WO2018199840A1

WO2018199840A1 - Microfluidic method and system for detecting basophil aller-gen threshold sensitivity (cd-sens) by using a gradient of at least one allergen

Info

Publication number: WO2018199840A1
Application number: PCT/SE2018/050419
Authority: WO
Inventors: Amam RUSSOM; Anna NOPP SCHERMAN; Joachim LUNDAHL; Gunnar Johansson
Original assignee: Russom Amam; Nopp Scherman Anna; Lundahl Joachim; Gunnar Johansson
Priority date: 2017-04-25
Filing date: 2018-04-24
Publication date: 2018-11-01

Abstract

The present invention relates to a microfluidic based method and system for detec- tion of basophil allergen threshold sensitivity (CD-sens). The method and system in- volves taking whole blood directly from the allergic patient and isolate the basophils for activation followed by image based detection of the degranulation markers. The method and system involves the isolation of basophils which express CD203c sur- face markers direct from whole blood, activating the isolated basophils with at least one activation composition comprising a gradient of anti-Fcɛ RI or a gradient of at least one allergen, capturing the activated basophils and detection of expression of CD63 degranulation markers expressed on the basophils by using microscopy.

Description

MICROFLUIDIC METHOD AND SYSTEM FOR DETECTING BASOPHIL ALLERGEN THRESHOLD SENSITIVITY (CD-SENS) BY USING A GRADIENT OF AT LEAST ONE ALLERGEN

TECHNICAL FIELD

The present invention relates to a microfluidic based method and system for detection of basophil allergen threshold sensitivity (CD-sens). The method and system involves taking whole blood directly from the allergic patient and isolating the basophils for activation followed by image based detection of the degranulation markers. The method and system involves the isolation of basophils which express CD203c surface markers direct from whole blood, activating the isolated basophils with an activation composition comprising a gradient of anti-Fes Rl or a gradient of at least one allergen, capturing the activated basophils and detection of expression of CD63 degranulation markers expressed on the basophils by using microscopy.

BACKGROUND OF INVENTION

Peripheral blood is the most frequently accessed biological material in the clinic and isolation of cells from blood is of broad clinical and scientific importance. Leukocytes are responsible for providing immunity [1 , 2], but also have an important role in the pathogenesis of inflammatory diseases such as allergic reactions.

Basophils represent a sub population of leukocytes and a granulocyte constituting of less than 1 % of the total peripheral circulating leukocytes [3, 4]. Typically, basophils contain granules consist of immune mediators that are involved in connecting both innate and adaptive immune responses during pathogenesis of many inflammatory diseases [5]. The basophil regulates CD4+ T helper cells (Th2) immune response by the release of interleukin-4 cytokine after activation, which promotes differentiation of B-cells into plasma cells to produce immunoglobulin E (IgE) that mediates the allergic reaction [6, 7]. Basophils are involved in the inflammatory responses of allergic reactions, mainly IgE-mediated allergic reaction. The IgE-mediated allergic reaction is provoked by re-exposure to a specific allergen. Switched activated plasma cells produce IgE-antibodies (IgE-ab) which predominantly bind to specific receptors (FcsRI) on the surface of mast cells and basophils.

Binding of the allergen to IgE-ab on basophils causes activation, degranulation, and release of a variety of immune-modulators such as histamine [8, 9]. Histamine leads to increase of vascular permeability and smooth muscle contraction that may develop into fatal clinical systemic condition, e.g. anaphylaxis [10].

Allergy is a worldwide medical problem. The prevalence of allergic reaction is estimated to be 25-30% [1 1 ]; and the rate of mainly food allergy is increasing, especially in young people. Understanding, mechanisms, and patient-specific risk factors constitute the key to improve the monitoring and treatment of individuals who have susceptibility to develop allergic reaction. Diagnosis of allergy mainly relied on patient history, analysis of IgE-antibodies (IgE-ab) and/or skin prick test (SPT) to the allergen in question. In order to achieve a more reliable diagnosis, in vivo challenges can be performed. However, in vivo challenge tests are less specific, less sensitive and always involve a risk for the patient to develop severe allergic reaction [12, 13]. Measurement of IgE-ab in serum has been used for allergy diagnosis by detection of allergy triggers using different methods such as ImmunoCAP.

The rareness of basophils in peripheral blood has resulted in scant yields and cliff i- culty in their isolation, and has hampered the study of basophil biology. The discovery of CD203c as a specific biomarker for basophil [14, 15], has improved the investigation techniques of basophil activation using flowcytometry. An example of a flow- cytometry based technique is described in US2010/01 12628. Basophil activation test (BAT) is a method used in the clinic to measure the expression of activator markers on basophil surface such as CD63 and CD203c, which are upregulated on the surface of blood basophil after activation [18]. CD63 is a degranulation marker present in the inner surface of cytoplasmic granules of basophil, and after activation of basophil, the outer surface of the granules merge to the inner surface of cell membrane and the granules release their contents into the extracellular space and CD63 be- comes exposed on the basophil surface to be detected by flowcytometry [16, 17]. BAT has been evaluated as a method for allergy diagnosis in clinic, and there has been a general agreement between the clinical presentation (systemic reaction versus large local reaction) and the results of BAT, suggesting that the BAT is a potential biomarker of allergy [18]. However, US2010/01 12628, as well as similar techniques described in US2012083007, are silent about detection of basophil allergen threshold sensitivity (CD-sens) as well as the use of a gradient. Hence there is a need of providing a method and system for the detection of basophil allergen threshold sensitivity.

Basophil activation test can also be used in the follow-up of patients undergoing allergen immunotherapy (AIT) and treatment with anti-lgE by performing of basophil aller- gen threshold sensitivity (CD-sens), which has been shown to correlate significantly with IgE-ab measurements, skin prick test (SPT) and nasal-, bronchial-, and oral- allergen challenges [19, 20]. However, the cost and technical requirements for operation and maintenance of flow cytometry as a technique for BAT and the cumbersome preprocessing and pre-labeling of the sample before flowcytometric analysis have limited their reach for clinical application.

The need for competent cell based diagnostic tools for various diseases has led to the development of novel microfluidic separation techniques. Microfluidics offer numerous advantages including reduced sample volumes, faster sample processing, high sensitivity, low cost, and increased portability [21 , 22]. To date, many microflu- idic cell isolation technologies have been developed employing immunoaffinity [23], size [24], and electrical properties for separation [25]. One example of microfluidics is described in WO2017/01 1819 which relates to a method of diagnosing urinary, gastric and pulmonary infections by detecting bacterial cells by using microdroplets based microfluidics. Among these techniques, immunoaffinity provides a more spe- cific method for cell enrichment, where antibodies against the cells of interest are immobilized onto the microfluidic surface for cell capture followed by optical detection [21 , 22]. While affinity-capture has been successfully used to capture leukocyte sub- populations such as neutrophils [26] and lymphocytes [27] directly from whole blood, the specific capture of activated basophils by using immobilized anti-CD203c anti- bodies together with anti CD63-antibodies has previously not been reported in a microfluidics based system. The FCERI signaling pathway, which is responsible for triggering allergic reactions, has been studied in a continuous flow microfluidic platform, using cultured RBL-2H3 cells [28]. The activation of basophil cell lines (KU-812 cells) was recently investigated using an integrated centrifugal microfluidic platform to screen agents that can block allergic activities [29, 30]. However, basophil cell line (KU-812) is a human leukemia cell line. Cancer cells undergo a variety of biological and morphological changes than normal cells, this will interfere with the cell function and regulation of surface marker expression either the effector and degranulation detection markers [31 ]. In the basophil activation test, typically cells are activated through FcsRI recep- tor, the expression of FcsRI dependent on the amount of IgE in the blood, absence of IgE around basophil cells leads to down regulation of FcsRI on the cell surface and suppress the activation of basophils [32, 35].

In order to overcome the above indicated disadvantages of the prior art techniques, the present invention, involving microfluidics based immunoaffinity approach that rap- idly isolate basophils direct from patient's blood to diagnose allergy, was developed. The microfluidic device captures CD203c positive cells (basophils) in single step directly from whole blood without pre-labeling and preprocessing of the sample. To validate the method, basophil activation test after capturing the cells using anti-FceRI antibody followed by detection of basophil degranulation marker CD63 using fluores- cent microscopy was performed and compare the results with an established with flow cytometric method.

OBJECT OF THE INVENTION

The object of the present invention is to provide a method and system for diagnosing allergy.

A further object of the invention is to provide a method and system for measuring basophils response.

A further object of the invention is to provide a method and system for measuring basophils response in whole blood. A further object of the invention is to provide a microfluidic method and system for measuring basophils response. A further object of the invention is to provide a microfluidic method and system for basophil activation test (imBAT).

