WO2019166544A1

WO2019166544A1 - Detection of blocking antibodies

Info

Publication number: WO2019166544A1
Application number: PCT/EP2019/054971
Authority: WO
Inventors: Cécile PAQUES; Thierry Legon; Sabine Pirotton; Nicolas BOVY
Original assignee: Asit Biotech Sa
Priority date: 2018-02-28
Filing date: 2019-02-28
Publication date: 2019-09-06

Abstract

A method for detecting blocking antibodies to an allergen in a serum sample comprising the steps: a) providing : allergens labeled with a linker or a first reporter allergen-specific IgE antibodies b) combining the allergens, the allergen-specific IgE antibodies and the serum sample to obtain a mixture c) adding anti-IgE antibodies labeled with a second reporter and, if the allergen is labeled with a linker, adding a first reporter having affinity for the linker d) measuring if first and second reporters are in proximity to each other in the mixture.

Description

Detection of blocking antibodies

The present invention is directed to a method for the detection of blocking an- tibodies.

Allergy or allergic disease describes a condition caused by hypersensitivity of the immune system to something in the environment that usually causes little or no problem in most people, called allergen.

These diseases include hay fever, food allergies, atopic dermatitis, allergic asthma and anaphylaxis.

Symptoms may include red eyes, sneezing, running nose, shortness of breath, up to severe reactions which could be life threatening. Treatment for allergies includes avoiding the allergen and the use of medications like antihistamines, and corticosteroids for treating the symptoms.

In the developed world about 20% of the population are affected by allergic rhinitis and the rate of allergic diseases appears to be increasing.

Allergies are related to IgE activity. One possible treatment of allergies is aller- gen-immunotherapy also called desensitisation or hyposensitisation wherein pa- tients are exposed to increasing amounts of allergen to change the patient ^'s immune system response. There are promising applications where hydrolyzed allergen preparations are applied; see for example WO 2008/000783 or WO 2012/172037.

Patients undergoing immunotherapy to treat their allergies develop blocking an- tibodies. These antibodies are highly relevant for assessing the success of the immunotherapy and can be used as a surrogate marker. The production and the protective role of blocking antibodies in response to immunotherapy were ob- served in many studies (Van Neerven RJJ, Knol EF, Ejrnaes A, WOrtzen PA. IgE- mediated allergen presentation and blocking antibodies: Regulation of T-cell ac- tivation in allergy. Int Arch Allergy Immunol. 2006; 141(2) : 119-129) and (Wachholz PA, Soni NK, Till SJ, Durham SR. Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy. J Allergy Clin Im- munol. 2003; 112(5) :915-922.). These antibodies prevent the activation of CD4⁺ T cell by inhibiting the binding between serum-IgEs and allergens.

The typical test for measuring blocking antibodies is the FAB assay described in Shamji et al. (Shamji, Mohamed H., Louisa K. Wilcock, Petra A. Wachholz, Rebecca J. Dearman, Ian Kimber, Peter A. Wurtzen, Mark Larche, Stephen R. Durham, and James N. Francis (2006) "The IgE-Facilitated Allergen Binding (FAB) Assay: Validation of a Novel Flow-Cytometric Based Method for the Detection of Inhibitory Antibody Responses." Journal of Immunological Methods 317 (1-2) : 71-79).

