WO2006084299A2 - Method for evaluating the allergen sensitivity of an individual - Google Patents

Abstract

The present invention discloses a method for evaluating the allergen sensitivity of an individual and/or the clinical efficacy of an allergen immunotherapy comprising the steps: providing at least two samples selected from the group consisting of blood or fractions thereof, connective tissue, nasal, bronchial, skin or gut biopsy material from an individual subjected or intended to be subjected to an immunotherapy with at least one pure allergen or derivative thereof, wherein the samples contain cells capable of releasing mediators in response to said allergen, contacting said sample with said allergen or derivative thereof, and determining the amounts of mediators released from said sample and evaluating the allergen sensitivity of the individual prior to therapy and/or the clinical efficacy of the immunotherapy by comparing said amounts.

Description

Method for evaluating the allergen sensitivity of an individual

The present invention relates to methods for monitoring the efficacy of an allergen immunotherapy.

An allergy is an immune malfunction wherein an individual is hypersensitised to react immunologically to typically per se harmless substances called allergens . The principal antibody which is involved in allergic reactions is IgE . Every individual has different IgE antibodies and each allergic substance stimulates production, of its own specific IgE . An IgE antibody binding a defined allergen will therefore react only against said allergen . The constant region ( Fc region) of IgE is able to bind to specific receptors of cells , which are able to release histamine or other inflammatory mediators , cytokines and/or proteases into the surrounding tissue . Histamine releasing cells are mainly mast and basophilic cells . The release of histamine is initiated when cell-bound IgE is contacted and cross-linked by the allergen .

Especially histamine causes the main allergic reactions . Histamine released in the nose, eyes , and sinuses, for example, stimulates sneezing, a runny nose, and itchy eyes ; released in the lungs it causes narrowing and swelling of the lining of the airways and the secretion of thick mucus; in the skin, rashes and hives ; and in the digestive system, stomach cramps and diarrhea .

Typical allergens are derived from plant pollens , like rye grass, ragweed, timothy grass and birch trees pollens , mold spores , drugs , like penicillins, sulfanomides, salicylates and local anaesthetics, foods, like nuts , seafood, egg, peas, beans , peanuts and other legumes, milk, insect products, like bee-sting venom, wasp sting venom, cockroach calyx and dust mites, and animal hair and dander .

There exists a number of medical treatments for allergies . Mainly three methods are regularly used in medical practice: chemotherapy, immunotherapy and alternative medical methods .

In chemotherapy antagonistic drugs are used to block the action of allergic mediators, preventing activation of cells and degranulation processes . They include antihistamines, cortisone, adrenalin (epinephrine) , theophylline and Cromolyn sodium. These drugs help alleviate the symptoms of allergy but play little role in chronic alleviation of the disorder . They can play an imperative role in the acute recovery of someone suffering from anaphylaxis .

In alternative medicine, a number of treatments are considered effective by practitioners in the treatment of allergies, particularly traditional Chinese medicine . However, none of these have been backed up by good quality evidence .

The most promising therapy form is probably immunotherapy. In the course of an immunotherapy where an individual is gradually vaccinated against progressively larger doses of the allergen in question . This can either reduce the severity or eliminate hypersensitivity altogether . Alternatively, monoclonal anti- IgE antibodies may be inj ected. These antibodies bind to free IgE signalling such sources for destruction . They do not bind to IgE already bound to the Fc receptor on basophils and mast cells as this would stimulate the allergic inflammatory response .

The proteins and glycoproteins used in allergen immunotherapy are usually extracted from materials such as pollens, molds, pelt and insect venoms . Based on the clinical evaluation, repeated subcutaneous inj ections of a solution of the disease- causing allergen or a derivative thereof are done once or twice a week in increasing doses until a maintenance dose is reached. This maintenance dose is then inj ected every 2 to 4 weeks .

In order to accomplish an immunotherapy in a successful manner monitoring of the progress of said therapy has to be performed.

For instance, in Wantke et al . (Clin Exp Allergy 23 ( 1993) 992-995 ) a method for monitoring an immunotherapy for allergic rhinoconj unctivitis is disclosed. Therein the authors analysed the spontaneous histamine release, i . e . , the release without addition of allergen, in patients prior and after the immunotherapy and showed that the - histamine release into the blood after exposure to the allergen was significantly reduced after four months of treatment . However, this method cannot be used to assess changes in sensitivity towards a particular allergen and specific efficacy of the treatment .

Stephan et al . (Allergy 44 ( 1989) 453-459) investigated the effect of bee venom immunotherapy over a period of more than five years by analysing the allergen induced histamine release in whole blood. However, the authors of this study did not correlate the results of histamine release with a clinical paramet- er, e . g. , skin sensitivity and hence no data were shown which would justify to use the assay to measure and reflect clinical sensitivity to a given allergen . Furthermore, no samples obtained before and after treatment were compared among each other .

Yuta et al . (Arerugi 51 (2002) 634-648 ) studied the histamine release from basophilic cells to evaluate an immunotherapy of allergic rhinitis . The authors analysed samples at the beginning of the treatment and at six months after starting immunotherapy and could show the positive effect of the therapy . In this article samples obtained before and after treatment were analysed and the authors could only show that the rush protocol leads to an exhaustion of the cells but does not show a reduction of histamine release . In this context it should be noted that rush immunotherapy works already before ,,blocking antibodies" are induced by immunotherapy, i . e . , sometimes after hours and few days . This may be interpreted as an exhaustion of cells . However, the assessment of the effect of blocking antibodies which appear after several weeks of treatment is important . Hence an assay where the IgG antibodies are still present, e . g . whole blood, has to be used. In contrast thereto, in Yuta et al . the cells were washed and hence the interference of blocking IgG could not be measured.