A further object of the invention is to provide a microfluidic immunoaffinity method and system for basophil activation test (imiBAT). The object of the present invention is to provide a method and system for diagnosing allergy which is fast and/or cheap.

A further object of the invention is to provide a method and system for detection of basophil allergen threshold sensitivity (CD-sens).

A further object of the invention is to provide a method and system for accurately measuring and monitoring the patient's actual allergen sensitivity.

A further object of the invention is to measure the treatment efficacy by following changes in allergen sensitivity during allergen-specific immunotherapy (ASIT) to allergens as well as anti-lgE (e.g. omalizumab) treatment.

SUMMARY OF THE INVENTION

The objects of the invention are attained by the first, second, third and fourth aspects of the invention. More importantly, the complex set of problems and disadvantages associated with prior art techniques are solved by said first, second, third and fourth aspects of the invention. In a first aspect of the invention, there is provided a microfluidic method for detection of basophil activation comprising the steps of: a. Providing a microfluidic device,

b. Modifying the surface of the microfluidic device,

c. Immobilizing a primary anti-CD203c antibody or fragment thereof on the sur- face of the microfluidic device,

d. Passing a sample through the microfluidic device, wherein the primary anti- CD203c antibody or fragment thereof which is immobilized on the surface of the microfluidic device binds CD203c surface markers of basophils and thereby captures basophils from the sample, wherein said basophils are either activated or not activated, e. If said basophils are not activated, activating the captured basophils on the mi- crofluidic device by using at least one activation composition comprising an activation molecule,

f. Applying an anti-CD63 antibody or fragment thereof for binding to the CD63 degranulation markers on the activated basophils, and

g. Providing a device for detecting the expression of CD203c surface markers and/or CD63 degranulation markers.

In one embodiment, said method for detection of basophil activation is a method of detection of basophil allergen threshold sensitivity (CD-sens).

In one embodiment, said activation molecules is at least one allergen, wherein the basophils are activated by said activation composition comprising a gradient of at least one allergen, wherein said gradient provides at least two concentrations of allergen, and wherein the concentrations of allergens are prepared outside said microflu- idic device or in said microfluidic device,

In one embodiment, the percentage of activated cells is fitted to a curve versus allergen concentration, wherein the eliciting concentration at which 25-75% of basophils respond, referred to as EC25-EC75, is determined, and wherein basophil allergen threshold sensitivity is calculated by inversion of EC25-EC75 and multiplication by 100.

In one embodiment, the percentage of activated cells is fitted to a curve versus allergen concentration, wherein the eliciting concentration at which 50% of basophils respond, referred to as EC50, is determined, and wherein basophil allergen threshold sensitivity (CD-sens) is calculated by inversion of EC50 and multiplication by 100. In one embodiment, the allergen gradient generation is provided by linear allergen gradient generation or none-linear allergen gradient generation.

In one embodiment, the allergen gradient generation is provided by a flow-based gradient generator or a diffusion-based gradient generator, wherein the temperature is lower than 37 °C when a diffusion-based gradient generator is used. In one embodiment, the concentration gradient is more than one concentration which is generated below 35 °C and then the temperature is raised to activate the basophils.

In one embodiment, the microfluidic device comprises a polymer surface, preferably PDMS surface, more preferably PDMS-glass surface or PDMS-plastic surface.

In one embodiment, wherein the modifying of the surface of the microfluidic device comprises the step of applying molecules having a silane group on the surface of the microfluidic device, more preferably the silane groups are organosilane groups.

In one embodiment, the modifying of the surface of the microfluidic device comprises the steps of (i) incubating the surface of the microfluidic device with 3-mercaptopropyl trimethoxysilane, and (ii) addition of 4-Maleimidobutyric acid N-hydroxysuccinimide ester to said surface after optional washing of said surface.

In one embodiment, the modifying of the surface of the microfluidic device comprises the step of applying a layer-by-layer (LbL) coating on the surface of the microfluidic device, preferably the layer-by layer coating comprises at least one layer of poly- cation and at least one layer of a polyanion, wherein said polycation is preferably selected from Poly(allylamine)[PAA], Poly(allylaminehydrochloride)[PAH], Polyani- line[PA], Poly(ethylenimine)[PEI] Poly(L-lysine hydrobromide)[PL], Poly(dimethyla- mino) ethylmethacrylate [PMA], chitosan, Diethylaminoethyl dextran[DEAED] and proteins such as peroxidase and horse hemoglobin, and wherein said polyanion is preferably selected from Poly(styrene sulfonate)[PSS], Poly(acrylic acid)[PAA] and biopolymers such as alginate, cellulose and hyaluronic acid[HA] or derivatives of as alginate, cellulose and hyaluronic acid[HA].

In one embodiment, wherein the layer-by-layer (LbL) coating comprises a first layer of PAH, a second layer of NFC, a third layer of PEI and a fourth layer of NFC.

In one embodiment, the modifying of the surface of the microfluidic device comprises the step of applying recombinant spider silk coating on the surface of the microfluidic device, wherein the recombinant modified silk preferably comprises Z-4RepCT domain for antibody binding. In one embodiment, a molecule having an affinity for biotin is added to the modified surface of the microfluidic device, wherein the molecule having an affinity for biotin is covalently or non-covalently linked to the modified surface.

In one embodiment, the molecule having an affinity for biotin is selected from avidin, streptavidin and/or neutravidin, or derivatives of avidin, streptavidin and/or neu- travidin.

In one embodiment, the anti-CD203c antibody is biotinylated.

In one embodiment, the sample is a blood sample collected from a subject.

In one embodiment, the basophils are activated by an activation composition com- prising anti-FcsRI antibody, preferably the activation is at 35-42 °C, more preferably 36-38, most preferably about 37 °C.

In one embodiment, the anti-FcsRI is preferably used as a positive control, most preferably a physiologically solution (such as the solution which the allergen is dissolved in) is used a negative control. In one embodiment, the basophils are activated by an activation composition comprising at least one allergen, preferably selected from food allergen, a peanut allergen, a cashew allergen, an apple allergen, a milk allergen, an environmental allergen, a cockroach allergen, a tree pollen allergen, a grass allergen, a mold allergen, a hay allergen, and a drug allergen, preferably the activation is at 35-42 °C, more pref- erably 36-38, most preferably about 37 °C.

In one embodiment, the basophils are activated by an activation composition comprising a gradient of anti-FcsRI or at least allergen, wherein the captured basophils are activated with either (i) lower and lower doses of anti-FcsRI antibody or at least allergen, or (ii) higher and higher doses of anti-FcsRI antibody or at least allergen. In one embodiment, the device for detecting the expression of CD203c surface markers and/or CD63 degranulation markers is a microscope, preferably a fluorescence microscope.

In one embodiment, the anti-CD63 antibody or fragment thereof is a fluorescently labelled anti-CD63 antibody or fragment thereof. In one embodiment, the primary anti-CD203c antibody or fragment thereof is conjugated with a secondary antibody or fragment thereof, wherein the secondary antibody or fragment thereof is preferably fluorescently labelled.

In one embodiment, the primary anti-CD203c antibody or fragment thereof is a fluo- rescently labelled anti-CD203c antibody or fragment thereof.

In one embodiment, an antibody fragment is selected from a Fab fragment, F(ab')2 fragment, single chain Fv fragment and monovalent IgG.

In one embodiment, the sample is passed through the microfluidic device with a flow rate of 0.5-1000 μΙ min-1 , preferably 1 -20 μΙ min-1 , more preferably 3-10 μΙ min-1 . In one embodiment, the sample is not subjected to pre-labelling and/or pre-processing before being applied to the microfluidic device.

In a second aspect of the invention, there is provided a microfluidic system for detection of basophil activation comprising, a. A microfluidic device having a modified surface,

b. A primary anti-CD203c antibody or fragment thereof configured for being immobilized on the modified surface of the microfluidic device,

c. A sample for passing through the microfluidic device, wherein the basophils in said sample are configured for being activated after that the anti-CD203c antibody or fragment thereof which is immobilized on the modified surface of the microfluidic device has bound the CD203c surface markers of basophils, d. At least one activation composition comprising of an activation molecule configured for activating basophils which are captured on the microfluidic device, e. An anti-CD63 antibody or fragment thereof configured for binding to the CD63 degranulation marker on the activated basophils,

f. A device for detecting the expression of CD203c surface marker and/or CD63 degranulation marker.

In one embodiment, said system for detection of basophil activation is a system of detection of basophil allergen threshold sensitivity (CD-sens). In one embodiment, said activation composition comprises a gradient of anti-FcsRI and/or a gradient of at least one allergen.

In one embodiment, said gradient comprises at least two concentrations of allergen, and wherein the concentrations of allergens are configured to be prepared outside said microfluidic device or in said microfluidic device.