This in vitro assay uses a B-cell line immortalized by the Epstein-Barr virus and overexpressing CD23, the IgE low-affinity receptor. An allergic patient serum rich in allergen-specific IgE (slgE) (reference serum) in competition with a test serum is pre-incubated with the allergens of interest. The allergen-sIgE com- plexes formed are then added to the immortalized B cells and preferentially bind to CD23. The allergen-sIgE binding to CD23 is measured by flow cytometry. In this design (figure 1), the production of blocking antibodies, as evidence of the effectiveness of immunotherapy, is assessed as follows: FAB measurement is performed using a reference serum, rich in slgE, from a patient not having un- dergone immunotherapy. The assay is performed with reference serum alone or in the presence of serum from a treated patient to evaluate the level of blocking antibodies. Thus, the presence of blocking antibodies in the serum of the treated patient inhibits the formation of allergen-IgE complexes and the binding of these complexes to the B cells. Binding is normalized to 100% when the reference serum is tested alone. The FAB assay involves at least five different steps which require a lot of time and can impact the reliability of the results. Moreover, this test includes eukaryotic cell culture which is time consuming and can be non- reproducible. The immunostaining step involves several washing steps which compromise the repeatability. Finally, the reading requires a flow cytometer which is an expensive and complicated to use device. A further test is called automated particle counting immune assay (PACIA), based on Djurup et al. (Djurup, R., C. G M Magnusson, U. Minuva, I. S0ndergaard, 0. 0sterballe, and B. Weeke (1983) "An Automated Particle Counting Immunoassay (PACIA) for Determination of Blocking Antibodies against Timothy Grass Pollen in Sera from Desensitized Allergies." Allergy 38 (3) : 173-82).

The PACIA method developed by Djurup and 0sterballe uses latex beads cova- lently coupled to antibodies specific for the allergen. The allergen, by binding to the antibodies present on the beads, induces the agglutination of the latter in the form of complexes. The amount of non-agglutinated beads is then measured using a specific device (AutoCounter). The higher the signal, the higher the level of specific antibodies. The test is carried out as follows (figure 2) : the serum from an allergic patient is incubated with the allergen to allow the formation of the antibody-allergen complexes. The latex beads coated with allergen specific- antibodies are then added and bind to the available allergen proteins (agglu- tinated beads) that are not engaged in specific antibody-allergen complexes. The non-agglutinated beads are then quantified. The concentration of blocking antibodies is expressed as a percentage of inhibition of the agglutination ac- cording to the maximum obtained with a reference serum, using this formula:

where Ps is the peak height for the sample, PM, the peak height in absence of serum (maximal agglutination) and PR the peak height for the reference serum (maximal agglutination inhibition). The measurement of blocking antibodies production is indirect. The peak is actually representative of non-agglutinated latex beads and is not a direct measure of the activity of blocking antibodies. Moreover, this test does not measure a competition due to the presence of blocking antibodies but detects the remaining free allergens after the binding with the test serum. A further test is the immunoglobulin E inhibition test (WOrtzen, P. A., G. Lund, K. Lund, M. Arvidsson, S. Rak, and H. Ipsen (2008) "A Double-Blind Placebo- Controlled Birch Allergy Vaccination Study II : Correlation between Inhibition of IgE Binding, Histamine Release and Facilitated Allergen Presentation." Clinical and Experimental Allergy 38 (8) : 1290-1301).

In this test, all samples are analyzed on an Advia Centaur instrument using two different assay procedures. According to the first method, the serum from an allergic patient is incubated with paramagnetic particles (PMPs) coupled with human anti-IgE antibodies. This incubation is followed by a washing step. The PMPs are then brought into contact with the biotinylated allergens and streptav- idin labeled with acridine. This method allows therefore to measure the amount of allergen-sIgE in the serum. In the second procedure, the washing step is not performed. Blocking antibodies are therefore present throughout the manipula- tion and interfere with the binding of IgE to the biotinylated allergens. They induce a decrease in the signal measured initially with the first method. The ratio between the readout from the two assay procedures is a measure of the inhibition by non-IgE antibodies on the binding of IgE to the allergens. This method involves duplicating the test for each sample, which is expensive in terms of material and time. The washing steps impact the reliability. Moreover, this test provides an indirect measure as the blocking antibody titers are calcu- lated on the difference between the results of two independent tests.

Despite these existing tests there is still a need for further developments, especially easier, quicker or more reliable tests, preferrably for tests that overcome at least some of the drawbacks of prior art.

The problem of the invention is to provide further test methods for blocking antibodies.