In addition to histamine release also other methods for the assessment of basophil and mast cell activation are known, which include measuring the release of leutrienes (Van Rooyen & Anderson, R. J. Immunol . Methods 2004 , 288 , 1-7 ) , tryptase (Taira M et al . , J . Asthma 2002 , 39 , 315-322 ) and other mast cell or basophil products which are released upon allergen-specific activation of the mast cells and basophils . Furthermore also the upregulation of activation markers such as CD63 and CD203c resulting from the exposure of an individual to an allergen can be measured by flow cytometry (Hauswirth A.W . , et al . J. Allergy Clin . Immunol . 2002 , 110 , 102-109 ) .

Therefore it is an object of the present invention to provide in vitro means and methods to monitor as close as pos^¬ sible clinical efficacy and the progress of an allergen immunotherapy and allergen sensitivity of an individual .

Therefore the present invention provides a method for evaluating the allergen sensitivity of an individual and/or the clin- ical efficacy of an allergen immunotherapy comprising the steps :

- providing at least two samples selected from the group consisting of blood or fractions thereof, connective tissue, nasal , bronchial , skin or gut biopsy material from an individual subj ected or intended to be subj ected to an immunotherapy with at least one pure allergen or derivative thereof, wherein the samples contain cells capable of releasing mediators in response to said allergen

- contacting said sample with said allergen or derivative thereof, and

- determining the amounts of mediators released from said sample and evaluating the allergen sensitivity of the individual prior to therapy and/or the clinical efficacy of the immunotherapy by comparing said amounts .

The evaluation of the allergen sensitivity of an individual and/or the clinical efficacy as well as the progress of an allergen immunotherapy is important in order to guarantee an effective treatment, e . g . by changing the dose and/or time intervals of the administered allergen . Therefore a reliable method to monitor the immunotherapy is required which directly reflects the sensitivity of an individual for a certain type of allergen prion and in the course of on immunotherapy. The measurement of the amount of IgE binding specifically to an allergen turned out to be not suited to determine the degree of sensitisation of an individual for a certain type of allergen, since there is no direct correlation between the amount of IgE present in an individual and the mediator release from mast and basophilic cells . Therefore the release of mediator of a sample of an individual comprising mediator releasing cells is preferred. It was surprisingly found that the method according to the present invention gave comparable, if not identical, results as the traditionally used skin sensitivity test .

The samples provided by an individual are preferably contacted with the same allergen, which is used for immunotherapy. However, it is also possible to perform the immunotherapy with an allergen extract and to monitor said therapy with substantially purified ("pure") allergens .

Of course the method according to the present invention may also be used to monitor the progress of an allergen immunotherapy by determining the allergen sensitivity of an individual in the course of the therapy.

"Allergens" according to the present invention are molecules or mixtures of molecules able to induce the production of specific antibodies (IgE) which are responsible to trigger mediator release of a mediator releasing cell and to cause consequently allergic effects in the individual . Of course, "allergens" are also capable to induce the production of antibodies other than IgE ( e . g . IgG) . However, the allergens used in the method according to the present invention are preferably purified, i . e . the allergens consist substantially of one single allergen molecule, whereby the degree of purity exceeds 90% (w/w) , preferably 95% (w/w) , most preferably 99% (w/w) . Due to the use of substantially purified or isolated allergens it is possible to determine and to dose in a reproducible manner the amount of allergen used in immunotherapy as well as used in a method according to the present invention . In contrast thereto allergen extracts contain varying concentrations of the specific allergen, depending on the specific purification conditions . Furthermore allergen extracts may also contain more than one allergen, which may be present in the extract in different concentrations (the amount of the allergen of interest is not definable in an accurate manner) and may further provoke cross reactions (see for instance Marth K et al . (2004 ) J. Allergy Clin . Immunol . 113 : 470- 474 ; Marth K et al . (2004 ) XXIII EAACI congress abstract book 597 : 181; Akkerdaas HJ et al . (2003 ) Arb . Paul Ehrlich Inst . Bundesamt Sera Impfstoffe Frankf . a . M. 94 : 87-95) . In addition, allergen extracts may contain contaminations or substance which may influence the stability of the extract . This problem can also be avoided by using substantially purified or "pure" allergens .

The term "derivative" allergen as used herein refers to modified (deleted, point mutated, truncated etc. ) allergens which still exhibit the same antigenic and IgE binding properties as the native allergen from which they are derived from.

According to a preferred embodiment of the present invention the mediators are selected from the group consisting of histamine, tryptase, prostaglandins, leukotrienes, especially cysteinyl leukotrienes, eosinophil cationic protein, cytokines , like interleukins (IL) , especially IL-2R, CD63 , CD203c and combinations thereof . The allergic response of an individual after the exposure of said individual to an allergen is primarily caused by the release of mediators by mast cells . These mediators produce the early symptoms of an allergic reaction (e . g. sneezing, itching) and stimulate the production and infiltration into local tissue of circulating leukocytes (e . g . eosinophils) . The mediators can be released from the cells by degranulation (histamine and proteases ) or after neosynthesis of said mediators (Quraishi S . A. et al . , JAOA Supplement 5 , 104 -. S7-S15 ) . According to the present invention also activation markers - besides mediators - can be determined (e . g . Yoshimura C , et al . , (2002 ) J Allergy Clin Immunol . 109 : 817-23 ) .