In one embodiment, the modified surface of the microfluidic device comprises molecules having silane groups, more preferably the silane groups are an organosilane group.

In one embodiment, the surface of the microfluidic device comprises the reaction product of 3-mercaptopropyl trimethoxysilane and 4-Maleimidobutyric acid N-hy- droxysuccinimide ester.

In one embodiment, wherein the modified surface of the microfluidic device com- prises a layer-by-layer (LbL) coating on the surface of the microfluidic device, preferably the layer-by layer coating comprises at least one layer of polycation and at least one layer of a polyanion, wherein said polycation is preferably selected from Poly(al- lylamine)[PAA], Poly(allylaminehydrochloride)[PAH], Polyaniline[PA], Poly(ethyl- enimine)[PEI] Poly(L-lysine hydrobromide)[PL], Poly(dimethylamino) ethylmethacry- late [PMA], chitosan, Diethylaminoethyl dextran[DEAED] and proteins such as peroxidase and horse hemoglobin, and wherein said polyanion is preferably selected from Poly(styrene sulfonate)[PSS], Poly(acrylic acid)[PAA] and biopolymers such as alginate, cellulose and hyaluronic acid[HA] or derivatives of as alginate, cellulose and hyaluronic acid[HA]. In one embodiment, the layer-by-layer (LbL) coating comprises a first layer of PAH, a second layer of NFC, a third layer of PEI and a fourth layer of NFC.

In one embodiment, the modified surface of the microfluidic device comprises recombinant spider silk coating, wherein the recombinant modified silk preferably comprises Z-4RepCT domain for antibody binding. In one embodiment, the modified surface further comprises a molecule having an affinity for biotin.

In one embodiment, the anti-CD203c antibody or fragment thereof is biotinylated.

In one embodiment, the activation composition comprises anti-FcsRI antibody.

In one embodiment, the activation composition comprises at least one allergen, preferably selected from food allergen, a peanut allergen, a cashew allergen, an apple allergen, a milk allergen, an environmental allergen, a cockroach allergen, a tree pollen allergen, a grass allergen, a mold allergen, a hay allergen, and a drug allergen.

In one embodiment, the activation composition comprises a gradient of anti-FcsRI antibody or at least one allergen having either (i) lower and lower doses of anti-FcsRI antibody or at least allergen, or (ii) higher and higher doses of anti-FcsRI antibody or at least allergen.

In one embodiment, the device for detecting is a microscope, preferably a fluorescence microscope.

In one embodiment, the anti-CD63 antibody or fragment thereof is a fluorescently labelled anti-CD63 antibody or fragment thereof. In one embodiment, the primary anti-CD203c antibody or fragment thereof has been conjugated with a secondary antibody or fragment thereof, wherein the secondary antibody or fragment thereof is preferably fluorescently labelled.

In one embodiment, the primary anti-CD203c antibody or fragment thereof is a fluorescently labelled anti-CD203c antibody or fragment thereof. In one embodiment, an antibody fragment is selected from Fab fragment, F(ab')2 fragment, single chain Fv fragment and monovalent IgG.

In one embodiment, the microfluidic device is configured to have a flow rate of 0.5- 1000 μΙ min-1 , preferably 1 -20 μΙ min-1 , more preferably 3-10 μΙ min-1 . In one embodiment, the sample has not been subjected to pre-labelling and/or preprocessing before being applied to the microfluidic device.

In a third aspect of the invention, there is provided a use of the method according to the first aspect of the invention or system according to the second aspect of the invention for detecting and quantifying basophil activation.

In one embodiment, the use is for basophil activation test (BAT) or basophil allergen threshold sensitivity (CD-sens).

In one embodiment, the use is for determining the subject's susceptibility to an aller- gic reaction.

In a fourth aspect of the invention, there is provided a kit of parts for detection of basophil activation comprising: a primary anti-CD203c antibody or fragment thereof, microfluidic device, compounds for modifying the surface of the microfluidic device, acti- vation composition comprising activation molecules, and anti-CD63 antibody or fragment thereof.

In one embodiment, said kit for detection of basophil activation is a kit for detection of basophil allergen threshold sensitivity (CD-sens).

In one embodiment, said activation composition comprises a gradient of anti-FcsRI and/or a gradient of at least one allergen.

In one embodiment, the microfluidic device has a polymer surface, preferably PDMS surface, more preferably PDMS-glass surface or PDMS-plastic surface.

In one embodiment, the compounds for modifying the surface of the microfluidic device comprise molecules having silane groups, more preferably the silane groups are organosilane groups.

In one embodiment, the compounds for modifying the surface of the microfluidic device are 3-mercaptopropyl trimethoxysilane and 4-Maleimidobutyric acid N-hydroxy- succinimide ester, wherein the surface of the microfluidic device is configured to be modified by (i) incubating the surface of the microfluidic device with 3-mercaptopropyl trimethoxysilane, and then (ii) adding of 4-Maleimidobutyric acid N-hydroxysuccin- imide ester to said sur-face after optional washing of said surface.

In one embodiment, the compounds for modifying the surface of the microfluidic device is layer-by-layer (LbL) coating compositions, wherein the layer-by layer coating compositions preferably comprises at least one composition comprising polycation and at least one composition comprising a polyanion, wherein said polycation is preferably selected from Poly(allylamine)[PAA], Poly(allylaminehydrochloride)[PAH], Pol- yaniline[PA], Poly(ethylenimine)[PEI] Poly(L-lysine hydrobromide)[PL], Poly(dimethyl- amino) ethylmethacrylate [PMA], chitosan, Diethylaminoethyl dextran[DEAED] and proteins such as peroxidase and horse hemoglobin, and wherein said polyanion is preferably selected from Poly(styrene sulfonate)[PSS], Poly(acrylic acid)[PAA] and biopolymers such as alginate, cellulose and hyaluronic acid[HA] or derivatives of as alginate, cellulose and hyaluronic acid[HA].

In one embodiment, the layer-by-layer (LbL) coating compositions are PAH, NFC and PEI, wherein the layer-by-layer (LbL) coating is configured to comprises a first layer of PAH, a second layer of NFC, a third layer of PEI and a fourth layer of NFC

In one embodiment, the coating composition comprises recombinant spider silk coating composition, wherein the recombinant modified silk preferably comprises Z- 4RepCT domain for antibody binding. In one embodiment, the compounds for modifying the surface of the microfluidic device further comprises a molecule having an affinity for biotin.

In one embodiment, the molecule having an affinity for biotin is selected from avidin, streptavidin and/or neutravidin, or derivatives of avidin, streptavidin and/or neu- travidin. In one embodiment, the anti-CD203c antibody or fragment thereof is biotinylated.

In one embodiment, the activation composition comprises anti-FcsRI antibody.

In one embodiment, the activation composition comprises at least one allergen, preferably selected from food allergen, a peanut allergen, a cashew allergen, an apple allergen, a milk allergen, an environmental allergen, a cockroach allergen, a tree pol- len allergen, a grass allergen, a mold allergen, a hay allergen, and a drug allergen. In one embodiment, the anti-CD63 antibody or fragment thereof is a fluorescently labelled anti-CD63 antibody or fragment thereof.

In one embodiment, the primary anti-CD203c antibody or fragment thereof is a fluorescently labelled anti-CD203c antibody or fragment thereof. In one embodiment, the kit of parts further comprises a secondary antibody or fragment thereof for conjugating the primary anti-CD203c or fragment thereof, wherein the secondary antibody or fragment thereof is preferably fluorescently labelled.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 . Schematic illustration of the surface functionality of the microfluidic device and activation of captured basophil on chip, (a) The activation of captured basophils after the cross linking of anti-FcsRI and IgE, mimicking allergen cross linking of IgE antibodies, on the surface of basophil, and the release of mediators from basophil granules in parallel to up regulation of CD203c and CD63 on the surface of basophils, (b) Illustrates the fluorescent signal on the surface of basophil cell line after activation (on glass slide). The green signal shows CD203c and the red signal show CD63. Scale bar: 20 μιη. Figure 2. Microfluidic setup and device characterization, (a) The setup consists of a syringe pump connected to the microfluidic device. The enlarged box shows the dimensions of the 25 mm long and 50 μιη x 4 mm (height x width) device with a total internal volume of 5 μΙ_. (b) Cell capture as a function of shear stress (flow rate). The bars representing the number of captured basophils from basophil cell line (KU812) distributed in each 5 mm of chip at different flow rates. The highest number of captured cells was at 3 μΙ min-1 , correspondence to shear stress 0.026 dyn cm-2. (c) Fluorescently stained captured basophils in the functionalized chip. Scale bar (50 μηπ).