In one embodiment, the alternative is a method for detecting blocking antibod- ies to an allergen in a serum sample comprising the steps: a) providing : allergens labeled with a linker or a first reporter

allergen-specific IgE antibodies b) combining the allergens, the allergen-specific IgE antibodies and the serum sample to obtain a mixture c) adding anti-IgE antibodies labeled with a second reporter and, if the aller- gen is labeled with a linker, adding a first reporter having affinity for the linker d) measuring if first and second reporters are in proximity to each other in the mixture.

In a second embodiment the problem is solved by a method for detecting block- ing antibodies to an allergen in a serum sample comprising the steps: a) providing :

allergens labeled with a linker or a first reporter

allergen-specific IgE antibodies labeled with a second reporter b) combining the allergens, the allergen-specific IgE antibodies and the serum sample to obtain a mixture c) if the allergen is labeled with a linker, adding a first reporter having affinity for the linker d) measuring if first and second reporters are in proximity to each other in the mixture.

Compared to the FAB test, the method takes place without cell culture, making it more flexible, faster and more reproducible. All the steps may occur in a single tube, which further mimics the reality of the in vivo allergen-IgE complex for- mation. Moreover, the results are read on a classic multiplate reader, cheap device that does not require special training. Furthermore, this method can be easily adapted to other allergens and to study sera from different animal spe cies. Compared to the IgE inhibition test described of WOrtzen, the method is faster and requires less material because it relies on only one reading.

Finally, compared to the PACIA assay, the test more directly evaluates the amount of blocking antibodies, since it is a direct measure of inhibition of slgE binding to allergens.

According to the invention, there is provided an allergen labeled with a linker or a first reporter. A linker would be a compound that can be used to provide a detection by a corresponding moiety, for example pairs like biotin/streptavidin. The second starting material, an allergen-sIgE antibody could be a purified antibody or a serum sample from a reference patient or a pool of reference serum from several patients or a reference serum from an animal model of allergy .

Preferably, the reference serum contains a concentration of allergen slgE higher than 30 IU/ml, preferably higher than 100 IU/ml. The allergen-sIgE antibody has preferably a strong affinity for allergen as characterized by a steep slope

(figure 5).

In the second embodiment, these allergen-sIgE antibodies are labeled with a second reporter.

In the next step, the allergens, the allergen-sIgE antibodies and the serum sample to be tested are mixed. Depending on the starting materials, the next step is the addition of an anti IgE antibody labeled with the second reporter (if the allergen-sIgE antibodies have been used unlabled). If the allergens were not labeled with the first reporter, a first reporter is added that has affinity for the linker.

In the next step, the proximity of the first and second reporters in the mixture is detected.

If the serum sample comprises blocking antibodies, they prevent the allergen- sIgE antibodies from binding to the allergens. In presence of high concentration of blocking antibodies, there are less first reporters in proximity to the second reporters, leading to a reduction of the signal.

In a very preferred embodiment, the first and second reporters are beads. Because the method avoids the drawbacks of using cell culture, it is faster, more flexible and more reproducible. It may be performed without washing steps.

According to the invention, both the allergens or the antibodies may be labeled covalently or through affinity binding.

One preferred embodiment for the detection of proximity is that the first reporter releases a singlet oxygen upon excitation and the other reporter emits light upon contact with singlet oxygen, so that the detection comprises exciting the mixture with light at a certain wavelength and measuring the light emitted by the mixture at another wavelength. In this setting, the excitation is typically at a wave length of 650 to 750 nm and detection is between 580 and 650 nm.

The method of the present invention is useful for measuring the production of blocking antibodies against a variety of allergens. Suitable allergens are selected from pollen allergens, weed allergens, grass allergens, tree allergens, shrub al- lergens, ragweed allergens, seed allergens, flower allergens, vegetable allergens, milk allergens, egg allergens, grain allergens, fungi allergens, fruit allergens, berry allergens, nut allergens, bean allergens, peanut allergens, fish allergens, shellfish allergens, seafood allergens, meat allergens, spices allergens, house dust mite allergens, insect allergens, mite allergens, mould allergens, animal allergens, pigeon tick allergens, worm allergens, soft coral allergens, animal dander aller- gens, nematode allergens, allergens of Hevea brasiliensis, venom allergens.