The sample is blood or fractions thereof (e . g . plasma, serum) , connective tissue, nasal, bronchial, skin or gut biopsy material .

Mediator releasing cells can be found in blood and fractions thereof, in connective and several other tissues . It was surprisingly found that the method according to the present invention closely mirrors cutaneous sensitivity when using pure allergens , especially when whole blood is used. In contrast thereto, measurements of specific IgE did not correlate with cutaneous sensitivity. Therefore the sample to be used in a method according to the present invention may be a blood sample (preferably heparinised blood) or connective tissue .

The mediator releasing cells used in the method according to the present invention may be isolated from the sample . Due to this isolation other possibly disturbing substances present in the sample may be removed. Especially considering that blood, for instance, may contain released mediator providing a high background during the determination of the amount of mediator released into the sample upon contact with an allergen . This problem may be avoided by measuring the amount of mediator present in the sample prior its exposure to the allergen . On the other hand experimental data revealed that substantially no correlation between histamine release and skin sensitivity, for instance, exists . Therefore, the samples to be used according to the present invention are not isolated or washed prior contacting the sample with the allergen or derivative thereof . This may be reasoned by the fact that when mediator releasing cells are washed all antibodies including those IgG antibodies which should be induced in the course of an allergen therapy and which would act as blocking antibodies in order to reduce the amount of IgE-allergen complexes (due to competition with IgE molecules) in the sample are removed (see e . g . Stahl-Skov et al . (1977 ) Clin . Exp. Immunol . 27 : 432-439)

Preferably said cells are mast and/or basophilic and/or eosinophilic cells .

Mast and basophilic cells are those cells which release most of the mediators , especially histamine, when exposed to an allergen . Mast cells are found in connective tissues of the skin, lung and gastrointestinal tract, whereas basophilic cells are found in blood. These cells can be isolated by known methods and be used in a method according to the present invention . Isolation protocols for mast cells can be found in Jamur MC et al . (J Histochem Cytochera. 1997 45 : 1715-1722) , Massey WA (J Immunol . 1991 147 : 1621-7 ) , isolation protocols for basophilic cells in Valent P. (Proc. Natl . Acad. Sci USA 1989, 86, 5542-5546) .

According to a preferred embodiment of the present invention the sample further comprises immunoglobulins (Ig) , especially immunoglobulin G (IgG) .

The procedure should preferably be carried out with samples containing IgG, e . g. whole blood samples . The presence of IgG in such samples is preferred since it allows the measurement of the interference of blocking IgG during the exposure of said cells to the allergen . In the course of an allergen immunotherapy IgGs directed to said allergen are produced. These IgGs bind to the allergen when an individual is contacted with said allergen and prevent that the allergen binds to IgE . Since the production of allergen binding IgGs is therefore directly involved in the response of an individual to an allergen and thus influencing the allergen sensitivity of an individual, the sample should preferably contain IgGs .

In order to evaluate the allergen sensitivity of an individual or the clinical efficacy of an allergen immunotherapy the samples are preferably provided before and after subj ecting said individual to an immunotherapy.

To monitor and to evaluate the efficacy of an immunotherapy it is necessary to determine the sensitivity of an individual to an allergen prior and in the course of the therapy . Therefore the mediator release is determined at various stages of the therapy. In the course of the therapy the sensitivity to an allergen ideally decreases . Furthermore, the determination of the mediator release at one or more time points before the immunotherapy may be useful for dosing the allergen in the course of the therapy.

According to another preferred embodiment of the present invention the samples are provided after subj ecting said individual to an immunotherapy.

Of course an immunotherapy may also be evaluated solely by analysing samples after the first administration of a medicament comprising an allergen .

Preferably the at least one sample is provided after a maximum of 1 hour, 2 hours , 6 hours , 12 hours , 24 hours , 5 days , 10 days, 4 weeks , 6 months , 12 months , 24 months and 36 months, after subj ecting said individual to an immunotherapy .

The sample to be analysed may be provided after a defined time period after the first administration of the allergen . Also the time intervals in between the single determinations of the mediator release may be preferably varied within the range of 1 hour, 2 hours, 6 hours , 12 hours , 2 days , 5 days, 1 week, 2 weeks, 4 weeks, 2 months, 4 months, 6 months, 12 months and 24 months .

According to a preferred embodiment said allergen is recom- binantly produced.

An efficient allergen immunotherapy and an accurate method to determine the release of mediator is preferably conducted with an allergen, which is recombinantly produced. Due to genetic engineering it is possible to produce a specific allergen in a high amount and to isolate said allergen . Allergens are usually isolated directly from the source which contains the allergen (e . g. pollen) and since the allergen is contained in an extract, said allergen is always part of a mixture of different allergenic and potential allergenic substances . Even purified "natural allergens" consist of several isoforms , some of them which may be even hypo or non-allergenic and hence give false test results ( Ferreira F. , et al . , J. Exp . Med. 1996, 183, 599- 609) . This problem can be avoided by the recombinant production of allergens . The allergen used for the administration to an individual may also be used in a method according to the present invention . Said allergen comprises preferably at least one deletion, at least one substitution or at least one insertion .

Also hypoallergenic allergen or derivatives thereof can be used when it comes to the question whether the patient may become sensitised to these derivatives during treatement .

According to a preferred embodiment of the present invention said allergen is modified by reshuffling the fragments of said allergen by genetic engineering .

The sample is preferably contacted with varying concentrations of said allergen .