Figure 3. Basophil capture from whole blood, (a) Cell capture yield at different flow rates. 72-65% of the basophil cells can be isolated from whole blood using a flow rate of 3 μΙ min-1 . The yield quickly drops to less than 55% out of this range. Each data point was repeated in at least 2 devices. The error bars represent standard deviations in measurements within each experiment, (b) The purity of basophils captured, calculated by the ratio of number of captured basophil to total number of leukocyte, in the chip was 40%. The majority of contaminating cells are non-specific bound, indicating that the antibody is specific to the basophil cells.

Figure 4. Basophil expression of CD203c in healthy and allergic patients, (a) Flow cytometry analysis of CD203c MFI in activated basophils compared with negative control. Scatter plots representing the range with whiskers and a middle line as the me- dian n=8, P value 0.0002. (b) CD203c expression of activated basophils corn-pared with non-activated cells in chip P=0.02.

Figure 5. Basophil expression of CD63 in microfluidic chip in blood from healthy and allergic individuals, (a) Percentage of CD63 expression of activated and non- activated basophils in microfluidic chip compared to flow cytometry analysis n=8. (b) His- tograms show CD63 MFI in activated basophils (red line) compared with non-activated basophils (blue line), (c) The fluorescent staining of activated basophil captured in chip. Scale bar (10 μιη), green signal represents CD203c Alexa488, blue signal represents nucleus staining Hoechst stain and the red signal represents CD63 Alexa647. Figure 6. CD63 expression in activated basophils comparing allergic patients with healthy controls, (a) Flow cytometric analysis of CD63 expression of anti-FcsRI activated basophils was significantly higher in allergic patients as compared to healthy controls to p=0.03., scatter plots representing the range with whiskers and a middle line as the median, (b) Significantly higher CD63 expression of anti-FcsRI activated basophils captured in microfluidic chip in allergic patients as compared healthy controls p=0.04. Scatter plots representing the range with whiskers, and a line as the median.

Figure 7. 4-channel gradient microfluidic device, (a) Microfluidic setup for 4-channel gradient chip, i.e. 4-channel gradient microfluidic device, (b) The bars representing the number of captured basophils from CD203+ (pos) and CD203- (neg) basophil cell line (KU812) distributed in 4-channel gradient microfluidic device at different flow rates. Figure 8. 8-channel gradient microfluidic device, (a) Microfluidic setup for 8-channel gradient chip, i.e. 8-channel gradient microfluidic device. Blood sample and washing is done from the inlet 1 and 2, while specific allergen concentration is introduced from each of the 8 outlets. The channels are divided to avoid cross-talk between the con- centrations. The setup consists of a syringe pump connected to the 8-channel microfluidic device, (c) The bars representing the number of captured basophils from CD203+ and CD203- basophil cell line (KU812) distributed in 8-channel gradient microfluidic device at different flow rates.

Figure 9. 16-channel gradient microfluidic device. A) Microfluidic setup for 16-channel gradient chip, i.e. 8-channel gradient microfluidic device Blood sample and washing is done from the inlet, while specific allergen concentration is introduced from each of the 8 outlets. Each concentration is measured in two cell capturing chambers, (b) The bars representing the number of captured basophils from CD203+ and CD203- basophil cell line (KU812) distributed in 8-channel gradient microfluidic device at dif- ferent flow rates.

Figure 10. Comparison of activation of single chip and FACS with allergen, (a) microfluidic device results, (b) FACS results.

Figure 1 1 . a) The fraction of CD63+ basophils is plotted against log allergen concentration. Basophil reactivity is the dose (range) at which maximal response occurs. Ba- sophil sensitivity is the dose at which half of the maximal response occurs. ^*At high allergen concentrations, basophil response may be suppressed, b) A Change in sensitivity toward higher allergen concentration is the most reproducible basophil bi- omarker for clinical sensitivity to allergen to date.

Figure 12. Flow-based allergen gradient generator. Figure 13. Diffusion based allergen gradient generation.

Figure 14. Flow based, universal allergen concentration generator. DETAILED DESCRIPTION

The present invention relates to a microfluidic affinity based method and system that takes whole blood directly from the allergic patient and isolates the basophils for activation followed by image based detection of the degranulation markers. The present invention may also be referred to as a microfluidic affinity based basophil activation test (imiBAT).

Conventionally, flow cytometry has been used for detection and quantification of basophil activation markers. However, as demonstrated in the below examples, the present invention relates to a basophil activation test using a microfluidic chip which has higher efficacy than flow cytometry.

In the present invention, the IgE dependent basophil activation pathway is initiated, when the allergen cross link IgE- FceRI receptors on the basophil surface (Fig. 1 a). Activation of FceRI receptor enhances several downstream signaling events in the basophil leading to regulation of intracellular Ca+ signaling which induces cell degranulation, concomitant release of immune mediators, and up regulation of activator markers (CD63 and CD203c) on basophil surface. As be seen in Fig.1 b, basophil activation level is possible to measure using fluorescent microscopy.

The method and system of the present invention is capable of isolating CD203c positive cells direct from whole blood for in-vitro activation of the cells for allergy diagno- sis. The captured basophils are activated and then the level of basophil activation is determined by detecting the expression of CD63.

Basophil allergen threshold sensitivity (CD-sens)

Basophils and mast cells are effector cells that initiate the IgE-mediated allergic reac- tion, hence making those cells interesting targets for allergy diagnostic tests. While mast cells are predominantly located in the tissue and therefore not suitable for in vitro analysis, basophils are readily accessible in the blood [39].

There are two common measures of basophil activity: basophil reactivity [39], the number of basophils that respond to a given stimulus and basophil sensitivity [39], the allergen concentration at which half of all reactive basophils respond (Figure 1 1 a) [39].

Basophil reactivity depends on the priming state of the basophil and the cellular translation of the IgE signal within the cell [40]. Basophil sensitivity is a function of re- activity and the compound affinity of cell-bound slgE for allergen and free competing immunoglobulin [40].

Measurements of basophil sensitivity requires measurement of reactivity at least 4-8 allergen concentrations [41 ]. If basophils are stimulated with serial dilutions of the allergen, the basophil allergen threshold sensitivity, CD-sens [42], can be determined. The graded response to allergen is fitted to a curve of reactivity (i.e. percentage of activated cells) versus allergen concentration, and the eliciting concentration at which 50% of basophils respond (EC50) is determined. EC50 can be expressed as 'CD- sens' by inversion and multiplication by 100 [41 ,42].

The CD-sens method can be seen as an in vitro allergen challenge that not only tells whether the patient's basophils react to the allergen or not, but also how sensitive they are to the allergen. The test is carried out with blood in test tubes and analyzed by flow cytometry. Previous studies show that CD-sens correlate well with the clinical allergen sensitivity in the target organ e.g. SPT titration [42], nasal-, bronchial- [42,43] and food challenge with plant allergens e.g. peanut, hazelnut and to wheat [44,45].

Previous studies indicate that CD-sens is a method that without side effects can be used to accurately measure and monitor the patient's actual allergen sensitivity. CD- sens is also used to measure the treatment efficacy by following changes in allergen sensitivity (Figure 1 1 b) during allergen-specific immunotherapy (ASIT) to birch, timo- thy [46-48] and OIT (oral immunotherapy) to peanut [49] as well as anti-lgE (omali- zumab) treatment [42, 50-52].

Gradient chip for CD-sens measurement

As already disclosed above, measurements of basophil sensitivity require measure- ment of reactivity at 4-8 allergen concentrations. Currently, the test is carried out in test tubes one concentration at a time and analyzed by flow cytometry. The present invention is related to the use of microfluidic device to capture the cells (basophils) from while blood and then expose these cells to a gradient of allergen concentrations on-chip for determining the allergen sensitivity. The allergen gradient generation can be either linear or non-linear. The present invention uses two types gradient forming principles: flow-based gradient generators and diffusion-based gradient generators. The invention provides first the specific capture of target cells using antibodies coated on the surface of the chip. This is then followed by cell activation using different concentrations of allergen. Briefly, the microfluidic device, coated with anti-CD203c, first capture basophils direct from whole blood. Following, the captured basophils are activated by allergen concentration and the expression of CD63 expression (degranulation marker) is measured for determination of the sensitivity.

There have been a number of microfluidic based gradient generating devices [53, 54, 55]. Majority of gradient devices are flow based methods and rely on diffusion for mixing adjacent streams of different concentrations, and exploit the small dimensions in microfluidic channels to deliver spatial and temporal concentrating of chemicals. The devices are based on passive mixing, such that no external forces are required to continuously mix two adjacent flows in the laminar flows. The present invention provides different types of gradient generators methods (flow-based and diffusion based) to expose the cells captured on the chip surface (figures 8a, 9a, 12, 13 and 14). The cells are activated at 37 °C. Hence the fluidic priming can effectively be done at lower temperature. This allow for generating different type concentration profiles (linear gradient and none-linear such as power, exponential etc.) prior heating to initiate the activation and degranulation of basophils. Experimental procedure:

1 . The microfluidic devices are coated with antibodies on the surface

2. Whole blood is flown over the surface, upon where the target cells (Basophils) are captured

3. A washing buffer is flown over the surface to remove non-captured cells from the surface

4. The desired allergen concentration gradient is generated, and the cells are exposed to the gradient 5. The temperature is increased to 37 °C and incubation takes place

6. A washing buffer is introduced to remove the allergen

7. Detection of activation markers (e.g. CD63 on the cell surface)

Step 1 ,2,5 & 6 are common to all allergen gradient generating devices. Step 3 differ depending on the devices used to generate the allergen gradient.

