emission parameters, which are obtained through excitation of different fluorochromes by the detection of electromagnetic radiation signals received by a detector afterwards of a flow cytometer. 14) Computer-readable medium or computer program product having computerexecutable instructions for performing the steps as identified in any one of claims 9 to 12. 15) The use of a test kit or kit of parts for determining the prevalence and/or severity of an allergy in an induvial suspected to be afflicted with allergy against an allergytriggering source comprising anti-CD-203c, anti-FceRla and anti-CD63 antibodies, each labelled with a distinct fluorochrome, optionally lysis buffer for erythrocytes and, optionally, washing buffer, and, in addition instruction on how to use the test or kit of parts in a method according to any one of claims 1 to 9, preferably further comprising at least the extract of the allergen triggering source, the major allergen, and one lipid- associated allergen being different to the major allergen. 16) A method according to any of the preceding claims wherein the CSTC index determined is used to guide behavioral recommendations and/or therapy decisions for the individual.

Description

BAT-based method for determining the prevalence and/or severity of an allergy in an individual and kits therefore The present invention relates in a first aspect to a method for determining the prevalence and/or severity of an allergy in an individual with or suspected to be afflicted with allergy against an allergy-triggering source whereby the method is a basophil activation test (BAT)- based method substituting the current, potentially risky method of allergy challenge considered as the gold standard of allergy diagnostic measures. In addition, the present invention relates to the method which can be conducted automatically and preferably in a computer-implemented method for fast and reliable determination of the prevalence and/or severity of allergy in said individual. In particular, the present method allows the calculation of a combined symptom and tolerance class (CSTC), which allows the individual to be categorized into different classes of allergy severity, e.g., to guide therapeutic decisions and/or behavioural recommendations for said individual. Moreover, the present invention provides a computer-implemented method for doing so and in a further aspect, a test kit, or a kit of parts for determining the prevalence and/or severity of an allergy according to the present invention. The use of the test kit includes the application of a basophil activation test following the method according to the present invention. Prior Art Prevalence to food allergy varies greatly from region to region but increases steadily, especially in Western industrial nations. Allergic disorders have emerged from a rare disease to one of the most common chronic diseases in Western countries, afflicting approximately 30% of the population. Moreover, the prevalence is still rising and will become a problem for the population of the world. It is predicted that 50% of the European population may be afflicted with allergy in the future. For example, allergy and allergic reactions to peanut have increased threefold in the US in the past decades, whereby allergy affects about 2% of children in the United Kingdom and the US and being the most common cause of life-threatening anaphylaxis in childhood. Therefore, correct diagnosis and grading is crucial to prevent potentially life-threatening allergic reactions against peanuts, as well as other e.g., food-borne, or environmental allergy-triggering sources (incl. e.g., wasp venom and others). The skin prick-tests (SPT) and immunoglobulin (lg)E antibody assays have been used to detect sensitization, but do not prove allergy. To detect allergy in unclear cases, challenge tests with suspected allergen sources are currently the diagnostic gold standard. For food allergies, these challenge tests are conducted as oral food challenge (OFC) to determine the prevalence and/or severity of allergy against the allergenic sources. However, challenge tests in general and OFC in particular are very time consuming and associated with inherent risks for the person tested. To be able to provide immediate medical attention in the case of any acute allergic reactions during challenge, the affected person must be constantly monitored, and hence an OFC is often associated with hospitalization. To avoid such dangers and burdens, an attempt is made to establish the diagnosis by thorough medical history and determining sensitivity based on measuring soluble IgE in the blood (serum) and/or via SPT. However, historybased diagnosis combined with measurement of IgE is often not sufficient to allow a clear determination of the prevalence and/or severity of an allergy. In the last decade, the basophil activation test has been discussed as a method to safely simulate allergic reactions with patient blood in vitro, and thus determine the presence and severity of allergy. In this context, several attempts have been made to utilize the BAT to distinguish individuals with asymptomatic sensitization from true allergic individuals and to predict the severity of allergy. However, each of these attempts had its shortcomings. For example, Santos A., et al., J. Allergy Clin. Immunol., 2015, 135 (1), 179 - 186 disclose distinguishing parameters of basophil activation test reflecting the severity and tolerance of allergic reactions to peanut. In addition, Schwager C., et al., report on peanut oleosins associated with severe peanut allergy and the importance of lipophilic allergens for comprehensive allergy diagnosis (see Schwager et al., J. Allergy Clin. Immunol., 2017, Doi: 10.10168/J.Jaci 2017.02.020). Wanich N., et al. (2009, Journal of Allergy and Clinical Immunology, 123, no. 4, 789- 794), and Ldtzsch B., et al. (Clinical and Translational Allergy, 2016, 6(1) 1-9) reports on possible differentiation between tolerogenic or sensitize individuals in comparison to allergic patients using a BAT to determine milk or hazelnut allergy. In both documents, allergy is determined as an IgE-dependent reaction of basophils. Both docume