A further embodiment of the invention is a kit for performing the method compri- sing a) an allergen labeled with a linker or a first reporter bl) allergen-specific IgE antibodies and anti-IgE antibodies labeled with a second reporter or b2) allergen-specific IgE antibodies labeled with a second reporter c) if the allergen is labeled with a linker, a first reporter with affinity for the linker.

In some embodiments, the kit further comprises d) a leaflet for conducting the method of the invention.

A preferred embodiment of the invention is the use of the method for the de- tection of blocking antibody production after allergy immunotherapy treatment in patients or to test the efficacy of new treatments in animal model.

In one preferred embodiment, the test uses the method of detection of slgE by AlphaLISA from PerkinElmer. It is a homogeneous phase test, without washing step, which relies on the use of two types of beads. Briefly, the serum to be tested is incubated with a biotinylated allergen, to allow the formation of IgE- allergen complexes. Acceptor beads, which are coupled with a human anti-IgE antibody, bind to the IgEs present in the serum. The donor beads coupled to the streptavidin are then added and bind to the allergens. If the IgE-allergen complex is formed, the two beads are close enough to produce a detectable signal. The test is performed with a reference serum rich in slgEs alone or in competition with a serum from an allergic patient to be tested. The presence of blocking antibodies in the latter, induced by an immunotherapy treatment for example, decreases the signal generated following the inhibition of the for- mation of IgE-allergen complexes. The measurement of this inhibition allows to evaluate the production of blocking antibodies following immunotherapy in a simple and fast in vitro test.

All references cited herein are incorporated by reference to the full extent to which the incorporation is not inconsistent with the express teachings herein. Figures

Figure 1 shows the Principle of the FAB assay. Step 1 : B cell line is EBV- transformed and the expression level of CD23 is checked; Step 2 : Reference serum alone or in combination with patient serum is incubated with allergens; Step 3 : These mixes are transposed on B cells; Step 4: B cells are washed and immuno-stained; Step 5: Detection of Ig E-labelling by flow cytometry.

Figure 2 shows the Principle of the PACIA test. Step 1 : patient serum is incu- bated with allergen extracts in excess. The anti-allergen antibodies from se- rum neutralize a part of the allergens; Step 2: Latex particles, coated with anti-allergen antibodies, are added in excess. The remaining allergen extracts agglutinate some of the particles; Step 3: The remaining free particles are counted in a modified AutoCounter. The value is read out as a peak height.

Figure 3 shows the Principle of the test of the invention.

Figure 4 shows the Titration of biotinylated proteins for the detection of grass pollen-sIgEs.

Figure 5 shows the Titration of serum dilution for the detection of grass pollen- slgEs in human reference serum and negative serum.

Figure 6 shows the Determination of optimal Acceptor beads/Donor beads ra- tio for the detection of grass pollen-sIgEs.

Figure 7 shows the detection of blocking antibodies in human serum produced after immunotherapy against grass pollen allergy.

Figure 8 shows the comparison of the production of blocking antibodies in aller- gic patients treated with placebo or with immunotherapy.

Figure 9 shows the correlation between the measurement of blocking antibodies with FAB method or with the method of the invention.

Figure 10 shows the correlation between the measurement of blocking antibod- ies with the method of the invention and the level of IgG₄ in human serum

Figure 11 shows the Determination of the optimal concentration of biotinylated Dermatophagoides pteronissimus (HDM) proteins for the detection of HDM- slgEs.

Figure 12 shows the Determination of the optimal serum dilution for the detec- tion of HDM-sIgEs.

Figure 13 shows the Detection of blocking antibodies in serum from HDM sensi- tised mice. Figure 14 shows the Detection of blocking antibodies in serum from peanut sen- sitised mice.

Examples

The invention is further explained by the following, non-limiting examples.