Different types of gradient generating devices are illustrated in figures 8a, 9a, 12, 13 and 14 and described briefly below.

• Allergen concentration generated off-chip:

Figures 9a and 8a show devices for analysis of 8 different allergen concentrations, where each concentration is prepared off-chip and introduces from one of the 8 outlets. In figure 9a (which is a manual allergen concentration generation device), blood sample and washing is done from the inlet, while specific allergen concentration is introduced from each of the 8 outlets. Each concentration is measured in two cell capturing chambers. In figure 8a (which is also a manual allergen concentration generation device), blood sample and washing is done from the inlet 1 and 2, while specific allergen concentration is introduced from each of the 8 outlets. The channels are divided to avoid cross-talk between the concentrations.

• Linear gradient generation:

Figure 12 and 13 show devices for on-chip linear gradient generation for flow- based allergen gradient generator and diffusion based gradient generation, respectively.

In figure 12, blood sample is introduced from inlet (1 ) and out through outlets (2 and 3). During this time, the gradient generating inlets (4, 5) are closed. For gradient generation, the desired allergen is introduced through one of the gradient generating inlets (either 4 or 5). The outlets (2,3) are initially opened for priming the gradient generation, and then closed so that the cells are exposed to the gradient. In figure 13, the sample and washing is done through inlet 1 and outlet 1 . Flowing allergen and buffer through inlet 2 and 3 respectively generates the gradient across the cell capture chamber. Molecules diffuse from the side channels into the cell capture chamber. In this constellation, the temperature is kept below 37 °C until a linear gradient is achieved.

• Universal gradient generation:

By introducing flow dividers inside a channel, it is possible to generate different type of gradient profile. Figures 14a and 14b show devices with flow divid- ers (see the zoomed box under figure 14a) to generate a specific concentration profile. Blood sample is introduced from inlet (1 ) and out through outlets (2 and 3). During this time, the gradient generating inlets (4, 5) are closed. For gradient generation, the desired allergen is introduced through one of the gradient generating inlets (either 4 or 5). The outlets (2,3) are initially opened for priming the gradient generation, and then closed so that the cells are exposed to the gradient. In figure 14a, the channel width is 4 mm while the channel width is 8 mm in figure 14b

EXAMPLES The results disclosed in the examples show the efficacy of our system in measuring the level of activation by quantifying the expression of CD63 in the microfluidic chip. The effortlessness of the method and system of the present invention provides a new and useful method for measurement of basophils activation level in micro-fluidic chip, which facilitate diagnosis and monitoring of allergic patients.

MATERIALS AND METHODS A. Device microfabrication

Two types of microfluidic devices have been used in the present invention. The first, the Hele-Shaw chip, was designed based on the equations derived by Usami et al [33]. The chip has a channel designed to have a linear reduction of shear stress along the chamber and was used to optimize the shear stress for maximum cell capturing. The fabricated flow chambers were 50 μιη height, width of 5 mm that increase along the channel length and total length 50 mm, respectively.

The second chip design has straight channel, where the width, height and length of the channel were 4 mm, 50μιη and 25 mm respectively.

The microfluidic devices were fabricated in polydimethylsiloxane (PDMS) using standard soft lithography techniques [34]. Briefly, channel replicas were produced us- ing a negative photoresist SU-8 (MicroChem) onto the silicon wafer using standard microelectromechanical systems (MEMs) technology. The height of the SU-8 pattern on the master was measured using a surface profilometer. The devices were produced by casting PDMS onto the patterned silicon wafers. The elastomeric PDMS (Dow Corning) was mixed with a cross-linker with a ratio of 10:1 (wt/wt), and poured onto the master used as a mold, degassed and cured at 65 °C for 6 hours. The curved PDMS with replicated channels was peeled off from the silicon wafer and channel holes were punched with a Harris Uni-CoreTM, Tip ID 0.75 mm. The PDMS replica was bonded to a glass slide (70mm ^χ 30 mm) after brief oxygen plasma treatment. Access tubing (Tygon; Saint-Globain PPL corp) of slightly larger diameter than the access holes was press-fitted into the holes.

B. Surface modification

3-mercaptopropyl trimethoxysilane (Sigma Aldrich, Germany) was infused and incubated for 1 hour. This was followed by washing with ethanol and addition of 4-Malei- midobutyric acid N-hydroxysuccinimide ester (GMBS) (Sigma Aldrich, Germany), a cross linking agent for 30 mins. The devices were washed again first with ethanol and then with PBS and Neutravidin (Sigma Aldrich, Germany) was added and the devices were stored at 4°C. Before experiments, the devices were incubated with bioti- nylated anti-CD203c (MACS, Miltenyi Biotech, Germany) and incubated overnight.

C. Blood sampling Venous blood samples from healthy donors (Blood Center, Stockholm, Sweden) and allergic patients (n=8) (Sachs's Children Hospital, Stockholm, Sweden) was collected in 10 ml Na+ Heparin vacutainer tubes (Vacutainer, Becton Dickinson, UK) and analyzed within 3 hours. The study was approved by the regional ethics committee in Stock-holm, Sweden Dnr. 2014/1630-31 /4.

D. Cell line KU812 culture

The KU812 cell line (basophil cell line) samples were cultured in RPMI1640 media containing 10% FBS (Fetal bovine serum) and 0.2 % non-essential amino acids (Sigma Aldrich, Germany). Cells were cultured in a CO2 incubator and the medium was renewed every two to three days through standard cell culture practice.

E. Cell capture in microfluidic chip

KU812 basophil cells were washed and resuspended in 1 xPBS for processing into the chip. The devices were washed by 1 %BSA (bovine serum albumin) in 1 xPBS PH 7.2 at 20 μΙ min-1 to wash out the unbounded antibody. 70μΙ of sample were pumped into the Hele-shaw chip at desired shear rates (3-15) μΙ min-1 using a syringe pump (Harvard apparatus, USA). The chips were washed with 1 % BSA (w/v) at 20μΙ min-1 for 10min to remove the un-bound cells. Captured cells were stained using nuclear staining (Hoechst stain) (Sigma Aldrich, Germany), fluorescent images have taken by fluorescent microscope for each point; three measurements were made, corresponding to three 1 mm2 squares in that vicinity. The same experimental procedure was followed for cell capture using the straight channel device with flow rates (1 -20) μΙ min-1 . For whole blood experiment, the sample was processed into chip at different flow rates (3-10) μΙ min-1 , and the chips were washed by 1 % BSA at a flow rate of 20 μΙ min-1 . The captured cells from whole blood were stained using nuclear staining (Hoechst stain). In addition, CD203c which is basophil specific marker has been used to stain captured basophils in chip. Cells were fixed by 4% paraformaldehyde PFA for 10 min at RT, followed by washing of chip. Captured cells incubated with anti-

CD203c (Abeam, UK) for 1 hour at RT followed by conjugation of primary anti-body with fluorescently conjugated PE anti-mouse secondary antibody (Abeam, UK). Finally the chips were visualized by Eclipse Ti Nikon microscope, images were acquired by Zyla 5.5 sCMOS Andor camera, and images were transferred using the Mi- croManager Version 1 .4 soft-ware, plug-in and processed using Imagej software.

F. Flowcytometry analysis

The flowcytometry experiments were performed to estimate the capturing efficiency and purity of basophils from whole blood. Depletion assays were done by counting basophil (CD203c) cells in the samples collected before and after the passage of blood through the microfluidic device. Samples were incubated with CD203c-PE (Abeam, UK), for 25min at +4oC. Following lysis of red blood cells (RBCs) with 2 ml cold isotonic solution (154 imM NH4CI, 10 imM KHCO3 supplemented with 0.1 imM EDTA, pH 7.2), and samples centrifuged for 5 min at 300 g at +4 °C. Cells were washed once with PBS and re-suspended in 300μΙ cold PBS, flow count beads (Beckman Coulter, Germany) were added to calculate the absolute number of basophils and leukocytes in the outlet aliquots using flow cytometry (Navios, Beckman Coulter Inc., Hialeah, FL, USA). Data were analyzed by the Kaluza Analysis Software (Beckman Coulter Inc., USA).