The amount of mediator released from a mediator releasing cell depends on the concentration of the allergen employed in the method according to the present invention . The higher the concentration of the allergen used to induce the release a distinct amount of mediator is, the lower is the sensitivity of the cells provided from an individual and vice versa . Therefore the determination of the amount of mediator released requires the use of varying concentrations of allergen .

Preferably the concentration of said allergen is selected within the range of lng/ml to lOOμg/ml, preferably within the range of lpg/ml to lOμg/ml .

According to a preferred embodiment the total amount of mediator of the cells contained in the sample provided by an individual is determined.

In order to determine the amount of total mediator present in the cells , these cells are lysed e . g . by several thawing and freezing cycles . The determined amount of mediator indicates the ^• mediator potentially releasable by said cells , which value may be employed to determining the degree of cellular sensitisation of the cells to a certain allergen .

A degree of cellular sensitisation is preferably defined by determining the concentration of said allergen inducing the release of 10% , preferably 30% , of the total amount of mediator of said cells .

The degree of cellular sensitisation is an indicator of the progress of the immunotherapy because it reveals the concentration, at which a cell releases 10% , preferably 20%, 25% , 30% , of the total amount of mediator present in the mediator releasing cell . In the course of a successful allergen immunotherapy ^• the concentration of the allergen employed should increase because a high concentration of allergen releasing a certain amount of mediator from said cells indicates that the cells are less sensitive than in a previous measurement . Also the dose inducing maximum release of the mediator may be evaluated . This allows to create a dose response curve and to measure the shifting of said curve in the course of an allergen immunotherapy.

Therefore, the allergen sensitivity of an individual and/or the clinical efficacy of the allergen immunotherapy is preferably evaluated by observing the degree of cellular sensitisation in the course of said immunotherapy.

According to a preferred embodiment of the present invention the mediator in the sample is determined by an immunological and/or a chromatographical method, preferably the method is selected from the group consisting of radioimmunoassay (RIA) , enzyme linked immunosorbent assay (ELISA) , high performance liquid chromatography (HPLC) , reverse transcriptase polymerase chain reaction, immunofluorescence flow cytometry and combinations thereof.

All of these methods have been established to come closer to clinical sensitivity . However, none of these methods has been used to look at a pure allergen in serology, basophil activation and skin sensitivity (e . g. Pierkes M. et al . , J Allergy Clin Immunol . (1999) 103 : 326-32 ; Di Lorenzo G. et al . , J Allergy Clin Immunol . ( 1997 ) 100 : 832-7 ) .

Preferred allergens to be used by the present invention include all maj or protein allergens available e . g . under www . allergen. org/List . htm. Specifically preferred groups of allergens according to the present invention include maj or allergens such as major birch pollen allergens, e . g . Bet v 1, major timothy grass pollen allergens , e . g . PhI p 1, PhI p 2 , PhI p 5 and PhI p 6 , major house dust mite allergens, e . g. Der p 1, Der p 2 , maj or cat allergen, e . g . FeI d 1, maj or bee and wasp allergens (see list) , other profilins , especially PhI p 12 , other birch allergens, especially Bet v 4 , storage mite allergens , especially Lep d 2 , and the allergens listed in table 1.

Table 1 : preferred allergen to be used by the present invention (including reference examples) ALLERGENS Species Name

Allergen Name Biochem. ID or MW cDNA or Reference, Obsolete name protein Ace .No .

Ambrosia artemisiifolia short ragweed

Rmb a 1 antigen E 8 C 8, 20

Amb a 2 antigen K 38 C 8, 21

Amb a 3 Ra3 11 C 22

Amb a 5 Ra5 5 C 11, 23

Amb a 6 Ra6 10 C 24 , 25

Amb a 7 Ra7 12 P 26

Ambrosia trifida giant ragweed

Amb t 5 Ra5G 4.4 C 9 , 10, 27

Artemisia vulgaris mugwort

Art v 1 27-29 C 28

Art v 2 35 P 28A

Art v 3 lipid transfer protein 12 P 53

Art v 4 profilin 14 C 29

Helianthus annuus sunflower

HeI a 1 34 29A

HeI a 2 profilin 15.7 C Y15210

Mercurialis annua

Mer a 1 profilin 14-15 C Y13271

Caryophyllales

Chenopodium album lamb ' s-quarters, pigweed,

Che a 1 17 C AY049012, 29B white goosefoot

Che a 2 profilin 14 C AY082337

Che a 3 polcalcin 10 C AY082338

Salsola kali

Russian-thistle

Sal k 1 43 P 29C Rosales

Humulus japonicus

Japanese hop

Hum j 4w C M335187

Parietaria judaica

Par j 1 lipid transfer protein 1 15 see list of isoaller- gens

Par j 2 lipid transfer protein 2 see list of isoaller- gens

Par j 3 profilin see list of isoaller- gens Parietaria officinalis

Par o 1 lipid transfer protein 15 29D

B . Grasses

Poales

Cynodon dactylon Bermuda grass

Cyn d 1 32 30, S83343

Cyn d 7 31 , X91256

Cyn d 12 profilin 14 31a, Y08390

Cyn d 15 9 AF517686

Cyn d 22w enolase data pending [

Cyn d 23 Cyn d 14 9 AF517685

Cyn d 24 Pathogenesis- related p . 21 pending Dactylis glomerata orchard grass

Dae g 1 AgDgI 32 P 32

Dae g 2 11 C 33 , S45354

Dae g 3 C 33A, 025343

Dae g 5 31 P 34

Festuca pratensis meadow fescue

Fes p 4w 60

Holcus lanatus velvet grass

HoI 1 1 Z27084

Lolium perenne rye grass

Iol p 1 group I 27 C 35 , 36 LoI p 2 group II 11 P 37 , 37A, X73363 LoI p 3 group III 11 P 38 LoI p 5 LoI p IX, LoI p Ib 31/35 C 34 , 39