Example 1: titration of biotinylated proteins for the detection of IgEs specific to grass pollen

The optimal concentration of a mix of biotinylated Lolium perenne proteins is determined by detecting grass pollen-sIgEs in a reference serum, rich in slgEs. This serum is a pool of 11 human immune sera from Lolium perenne allergic patients.

Several concentrations of biotinylated allergen proteins (1.2 ng/ml; 4 ng/ml; 12 ng/ml; 40 ng/ml; 120 ng/ml; 400 ng/ml and 1200 ng/ml) are tested. For each concentration, 10 pi of proteins are mixed with 5 mI of reference serum diluted at 1/100, 1/300 or 1/1000. These mixes are incubated 1 hour at 37°C to allow the formation of IgE-allergen complexes. Then anti-IgE Acceptor Beads were added and incubated 2 hours at room temperature (RT) in the dark. Finally, streptavidin-Donor Beads were added, and the signal generated by the for- mation of IgE-allergen complexes is measured after 30 minutes by using the device Spectramax i3x (Molecular Devices) equipped with the Alphascreen car- tridge, a multiplate reader equipped with the appropriate laser and detection filters. The signal increases proportionally with increasing concentrations and peaked at 120 ng/ml. Beyond this concentration, the signal decreases under the hook effect, as shown in figure 4.

Example 2: titration of serum dilution for the detection of grass pollen- slgEs

The optimal serum dilution is determined by testing several dilutions of refer- ence serum. The optimal dilution is one that allows to reach the highest signal. Serum from non-allergic subjects is used as negative serum.

Several dilutions of reference serum or negative serum (1/1, 1/2, 1/3, 1/5, 1/10, 1/20 and 1/30) were mixed with biotinylated allergens (120 ng/ml) and incubated 1 hour at 37°C. Then anti-IgE Acceptor Beads (10 pg/ml) were added and incubated 2 hours at RT in the dark. Finally, streptavidin-Donor Beads were added, and the signal generated by the formation of IgE-allergen complexes is measured after 30 minutes. Figure 5 shows that the signal reaches the maximal value at 1/2 serum dilution. The optimal dilution is 1/5 in the ascending part of the curve.

Example 3: Determination of optimal Acceptor beads/Donor beads ratio for the detection of grass pollen-sIgEs

Several concentrations of Acceptor and Donor beads are tested with the optimal serum dilution. Allergen concentration is adapted following the Donor beads concentration. The different concentrations used were 10 pg/ml; 20pg/ml; 30 pg/ml and 40 pg/ml of each types of beads. For the different concentrations of Donor beads, the allergen concentration is adjusted as follows: 30 ng/ml of allergen for 10 pg/ml of Donor beads; 60 ng/ml of allergen for 20 pg/ml of Donor beads; 90 ng/ml of allergen for 30 pg/ml of Donor beads and 120 ng/ml of allergen for 40 pg/ml of Donor beads. Figure 6 illustrates the results nor- malized to the negative serum. The optimal ratio between the two types of beads is 10 pg/ml of Donor beads and 10 pg/ml of Acceptor beads.

Example 4: detection of blocking antibodies in human serum

The efficacy of immunotherapy allergy treatment is assessed by the production of blocking antibodies in sera of patients allergic to grass pollen before and after a treatment. The treatment comprised administration of increasing amounts of allergen-derived peptides having a molecular weight between 1,000 and 10,000 Da. This production of blocking antibodies is evaluated by the percentage of inhibition of the slgE-allergen complexes formation in reference serum.

The test was performed in vitro on human sera samples from grass pollen aller- gic patients. Blood samples are collected at visit 1 (before treatment) and at visit 2 (post-treatment) for 10 patients. Human serum allergic to grass pollen (1/5) was added to the reference serum and to a mix of biotinylated Lolium perenne proteins (30 ng/ml) and incubated 1 hour at 37°C. Anti-IgE Acceptor Beads were added and incubated 2 hours at RT in the dark. Streptavidin-Donor Beads were added, and the signal generated by the formation of IgE-allergen complexes is measured after 30 minutes. The presence of blocking antibodies in the serum from allergic patient decreases the signal from the formation of IgE-allergen complexes in the reference serum. As shown in figure 7, the per- centage of the IgE-complexes formation decreased between visit 1 (before treatment - signal normalized to 100%) and visit 2 (post-treatment). These data mean that the immunotherapy treatment was effective in both patients.