G. Activation of basophils in microfluidic chip

The on-chip captured basophils were activated with anti-FcsRI antibody. Three μg/ml of anti-FcsRI were added into the chips and incubated at 37oC for 20 min in humidified chamber. Chips were washed by 1 % BSA. Cells were fixed and incubated for 30 min at RT with CD63 Alexa-647 (Abeam, UK). Finally, chips washed by 1 %BSA and imaged by fluorescent microscope, the experiments per-formed with healthy and allergic patient samples.

H. Statistical analysis

Scatter plots were prepared by GraphPad Prism 5, representing the range with whiskers and a middle line as the median. Statistical analysis was done in GraphPad Prism 5. Since the study population was not normally distributed, comparison between the groups was performed by the non-parametric Kruskal-Wallis test. Significant differences between groups were analyzed using the post hoc Mann Whitney test. A p value of <0.05 was considered significant.

EXAMPLE 1 - Microfluidic chip design and characterization

A device that could capture basophils from whole blood was designed and characterized. The surface was modified using chemistry for immobilization of CD203c antibody (basophil specific marker) to capture basophils. Using a basophil cell line (KU812), the functionality of chip sur-face to specifically capture CD203+ cells was examined.

Initially, a Hele-Shaw device [33] was used, which allows for an analysis of cell adhesion over a range of shear stresses for the cell capture and washing flow rates. The optimum shear stress for the highest capture efficiency for basophils was found to be 0.026 dyn cm-2.

The optimal shear stress using the straight channel (50 μηη χ 4 mm height and width) corresponded to 3 μΙ_/ιτπη (Fig.2a). The optimal flow rate was experimentally confirmed using the straight channel (Fig.2b).

When the shear stress is increased, the cell-capture efficiency drops. This observa- tion suggests that when target cells come into contact with the surface, cell-substrate adhesion is started. The sudden drop of cells captured at a higher flow rate indicates less time for antibody-cell contact. Once captured, the cells can withstand higher washing flow rate. In an embodiment of the invention the flow rate was 20 μΙ_/ιτπη for the washing step. The captured cell coverage is relatively uniform over the width of the channel, while there is difference in the cell capture along the length of the channel where the maximum adhesion of cells were at 10mm of chip length. The captured cells were stained with anti-CD203c fluorescent conjugated antibody to count the number of CD203c positive cells as shown in Fig.2c. Moreover, the purity and sensitivity of cell chip cap- ture were assessed by control chip (without anti-CD203c coating), where there was barley basophil captured. Based on this, the basophil capture direct from whole blood was examined.

EXAMPLE 2 - Basophils isolation from whole blood To investigate immunoaffinity capture of basophils from whole blood, 200 μΙ of whole blood was processed through the channel using syringe pump. As shown in Fig. 3a, the highest basophil capture yield was obtained for flow rate of 3 μΙ_/ιτπη, which is in agreement with the cell line based results. When the flow rate is increased to 5 μΙ_/ιτπη, the yield decreased from 64% to 49%. The optimal flow rate of 3 μΙ_/ιτπη was therefore chosen for all subsequent experiments.

The yield was analyzed by flow cytometric analysis of the blood basophil cell counts before and after flowing of blood sample through the chip channel. Following, on-chip imaging was used to fully characterize the microfluidic affinity chip capture in terms of purity and specificity of the antibody (Fig. 3b). The purity, calculated as the ratio be- tween the CD203+ cells to total leukocyte, was approx. 40%. Furthermore, it was confirmed that only non-significant number basophils were captured when blood was flown through an unmodified chip while the total number of leukocytes in control chip compared to CD203c+ chip did not differ significantly (Fig. 3b). This indicates that the binding of other leukocyte subpopulations is un-specific. Normally, for flow-cytometry based basophil activation test for allergy diagnosis, sufficient number of basophils gated from the sample to quantify CD63 expression is 200 basophils [36]. Surprisingly, the microfluidic devices of the present invention were capable of capturing more than 200 basophils without further optimization. This fact proves the capability of the device of the present invention to capture significant num- ber of basophil cells to measure the level of CD63 expression in basophils for clinical use.

The non-specific binding of other leukocytes can be further reduced by optimizing the device geometry and flow condition. Among the subpopulation, monocytes can express low level of CD63. However, the activation mechanism of the FcsRI pathway in monocytes is different and require very high concentration of stimuli to cross link FCERI and longtime incubation compared to in basophils [37]. Furthermore, since captured basophils are stained specifically with CD203c, this gives assurance to exclude other CD63 signal than CD203c + cell.

EXAMPLE 3 - Basophil expression of CD203c in healthy and allergic individuals In order to evaluate the imiBAT assay, the captured basophils were stimulated by anti-FceRI antibody to induce the degranulation of basophils. This was followed by detection of CD203c expression and CD63 (degranulation marker) using fluorescent microscopy. It was observed that the CD203c mean fluorescence intensity (MFI) in captured activated basophils is significantly higher than the non-activated basophils in healthy individuals p=0.02 (Fig. 4b), which parallel with flow cytometry analysis of CD203c MFI p=0.0002 (Fig. 4a). CD203c is a glycosylated type II transmembrane molecule, which is expressed constitutively on basophils. Moreover, the expression intensity of the CD203c is low on resting basophils, but becomes upregulated up on activation. Therefore, CD203c can be regarded as both an identification and activa- tion marker (piece-meal degranulation marker, PMD) for basophil. PMD exhibit empty or partially empty granule chambers that do not fuse with each other or with the plasma membrane. In addition, basophil contains numerous small cytoplasmic vesicles, some of which are fused to granule or plasma membranes. As partially depleted granules exhibit focal pieces or packets of lost granule particles this kinetic changes in the granules can be read out by the expression level of CD203c in basophil surface [15]. To further evaluate our device to perform imiBAT for allergy diagnosis, experiments were performed using samples from allergic patients to quantify the difference of CD203c MFI in activated and non-activated basophils compared with flow cytometry analysis. As for healthy individuals, the significant difference of CD203c ex- pression before and after activation using microfluidic chip p=0.04 (Fig. 4a-b). Together the results demonstrate the overexpression of CD203c after on-chip activation could be used as an activation marker on the microfluidic device. EXAMPLE 4 - Basophil expression of CD63 in healthy and allergic individuals

Expression of CD63 in activated captured basophils was compared with non-activated basophils analyzed in chip. It was determined that CD63% (number of CD63 positive cells from total CD203c+ cells) in (negative control) non-activated captured basophils ranges 20-25 %, comparing with 50-70% of CD63 expression in activated basophils. This parallels to activated basophils from healthy individuals and allergic patients analyzed by flowcytometry (Fig. 5a). It has been shown there are several causes likely to be responsible in vitro for a high basal value, particularly pyro-gens and endotoxins that could contaminate the materials used in the technique like plas- tic tubes or syringes. It is therefore important to work in sterile environment [36].

Further analysis of CD63 expression in basophils was conducted by measuring the CD63 MFI of basophils activated with anti-FcsRI compared to non-activated captured basophils. The difference in CD63 MFI was significantly higher in activated basophils than in non-activated captured cells (Fig. 5b), suggesting that the expression level of CD63 in activated basophils is higher than in non-activated cells. The threshold level of CD63 MFI in non-activated basophil compared to activated basophils is about 100, which can be considered as a background to measure the activation level in activated basophils.

Moreover, the difference of CD63 expression was significantly higher in allergic pa- tients compared to healthy controls (p=0.04 Fig. 6b). This result was parallel to flowcytometry analysis of CD63 expression comparing both groups (Fig. 6a P=0.03). Altogether, these data show the ability of the present invention to be used to measure the intensity of degranulation in captured basophils compared with flow-cytometry measurements. Analyses of CD63 expression on the basophil cell surface by micro- fluidic chip is a fast readout, mimicking of histamine release, from basophils. This has been shown to correlate to degranulation due to activation of basophils by allergens [38]. Degranulation of basophils has previously been studied before in centrifugal mi- crofluidic platform, where the morphological changes in intracytoplasmic granules after activation over time [30]. EXAMPLE 5

The embodiments described in Examples 1 , 2, 3, and 4 were repeated in microfluidic devices comprising 4, 8 and 16 channels. Moreover, the basophils were activated by an activation composition comprising a gradient of anti-FcsRI antibody. The microflu- idic devices as well as some of the results are disclosed in Figures 7-9.

EXAMPLE 6

Although the embodiments in Examples 1 -6 relate to the use of CD63 and CD203c antibodies, the present invention may also be conducted with an antibody fragment selected from a Fab fragment, F(ab')2 fragment and single chain Fv fragment. Moreover, a monovalent IgG may also be used.