LoI p 11 hom: trypsin inhibitor 16 39A Phalaris aquatica canary grass

Pha a 1 40 , S80654

Phleum pratense timothy PhI p i 27 X78813

PhI p 2 C X75925 , 41

PhI p 4 P 4la

PhI p 5 Ag25 32 C 42

PhI p 6 C Z27082 , 43

PhI p 11 trypsin inhibitor hom. 20 C AF521563 , 43A

PhI p 12 profilin C X77583, 44

PhI p 13 polygalacturonase 55-60 C AJ238848 Poa pratensis Kentucky blue grass

Poa p 1 group I 33 P 46

Poa p 5 31/34 C 34 , 47

Sorghum halepense Johnson grass

Sor h 1 48

C . Trees

Arecales

Phoenix dactylifera date palm

Pho d 2 profilin 14.3 C Asturias p . c .

Fagales

Alnus glutinosa alder

AIn g 1 17 C S50892

Betula verrucosa birch Bet v 1 17 C see list of isoaller- gens

Bet v 2 profilin 15 C M65179

Bet v 3 C X79267

Bet v 4 8 C X87153 , S54819

Bet v 6 h : isoflavone reductase 33.5 C see list of isoaller- gens

Bet v 7 cyclophilin 18 P P81531 Carpinus betulus hornbeam

Car b 1 17 see list of isoaller- gens

Castanea sativa chestnut

Cas Ξ 1 22 52

Cas S 5 chitinase

Cas S 8 lipid transfer protein 9.7 53

Corylus avellana hazel Cor a 1 17 see list of isoaller- gens

Cor a 2 profilin 14 C Cor a 8 lipid transfer protein 9 C Cor a 9 IIS globulin-like protein 40/? C Beyer p . c . Cor a 10 luminal binding prot . 70 C AJ295617 Cor a 11 7S vicilin-like prot . 48 C AF441864

Quercus alba White oak

Que a 1 17 54

Lamiales

Oleaceae

Fraxinus excelsior ash Fra e 1 20 58A, AF526295

Ligustrum vulgare privet Lig v 1 20 P 58A

Olea europea olive Ole e 1 16 C 59, 60

Ole e 2 profilin 15-18 C 6OA

Ole e 3 9.2 6OB

Ole e 4 32 P P80741

Ole e 5 superoxide dismutase 16 P P80740

Ole e 6 10 C 6OC, U86342

Ole e 7 ? P 6OD, P81430

Ole e 8 Ca2+-binding protein 21 C 6OE, AF078679

Ole e 9 beta-1 , 3-glucanase 46 ^• C AF249675

Ole e 10 glycosyl hydrolase hom. 11 C 6OF, AY082335

Syringa vulgaris lilac Syr v 1 20 P 58A

Plantaginaceae Plantago lanceolata English plantain

PIa 1 1 18 P842242

Pinales

Cryptomeria japonioa sugi Cry j 1 41-45 C 55, 56

Cry j 2 C 57 , D29772

Cupressus arisonica cypress

Cup a 1 43 A1243570

Cupressus sempervirens common cypress

Cup s 1 43 see list of isoaller- gens

Cup s 3w 34 ref pending Juniperus ashei mountain cedar

Jun a 1 43 P P81294

Jun a 2 C 57A, AJ404653

Jun a 3 30 P 57B, P81295

Juniperus oxycedrus prickly juniper

Jun o 4 horn: calmodulin 29 57C, AF031471 Juniperus sabinoides mountain cedar

Jun s 1 50 58

Juniperus virginiana eastern red cedar

Jun v 1 43 P81825 , 58B

Platanaceae Platanus acerifolia London plane tree

PIa a 1 18 P P82817

PIa a 2 43 P P82967

PIa a 3 lipid transfer protein 10 P Iris p . c .

D. Mites

Acarus siro arthropod mite Aca s 13 fatty acid binding prot . 14* RJ006774

Blomia tropicalis mite BIo t 1 cysteine protease 39 C &F277840

BIo t 3 trypsin 24* C Cheong p . c .

BIo t 4 alpha amylase 56 C Cheong p . c .

BIo. t 5 C 059102

BIo t 6 chymotrypsin 25 C Cheong p . c .

BIo t 10 tropomyosin 33 C 61

Bio t 11 paramyosin HO C AF525465, 61A

BIo t 12 BtUa C 027479

BIo t 13 Bt6, fatty acid bind prot . C 058106

BIo t 19 anti-microbial pep . hom. 7.2 C Cheong p . c . Dermatophagoides farinae American house dust mite

Der f 1 cysteine protease 25 C 69

Der f 2 14 C 70, 7OA, see list of isoallergens

Der f 3 trypsin 30 C 63

Der f 7 24-31 C SW: Q26456, 71

Der f 10 tropomyosin C 72

Der f 11 paramyosin 98 C 72A

Der f 14 mag3 , apolipophorin C D17686

Der f 15 98k chitinase 98 C AF178772

Der f 16 gelsolin/villin 53 C 71A

Der f 17 Ca binding EF protein 53 C 7IA.