Example 5: detection of blocking antibodies in human serum treated with placebo or with allergen derived peptides

The results obtained in example 4 were compared with the measurement of blocking antibodies observed in sera from allergic patients (n = 6) treated with a placebo. Figure 8 shows that treatment with increasing amounts of allergen- derived peptides induces the production of blocking antibodies compared to pla- cebo leading to a reduced percentage of allergen-IgE complexes. Additionally, a correlation analysis was performed between the level of blocking antibodies measured by FAB method or by the method of the invention (Figure 9). The R Pearson parameter obtained is 0.69 (p value < 0.0001), indicating a good cor- relation between both methods. The same analysis was performed to correlate data of the method of the invention with IgG4 measurement (subfamily of IgG antibodies containing blocking antibodies) in sera from the same patients (Figure 10). The R Pearson parameter obtained is 0.84 (p value < 0.0001), which indicates a strong correlation between blocking antibodies production measured by the method of the invention and IgG4 production.

Example 6: titration of biotinylated proteins for the detection of house dust mites (HDM)-sIgEs

Serum reference is a serum from a patient allergic to house dust mites (HDM), rich in HDM-sIgEs. This serum is tested at different dilutions (1/3; 1/10; 1/30; 1/100 and 1/300) and several concentrations of a mix of biotinylated Der- matophagoides pteronissimus (HDM) proteins (1.2 ng/ml; 4 ng/ml; 12 ng/ml; 40 ng/ml; 120 ng/ml; 400 ng/ml and 1200 ng/ml) are tested. This experiment is performed with 10 pg/ml of each type of beads. Human reference serum is added to allergen proteins and incubated 1 hour at 37°C. Anti-IgE Acceptor beads were added and incubated 2 hours at RT in the dark. Finally, streptavidin- Donor Beads were added, and the signal generated by the formation of IgE- allergen complexes is measured after 30 minutes. The results are illustrated in figure 11 and show that the optimal concentration of allergen proteins is 40 ng/ml.

Example 7: titration of serum dilution for the detection of HDM-sIgEs

Several dilutions of reference serum are tested (1/1; 1/3; 1/5; 1/10; 1/30; 1/50 and 1/100). Serum from non-allergic subjects is used as negative serum. These different dilutions are added to biotinylated allergens (40 ng/ml) and incubated 1 hour at 37°C. Anti-IgE Acceptor beads are then added for 2 hours at room temperature in the dark. Finally, streptavidin-Donor Beads were added, and the signal is measured after 30 minutes of incubation. Figure 12 shows that the dilution 1/10 is optimal for further experiments.

Example 8: detection of blocking antibodies in HDM-sensitised mouse serum

The efficacy of an immunotherapy treatment can also be assessed in a mice model by measuring blocking antibodies production in the sera of HDM sensi- tised mice (HDM).

In this test, serum from mouse sensitised to house dust mites are collected before and after immunotherapy treatment. Several dilutions of mouse serum (1/10; 1/200; 1/2000 and 1/20000) were mixed with human reference serum (1/10) and a mix of biotinylated Dermatophagoides pteronissimus proteins (20 ng/ml; 40 ng/ml or 80 ng/ml) and incubated 1 hour at 37°C. Anti-IgE Acceptor Beads (10 pg/ml) were added and incubated 2 hours in the dark. Streptavidin- Donor Beads (10 pg/ml) were added, and the signal generated by the formation of IgE-allergen complexes is measured after 30 minutes. The signal of reference serum was decreased in the presence of immunotherapy treated mouse serum (1/20) (figure 13). The effect is dilution dependent. The decrease of signal is due to the inhibition of human IgE-allergen complexes formation by blocking antibodies produced after immunotherapy and present in mouse serum. Example 9: detection of blocking antibodies in peanut-sensitized mouse serum

The efficacy of an immunotherapy treatment can also be assessed in a mice model by measuring blocking antibodies production in the sera of peanut-sen- sitised mice.