EXAMPLE 7

Although the embodiments in Examples 1 -6 relate to the use of anti-FcsRI antibody, the present invention may also be conducted with at least one allergen. The allergen is preferably selected from food allergen, a peanut allergen, a cashew allergen, an apple allergen, a milk allergen, an environmental allergen, a cockroach allergen, a tree pollen allergen, a grass allergen, a mold allergen, a hay allergen, and a drug allergen.

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Claims

1 . Microfluidic method for detection of basophil allergen threshold sensitivity (CD- sens) comprising the steps of:

a. Providing a microfluidic device, wherein the microfluidic device preferably has at least one channel, more preferably 4, 8 or 16 channels, b. Modifying the surface of the microfluidic device,

c. Immobilizing a primary anti-CD203c antibody or fragment thereof on the surface of the microfluidic device,

d. Passing a sample through the microfluidic device, wherein the primary anti-CD203c antibody or fragment thereof which is immobilized on the surface of the microfluidic device binds CD203c surface markers of basophils and thereby captures basophils from the sample, e. activating the captured basophils on the microfluidic device by using at least one activation composition comprising an activation molecule, the activation molecules is at least one allergen, wherein the basophils are activated by an activation composition comprising a gradient of at least one allergen, wherein said gradient provides at least two concentrations of allergen, and wherein the concentrations of allergens is prepared outside said microfluidic device or in said microfluidic device, f. Applying an anti-CD63 antibody or fragment thereof for binding to the CD63 degranulation markers on the activated basophils, and g. Providing a device for detecting the expression of CD203c surface

markers and/or CD63 degranulation markers.

2. Method according to the previous claim, wherein the percentage of activated cells is fitted to a curve versus allergen concentration, wherein the eliciting concentration at which 25-75% of basophils respond, referred to as EC25- EC75, is determined, and wherein basophil allergen threshold sensitivity is calculated by inversion of EC25-EC75 and multiplication by 100.

3. Method according to anyone of the previous claims, wherein the percentage of activated cells is fitted to a curve versus allergen concentration, wherein the eliciting concentration at which 50% of basophils respond, referred to as EC50, is determined, and wherein basophil allergen threshold sensitivity (CD- sens) is calculated by inversion of EC50 and multiplication by 100.

4. Method according to any one of the previous claims, wherein the allergen gradient generation is provided by linear allergen gradient generation or none-linear allergen gradient generation.

5. Method according to any one of the previous claims, wherein the allergen gradient generation is provided by a flow-based gradient generator or a diffusion- based gradient generator, wherein the temperature is lower than 37 °C when a diffusion-based gradient generator is used.

6. Method according to any one of the previous claims, wherein the concentration gradient is more than one concentration which is generated below 35 °C and then the temperature is raised to activate the basophils. .

7. Method according to any one of the previous claims, wherein the microfluidic device comprises a polymer surface, preferably PDMS surface, more preferably PDMS-glass surface or PDMS-plastic surface.

8. Method according to any one of the previous claims, wherein the modifying of the surface of the microfluidic device comprises the step of applying molecules having a silane group on the surface of the microfluidic device, more preferably the silane groups are organosilane groups.

9. Method according to the previous claim, wherein the modifying of the surface of the microfluidic device comprises the steps of (i) incubating the surface of the microfluidic device with 3-mercaptopropyl trimethoxysilane, and (ii) addition of 4-Maleimidobutyric acid N-hydroxysuccinimide ester to said surface after optional washing of said surface.

10. Method according to any one of the previous claims, wherein the modifying of the surface of the microfluidic device comprises the step of applying a layer- by-layer (LbL) coating on the surface of the microfluidic device, preferably the layer-by layer coating comprises at least one layer of polycation and at least one layer of a polyanion, wherein said polycation is preferably selected from Poly(allylamine)[PAA], Poly(allylaminehydrochloride)[PAH], Polyaniline[PA], Poly(ethylenimine)[PEI] Poly(L-lysine hydrobromide)[PL], Poly(dimethylamino) ethylmethacrylate [PMA], chitosan, Diethylaminoethyl dextran[DEAED] and proteins such as peroxidase and horse hemoglobin, and wherein said polyanion is preferably selected from Poly(styrene sulfonate)[PSS], Poly(acrylic acid)[PAA] and biopolymers such as alginate, cellulose and hyaluronic acid[HA] or derivatives of as alginate, cellulose and hyaluronic acid[HA].

1 1 . Method according to the previous claim, wherein the layer-by-layer (LbL) coating comprises a first layer of PAH, a second layer of NFC, a third layer of PEI and a fourth layer of NFC.

12. Method according to any one of the previous claims, wherein the modifying of the surface of the microfluidic device comprises the step of applying recombinant spider silk coating on the surface of the microfluidic device, wherein the recombinant modified silk preferably comprises Z-4RepCT domain for antibody binding.

13. Method according to any one of the previous claims, wherein a molecule having an affinity for biotin is added to the modified surface of the microfluidic device, wherein the molecule having an affinity for biotin is covalently or non-co- valently linked to the modified surface.

14. Method according to the previous claim, wherein the molecule having an affinity for biotin is selected from avidin, streptavidin and/or neutravidin, or derivatives of avidin, streptavidin and/or neutravidin.

15. Method according to any one of the previous claims wherein the anti-CD203c antibody is biotinylated.

16. Method according to the previous claim, wherein the sample is a blood sample collected from a subject.

17. Method according to any one of the previous claims, wherein the basophils are activated by an activation composition comprising anti-FcsRI activated at 35-42 °C, preferably at 36-38 °C, more preferably about 37 °C, wherein the anti-FcsRI is preferably used as a positive control, most preferably a physiologically solution (such as the solution which anti-FcsRI is dissolved in) is used a negative control.

18. Method according to any one of the previous claims, wherein the basophils being activated by an activation composition comprising at least one allergen is selected from food allergen, a peanut allergen, a cashew allergen, an apple allergen, a milk allergen, an environmental allergen, a cockroach allergen, a tree pollen allergen, a grass allergen, a mold allergen, a hay allergen, and a drug allergen, and preferably the activation is at 35-42 °C, more preferably 36- 38 °C, most preferably about 37 °C.

19. Method according to any one of the previous claims, wherein the basophils are activated by an activation composition comprising a gradient of anti-FcsRI antibody and/or a gradient of at least one allergen, wherein the captured basophils are activated with either (i) lower and lower doses of anti-FcsRI antibody or at least one allergen, or (ii) higher and higher doses of anti-FcsRI antibody or at least one allergen.

20. Method according to any one of the previous claims, wherein the device for detecting the expression of CD203c surface markers and/or CD63 degranula- tion markers is a microscope, preferably a fluorescence microscope.

21 . Method according to any one of the previous claims, wherein the anti-CD63 antibody or fragment thereof is a fluorescently labelled anti-CD63 antibody or fragment thereof.

22. Method according to any one of the previous claims, wherein the primary anti- CD203c antibody or fragment thereof is conjugated with a secondary antibody or fragment thereof, wherein the secondary antibody or fragment thereof is preferably fluorescently labelled.

23. Method according to any one of the previous claims, wherein the primary anti- CD203c antibody or fragment thereof is a fluorescently labelled anti-CD203c antibody or fragment thereof.

24. Method according to any one of previous claims, wherein an antibody fragment is selected from a Fab fragment, F(ab')2 fragment, single chain Fv fragment and monovalent IgG.

25. Method according to any one of the previous claims, wherein the sample is passed through the microfluidic device with a flow rate of 0.5-1000 μΙ ιτπη , preferably 1 -20 μΙ min^-1 , more preferably 3-10 μΙ min^-1.

26. Method according to any one of the previous claims, wherein said sample is not subjected to pre-labelling and/or pre-processing before being applied to the microfluidic device.

27. Microfluidic system for detection of basophil allergen threshold sensitivity comprising,

a. A microfluidic device having a modified surface, wherein the microfluidic device preferably has at least one channel, more preferably 4, 8 or 16 channels,

b. A primary anti-CD203c antibody or fragment thereof configured for being immobilized on the modified surface of the microfluidic device, c. A sample for passing through the microfluidic device, wherein the basophils in said sample are configured for being activated after that the anti-CD203c antibody or fragment thereof which is immobilized on the modified surface of the microfluidic device has bound the CD203c surface markers of basophils,

d. At least one activation composition comprising an activation molecule configured for activating basophils which are captured on the microfluidic device, wherein said activation composition comprises a gradient of anti-FcsRI and/or a gradient of at least one allergen, e. An anti-CD63 antibody or fragment thereof configured for binding to the CD63 degranulation marker on the activated basophils,

28. System according to claims 27, wherein said gradient comprises at least two concentrations of allergen, and wherein the concentrations of allergens is configured to be prepared outside said microfluidic device or in said microfluidic device.

29. System according to claims 27 or 28, wherein the microfluidic device comprises a polymer surface, preferably PDMS surface, more preferably PDMS- glass surface or PDMS-plastic surface.