Der f 18w 60k chitinase 60 C Weber p . c . Dermatophagoides microceras house dust mite

Der m 1 cysteine protease 25 68 Dermatophagoides pteronyssinus European house dust mite

Der p 1 antigen Pl, cysteine protease 25 62 , see list of isoallergens

Der p 2 14 62A-C, see list of isoallergens

Der p 3 trypsin 28/30 C 63 Der p 4 amylase 60 P 64 Der p 5 14 C 65 Der p 6 chymotrypsin 25 P 66

Der p 7 22/28 C 67

Der p 8 glutathione transferase C 67A

Der p 9 collagenolytic serine pro . P 67B

Der p 10 tropomyosin 36 C Y14906

Der p 14 apolipophorin like prot . C Epton p . c .

Buroglyphus maynei mite Eur m 2 C see list of isoaller- gens

Eur m 14 apolipophorin 177 C AF149827

Glycyphagus domesticus storage mite

GIy d 2 C 72B, see isoallergen list

Lepidoglyphus destructor storage mite

Lep d 2 Lep d 1 15 C 73, 74 , 74A, see isoallergen list

Lep d 5 C 75 , AJ250278

Lep d 7 C 75, AJ271058

Lep d 10 tropomyosin C 75A, AJ250096

Lep d 13 C 75, AJ250279

Tyrophagus putrescentiae storage mite

Tyr p 2 C 75B, Y12690

E . Animals

Bos domesticus domestic cattle

Bos d 2 Ag3, lipocalin 20 C 76, see isoallergen list

(see also foods )

Bos d 3 Ca-binding SlOO hom. 11 C L39834

Bos d 4 alpha-lactalbumin 14.2 C M18780

Bos d 5 beta-lactoglobulin 18.3 C X14712

Bos d 6 serum albumin 57 C M73993

Bos d 7 immunoglobulin 160 77

Bos d 8 caseins 20-30 77

Canis familiaris

(Canis domesticus )

Can f 1 25 C 78, 79 dog Can f 2 27 78, 79

Can f 3 albumin S72946

Can f 4 18 A59491

Equus oaballus domestic horse

Equ c 1 lipocalin 25 C 070823

Equ c 2 lipocalin 18.5 P 79A, 79B

Equ c 3 Ag3 - albumin 67 C 79C, X74045

Equ c 4 17 P 79D

Equ c 5 AgX 17 P Goubran Botros p . c .

Felis dortiesticus cat ( saliva)

FeI d 1 cat-1 38 15

FeI d 2 albumin 79E, X84842

FeI d 3 cystatin 11 79F, AF238996

FeI d 4 lipocalin 22 AY497902

FeI d 5w immunoglobulin A 400 Adedoyin p . c . FeI d 6w immunoglobulin M

800-

1000

Adedoyin p . c .

FeI d 7w immunoglobulin G 150 Adedoyin p . c . Cavia porcellus guinea pig

Cav p 1 lipocalin homologue 20 SW: P83507 , 80

Cav p 2 17 SW: P83508

Mus musculus mouse (urine)

Mus m 1 MUP 19 81 , 81A

Rattus norvegius rat (urine!

Rat n 1 17 82 , 83

F . Fungi (moulds) 1. Ascomycota

1.1 Dothideales Alternaria alternata Alt a 1 28 C U82633

Alt a 2 25 C 83A, U62442

Alt a 3 heat shock prot . 70 C U87807 , U87808

Alt a 4 prot . disulfideisomerase 57 C X84217

Alt a 6 acid ribosomal prot . P2 11 C X78222, U87806

Alt a 7 YCP4 protein 22 C X78225

Alt a 10 aldehyde dehydrogenase 53 C X78227, P42041

Alt a 11 enolase 45 C U82437

Alt a 12 acid ribosomal prot . Pl 11 C X84216 Cladosporium herbarum

CIa h 1 13 83B, 83C

CIa h 2 23 83B, 83C

CIa h 3 aldehyde dehydrogenase 53 C X78228

CIa h 4 acid ribosomal prot . P2 11 C X78223

CIa h 5 YCP4 protein 22 C X78224

CIa h 6 enolase 46 C X78226

CIa h 12 acid ribosomal prot . Pl 11 C X85180 1.2 Eurotiales Aspergillus flavus

Asp fl 13 alkaline serine protease 34 84 Aspergillus fumigatus

Asp f 1 18 C M83781, S39330

Asp f 2 37 C U56938

Asp f 3 peroxisomal protein 19 C U20722

Asp f 4 30 C AJ001732

Asp f 5 metalloprotease 40 C Z30424

Asp f 6 Mn superoxide dismut . 26.5 C U53561

Asp f 7 12 C AJ223315

Asp f 8 ribosomal prot . P2 11 C AJ224333

Asp f 9 34 C AJ223327

Asp f lOaspartic protease 34 C X85092

Asp f 11 peptidyl-prolyl isomeras 24 84A

Asp f 12 heat shock prot . P90 90 C 85

Asp f 13 alkaline serine protease 34 84B

Asp f 15 16 C AJ002026

Asp f 16 43 C g3643813

Asp £ IT C AJ224865

Asp f 18 vacuolar serine protease 34 84C

Asp f 22w enolase 46 C AF284645

Asp f 23 I>3 ribosomal protein 44 C 85A, AF464911 Aspergillus niger

Asp n 14 beta-xylosidase 105 C AF108944

Asp n 18 vacuolar serine protease 34 C 84B

Asp n 25 3-phytase B 6S-3.OO G 85B, P34754

Asp n ? 85 C Z84377

Aspergillus oryzae

Asp o 13 alkaline serine protease 34 C X17561

Asp o 21 TAKA-amylase A 53 C D00434 , M33218

Penicillium brevicompactum

Pen b 13 alkaline serine protease 33 86A

Penicillium chrysogenum

(formerly P . notatum)