In this test, serum from mouse sensitised to peanut are collected before and after immunotherapy treatment. Mouse serum (1/10) was mixed with human reference serum (1/10) and a mix of biotinylated Arachis hypogaea proteins (40 ng/ml) and then incubated 1 hour at 37°C. Anti-IgE Acceptor Beads (10 pg/ml) were added and incubated 2 hours in the dark. Streptavidin-Donor Beads (10 pg/ml) were added, and the signal generated by the formation of IgE-allergen complexes is measured after 30 minutes. The signal of reference serum was decreased in the presence of immunotherapy treated mouse serum. Figure 14 shows that peanut immunotherapy induces increasing production of blocking antibodies over time. The decrease of signal is due to the inhibition of human IgE-allergen complexes formation by blocking antibodies produced after immu notherapy and present in mouse serum.

Claims

1. A method for detecting blocking antibodies to an allergen in a serum sam- ple comprising the steps: a) providing : allergens labeled with a linker or a first reporter allergen-specific IgE antibodies b) combining the allergens, the allergen-specific IgE antibodies and the serum sample to obtain a mixture c) adding anti-IgE antibodies labeled with a second reporter and, if the allergen is labeled with a linker, adding a first reporter having affinity for the linker d) measuring if first and second reporters are in proximity to each other in the mixture.

2. A method for detecting blocking antibodies to an allergen in a serum sam- pie comprising the steps: a) providing : allergens labeled with a linker or a first reporter allergen-specific IgE antibodies labeled with a second reporter b) combining the allergens, the allergen-specific IgE antibodies and the serum sample to obtain a mixture c) if the allergen is labeled with a linker, adding a first reporter having affinity for the linker d) measuring if first and second reporters are in proximity to each other in the mixture.

3. The method of claim 1 or 2 wherein the first and second reporter are beads.

4. The method of any one of claims 1 to 3 wherein labeling of the allergen is covalently or through affinity binding.

5. The method of any one of claims 1 to 4 wherein labeling of the antibodies is covalently or through affinity binding.

6. The method of any one of claims 1 to 5, wherein one of the first and second reporter releases singlet oxygen upon excitation and the other reporter of the first and second reporters emit light upon contact with singlet oxygen and measuring comprises the steps

- exciting the mixture with light

- measuring light emission of the mixture.

7. The method of claim 6, wherein the mixture is excited with light having a wavelength between 650 and 750 nm.

8. The method of claim 6 to 7, wherein the light emission is measured at a wavelength between 580 and 650 nm.

9. The method of any one of claims 1 to 8, wherein the allergen-specific IgE antibodies are a reference serum sample from an allergic patient or a pool of serum from several patients.

10. The method of any of claims 1 to 9, wherein the allergen is selected from pollen allergens, milk allergens, venom allergens, egg allergens, weed aller- gens, grass allergens, tree allergens, shrub allergens, flower allergens, veg- etable allergens, grain allergens, fungi allergens, fruit allergens, berry aller- gens, nut allergens, seed allergens, bean allergens, peanut allergens, rag- weed allergens, fish allergens, shellfish allergens, seafood allergens, meat allergens, spices allergens, house dust mite allergens, insect allergens, mite allergens, mould allergens, animal allergens, pigeon tick allergens, worm al- lergens, soft coral allergens, animal dander allergens, nematode allergens, allergens of Hevea brasiliensis.

11. A kit for performing the method of any one of claims 1 to 10 comprising a) an allergen labeled with a linker or a first reporter bl) allergen-specific IgE antibodies and anti-IgE antibodies labeled with a second reporter or b2) allergen-specific IgE antibodies labeled with a second reporter c) if the allergen is labeled with a linker, a first reporter with affinity for the linker.

12. The kit of claim 11 further comprising d) a leaflet for conducting the method of any one claims 1 to 10.