30. System according to any one of the previous claims 27-29, wherein the modified surface of the microfluidic device comprises molecules having silane groups, more preferably the silane groups are an organosilane group.

31 . System according to any one of the previous claims 27-30, wherein the surface of the microfluidic device comprises the reaction product of 3-mercapto- propyl trimethoxysilane and 4-Maleimidobutyric acid N-hydroxysuccinimide ester.

32. System according to any one of previous claims 27-31 , wherein the modified surface of the microfluidic device comprises a layer-by-layer (LbL) coating on the surface of the microfluidic device, preferably the layer-by layer coating comprises at least one layer of polycation and at least one layer of a polyan- ion, wherein said polycation is preferably selected from Poly(allylamine)[PAA], Poly(allylaminehydrochloride)[PAH], Polyaniline[PA], Poly(ethylenimine)[PEI] Poly(L-lysine hydrobromide)[PL], Poly(dimethylamino) ethylmethacrylate

[PMA], chitosan, Diethylaminoethyl dextran[DEAED] and proteins such as peroxidase and horse hemoglobin, and wherein said polyanion is preferably se- lected from Poly(styrene sulfonate)[PSS], Poly(acrylic acid)[PAA] and biopoly- mers such as alginate, cellulose and hyaluronic acid[HA] or derivatives of as alginate, cellulose and hyaluronic acid[HA].

33. System according to the previous claim, wherein the layer-by-layer (LbL) coating comprises a first layer of PAH, a second layer of NFC, a third layer of PEI and a fourth layer of NFC.

34. System according to any one of previous claims 27-33, the modified surface of the microfluidic device comprises recombinant spider silk coating, wherein the recombinant modified silk preferably comprises Z-4RepCT domain for antibody binding.

35. System according to any one of the previous claims 27-34, wherein the modified surface further comprises a molecule having an affinity for biotin.

36. System according to the previous claim, wherein the molecule having an affinity for biotin is selected from avidin, streptavidin and/or neutravidin, or derivatives of avidin, streptavidin and/or neutravidin.

37. System according to any one of the previous claims 27-36, wherein the anti- CD203c antibody or fragment thereof is biotinylated.

38. System according to any one of the previous claims 27-37, wherein the activation composition comprises anti-FcsRI.

39. System according to any one of the previous claims 27-38, wherein the activation composition comprises at least one allergen, preferably selected from food allergen, a peanut allergen, a cashew allergen, an apple allergen, a milk allergen, an environmental allergen, a cockroach allergen, a tree pollen allergen, a grass allergen, a mold allergen, a hay allergen, and a drug allergen.

40. System according to any one of the previous claims 27-39, wherein the activation composition comprises a gradient of anti-FcsRI or a gradient of at least one allergen having either (i) lower and lower doses of anti-FcsRI or at least one allergen, or (ii) higher and higher doses of anti-FcsRI antibody or at least one allergen.

41 . System according to any one of the previous claim 27-40, wherein the device for detecting is a microscope, preferably a fluorescence microscope.

42. System according to any one of the previous claims 27-41 , wherein the anti- CD63 antibody or fragment thereof is a fluorescently labelled anti-CD63 antibody or fragment thereof.

43. System according to any one of the previous claims 27-42, wherein the primary anti-CD203c antibody or fragment thereof has been conjugated with a secondary antibody or fragment thereof, wherein the secondary antibody or fragment thereof is preferably fluorescently labelled.

44. System according to any one of the previous claims 27-43, wherein the primary anti-CD203c antibody or fragment thereof is a fluorescently labelled anti- CD203c antibody or fragment thereof.

45. System according to any one of previous claims 27-44, wherein an antibody fragment is selected from Fab fragment, F(ab')2 fragment, single chain Fv fragment and monovalent IgG.

46. System according to any one of the previous claims 27-45, wherein the micro- fluidic device is configured to have a flow rate of 0.5-1000 μΙ min i , preferably 1 -20 μΙ min^"1, more preferably 3-10 μΙ min^"1.

47. System according to any one of the previous claims 27-46, wherein said sample has not been subjected to pre-labelling and/or pre-processing before being applied to the microfluidic device.

48. Use of method or system according to any one of the previous claims 1 -47 for basophil allergen threshold sensitivity (CD-sens).

49. Use according to previous claim 48 for determining the subject's susceptibility to an allergic reaction.

50. Kit of parts for detection of basophil activation and basophil allergen threshold sensitivity comprising: a primary anti-CD203c antibody or fragment thereof, microfluidic device preferably having at least one channel, compounds for modifying the surface of the microfluidic device, at least one activation composition comprising activation molecules wherein said activation composition comprises a gradient of anti-FcsRI and/or a gradient of at least one allergen, and anti-CD63 antibody or fragment thereof.

51 . Kit of parts according to claim 50, wherein the microfluidic device has a polymer surface, preferably PDMS surface, more preferably PDMS-glass surface or PDMS-plastic surface.

52. Kit of parts according to claim 50 or 51 , wherein the compounds for modifying the surface of the microfluidic device comprise molecules having silane groups, more preferably the silane groups are organosilane groups.

53. Kit of parts according to any one of the previous claims 50-52, wherein the compounds for modifying the surface of the microfluidic device are 3-mercap- topropyl trimethoxysilane and 4-Maleimidobutyric acid N-hydroxysuccinimide ester, wherein the surface of the microfluidic device is configured to be modified by (i) incubating the surface of the microfluidic device with 3-mercaptopro- pyl trimethoxysilane, and then (ii) adding of 4-Maleimidobutyric acid N-hydroxysuccinimide ester to said sur-face after optional washing of said surface.

54. Kit of parts according to any one of the previous claims 50-53, wherein the compounds for modifying the surface of the microfluidic device is layer-by- layer (LbL) coating compositions, wherein the layer-by layer coating compositions preferably comprises at least one composition comprising polycation and at least one composition comprising a polyanion, wherein said polycation is preferably selected from Poly(allylamine)[PAA], Poly(allylaminehydrochlo- ride)[PAH], Polyaniline[PA], Poly(ethylenimine)[PEI] Poly(L-lysine hydrobro- mide)[PL], Poly(dimethylamino) ethylmethacrylate [PMA], chitosan, Diethyla- minoethyl dextran[DEAED] and proteins such as peroxidase and horse hemoglobin, and wherein said polyanion is preferably selected from Poly(styrene sulfonate)[PSS], Poly(acrylic acid)[PAA] and biopolymers such as alginate, cellulose and hyaluronic acid[HA] or derivatives of as alginate, cellulose and hyaluronic acid[HA].

55. Kit of parts according to the previous claim, wherein the layer-by-layer (LbL) coating compositions are PAH, NFC and PEI, wherein the layer-by-layer (LbL) coating is configured to comprises a first layer of PAH, a second layer of NFC, a third layer of PEI and a fourth layer of NFC

56. Kit of parts according to any one of previous claims 50-55, wherein the coating composition comprises recombinant spider silk coating composition, wherein the recombinant modified silk preferably comprises Z-4RepCT domain for antibody binding.

57. Kit of parts according to any one of the previous 50-56, wherein the compounds for modifying the surface of the microfluidic device further comprises a molecule having an affinity for biotin.

58. Kit of parts according to the previous claims 50-57, wherein the molecule having an affinity for biotin is selected from avidin, streptavidin and/or neutravidin, or derivatives of avidin, streptavidin and/or neutravidin.

59. Kit of parts according to any one of the previous claims 50-58, wherein the anti-CD203c antibody or fragment thereof is biotinylated.

60. Kit of parts according to any one of the previous claims 50-59, wherein the activation composition comprises anti-FcsRI antibody.

61 . Kit of parts according to any one of the previous claims 50-60, wherein the activation composition comprises at least one allergen, preferably selected from food allergen, a peanut allergen, a cashew allergen, an apple allergen, a milk allergen, an environmental allergen, a cockroach allergen, a tree pollen allergen, a grass allergen, a mold allergen, a hay allergen, and a drug allergen.

62. Kit of parts according to any one of the previous claims 50-61 , wherein the anti-CD63 antibody or fragment thereof is a fluorescently labelled anti-CD63 antibody or fragment thereof.

63. Kit of parts according to any one of the previous claims 50-62, wherein the primary anti-CD203c antibody or fragment thereof is a fluorescently labelled anti- CD203c antibody or fragment thereof.

64. Kit of parts according to any one of the previous claims 50-63, further comprising a secondary antibody or fragment thereof for conjugating the primary anti- CD203c or fragment thereof, wherein the secondary antibody or fragment thereof is preferably fluorescently labelled.

65. Kit of parts according to any one of the previous claims 50-64, wherein an antibody fragment is selected from a Fab fragment, F(ab')2 fragment, single chain Fv fragment and monovalent IgG.