Pen ch 13 alkaline serine protease 34 87

Pen ch 18 vacuolar serine protease 32 87

Pen ch 20 N-acetyl glucosaininiclas 68 87A

Penicillium citrinum

Pen c 3 peroxisomal mem. prot . 18 86B

Pen c 13 alkaline serine protease 33 86A

Pen c 19 heat shock prot . P70 70 C D64207

Pen c 22w enolase 46 C AF254643

Pen c 24 elongation factor 1 beta C AY363911

Penicillium oxalicum

Pen o 18 vacuolar serine protease 34 87B

1.3 Hypocreales Fusarium culmorum

Fus c 1 ribosomal prot . P2 11* AY077706 Fus c 2 thioredoxin-like prot . 13* AY077707

1.4 Onygenales Trichophyton rubEum

Tri r 2

Tri r 4 serine protease 88 Trichophyton tonsurans

Tri t 1 30 88A

Tri t 4 serine protease 83

1.5 Saccharomycetales

Candida albicans

Cand a 1 40 C 89

Cand a 3 peroxisomal protein 29 C AY136739

Candida boidinii

Cand b 2 20 C J04984 , J04985

2. Basidiomycotina

2.1 Hymenomycetes Psilocybe cubensis

Psi c 1 Psi c 2 cyclophilin 16 89A

Coprinus comatus shaggy cap

Cop c 1 leucine zipper protein 11 AJ132235 Cop c 2 AJ242791 Cop c 3 AJ242792 Cop c 5 AJ242793 Cop c 7 AJ242794

2.2 Urediniomycetes Rhodotorula mucilaginosa

Rho m 1 enolase 47 C 89B

Rho m 2 vacuolar serine protease 31 C RY547285

2.3 ϋstilaginomycetes Malassezia furfur

Mala f 2 MFl , peroxisomal 21 C AB011804 , 90 membrane protein Mala f 3 MF2 , peroxisomal 20 C AB011805, 90 membrane protein

Mala f 4 mitochondrial malate dehydrogenase 35 AF084828 , 9OA Malassezia sympodialis

Mala s i C X96486, 91

Mala s 5 18* C AJ011955

Mala s 6 17* C AJ011956

Mala s 7 C AJ011957 , 91A Mala s 8 19* C AJ011958 , 91A Mala s 9 37* C AJ011959, 91A

Mala s 10 heat shock prot . 70 86 C AJ428052 Mala s 11 Mn superoxide dismut . 23 C AJ548421 3. Deuteromycotina

3.1 Tuberculariales Epicoccum purpurascens ( formerly E . nigrum)

Epi p 1 serine protease 30 SW : P83340, 91B

G . Insects

Aedes aegyptii mosquito

Aed a 1 apyrase 68 C L12389

Aed a 2 37 C M33157

Apis mellifera honey bee

Api m 1 phospholipase A2 16 C 92

Api m 2 hyaluronidase 44 C 93

Api m 4 melittin 3 C 94

Api m 6 7-8 P Kettner p . c .

Api m 7 CUB serine protease 39 C AY127579

Bombus pennsylvanicus bumble bee

Bom p 1 phospholipase 16 P 95

Bom p 4 protease E 95

Blattella germanica

German cockroach

BIa g 1 Bd90k C

BIa g 2 aspartic protease 36 C 96

BIa g 4 calycin 21 C 97

BIa g 5 glutathione transferase 22 C 98

BIa g 6 troponin C 27 C 98

Periplaneta americana

American cockroach

Per a 1 Cr-PII C

Per a 3 Cr-PI 72-78 C 98A

Per a 7 tropomyosin 37 C Y14854

Chironomus kiiensis midge Chi k 10 tropomyosin 32.5* AJ012184 Chironomus thummi thummi midge Chi t 1-9 hemoglobin 16 C 99 Chi t 1.01 component III 16 C P02229 Chi t 1.02 component IV 16 C P02230 Chi t 2.0101 component I 16 C P02221 Chi t 2.0102 component IA 16 C P02221 Chi t 3 component II-beta 16 C P02222 Chi t 4 component IIIA 16 C P02231 Chi t 5 component VI 16 C P02224 Chi t 6.01 component VIIA 16 C P02226 Chi t 6.02 component IX 16 C P02223 Chi t 7 component VIlB 16 C P02225 Chi t 8 component VIIl 16 C P02227 Chi t 9 component X 16 C P02228

Ctenocephalides felis felis cat flea

Cte f 1 Cte f 2 MIb 27 AF231352 Cte f 3 25

Thaumetopoea pityocampa pine processionary moth Tha p 1 15 PIR:A59396, 99A

Lepisma saccharina silverfish

Lep s 1 tropomyosin 36 AJ309202

Dolichovespula maoulata white face hornet

DoI m 1 phospholipase Al 35 C 100 DoI m 2 hyaluronidase 44 C 101 DoI m 5 antigen 5 23 C 102 , 103

Dolichovespula arenaria yellow hornet

DoI a 5 antigen 5 23 104

Polistes annularies wasp Pol a 1 phospholipase Al 35 105 Pol a 2 hyaluronidase 44 105 Pol a 5 antigen 5 23 104

Polistes dominulus

Mediterranean paper wasp Pol d 1 Hoffman p , c .

Pol d 4 iserine protease 32-34 C Hoffman p . c .

Pol d 5 P81656

Polistes exclamans wasp Pol e 1 phospholipase Al 34 P 107

Pol e 5 antigen 5 • 23 C 104

Polistes fuscatus wasp Pol f 5 antigen 5 23 C 106

Polistes gallicus wasp Pol g 5 antigen 5 24 C P83377

Polistes metricus wasp Pol m 5 antigen 5 23 C 106

Vespa crabo

European hornet

Vesp c 1 phospholipase 34 P 107

Vesp c 5 antigen 5 23 C 106

Vespa mandarina giant asian hornet

Vesp m 1 Hoffman p . c

Vesp in 5 P81657

Vespula flavopilosa yellowjacket Ves f 5 antigen 5 23 C 106

Vespula germanica yellowjacket Ves g 5 antigen 5 23 C 106

Vespula maculifrons yellowjacket

Ves m 1 phospholipase Al 33.5 C 108

Ves m 2 hyaluronidase 44 P 109

Ves m 5 antigen 5 23 C 104

Vespula pennsylvanica yellowjacket

Ves p 5 antigen 5 23 C 106

Vespula squamosa yellowjacket

Ves s 5 antigen 5 23 C 106

Vespula vidua wasp Ves vi uj antigen 5 23 C 106

Vespula vulgaris yellowjacket

Ves v 1 phospholipase ! Al 35 C 105R Ves v 2 hyaluronidase 44 105A Ves v 5 antigen 5 23 104

Myrmecia pilosula Australian jumper ant Myr p 1 X70256 Myr p 2 S81785

Solenopsis geminata tropical fire ant

Sol g 2 Hoffman P . C Sol g 4 Hoffman P . C

Solenopsis invicta fire ant Sol i 2 13 C 110 , 111 Sol i 3 24 C 110 Sol i 4 13 C 110

Solenopsis saevissima Brazilian fire ant

Sol s 2 Hoffman p . c .

Triatoma protracta California kissing bug Tria p 1 Procalin 20 AF179004 , 11IA.

H. Foods

Gadus callarias cod

Gad c 1 allergen M 12 112 , 113 Salmo salar Atlantic salmon

Sal s 1 parvalbumin 12 X97824

Bos domesticus domestic cattle

Bos d 4 alpha-lactalbumin 14.2 C M18780

(milk) Bos d 5 beta-lactoglobulin 18.3 C X14712 see also animals

Bos d 6 serum albumin 67 C M73993

Bos d 7 immunoglobulin 160 77

Bos d 8 caseins 20-30 77

Gallus domesticus chicken

Gal d 1 ovomucoid 28 114 , 115 Gal d 2 ovalbumin 44 C 114 , 115

Gal d 3 Ag22, conalbumin 78 C 114 , 115

Gal d 4 lysozyme 14 C 114 , 115

Gal d 5 serum albumin 69 C X6Q688

Metapenaeus ensis shrimp Met e 1 tropomyosin C U08008

Penaeus aztecus shrimp Pen a 1 tropomyosin 36 P 116

Penaeus indicus shrimp Pen i 1 tropomyosin 34 C 116A

Penaeus monodon black tiger shrimp

Pen m 1 tropomyosin 38 C

Pen m 2 arginine kinase 40 C AF479772, 117

Todarodes pacificus squid Tod p 1 tropomyosin 38 P 117A

Helix aspersa brown garden snail

HeI as 1 tropomyosin 36 C Y14855, 117B

Haliotis midae abalone

Hal m 1 49 117C

Rana esculenta edible frog

Ran e 1 parvalbumin alpha 11.9* C AJ315959

Ran e 2 parvalbumin beta 11.7* C AJ414730

Brassica juncea oriental mustard

Bra j 1 2S albumin 14 C 118

Brassica napus rapeseed

Bra n 1 2S albumin 15 P 118A, P80208

Brassica rapa turnip Bra r 2 hom: prohevein 25 P81729

Hordeum vulgare barley Hor v 15 BMAI-I 15 C 119

Hor v 16 alpha-amylase

Hor v 17 beta-amylase

Hor v 21 gamma-3 hordein 34 C 119A, SW: P80198

Secale cereale rye sec c 20 secalin see isoall . list

Triticum aestivum wheat Tri a 18 agglutinin

Tri a 19 omega-5 gliadin 65 PIR:A59156 Zea mays maise, corn

Zea m 14 lipid transfer prot . P19656 Oryza sativa rice Ory s 1 119B, 031771 Apium graveolens celery Api g 1 hom: Bet v 1 16* Z48967

Api g 4 profilin AF129423

Api g 5 55/58 P81943 Daucus carota carrot Dau c 1 hom: Bet v 1 16 117D, see isoallergen list

Dau c 4 profilin AF456482

Corylus avellana hazelnut

Cor a 1.04 hom: Bet v 1 17 see list of isoaller- gens

Cor a 2 profilin 14 AF327622

Cor a 8 lipid transfer protein 9 AF329829 Malus domestica apple MaI d 1 hom: Bet v 1 see list of isoaller- gens

MaI d 2 hom: thaumatin C AJ243427

MaI d 3 lipid transfer protein 9 C Pastorello p . c .

MaI d 4 profilin 14.4* C see list of isoaller- gens

Pyrus communis pear Pyr c 1 hom: Bet v 1 18 C AF05730

Pyr c 4 profilin 14 C AF129424

Pyr c 5 hom: isoflavone reductas 33.5 C AF071477 Persea americana avocado Pers a 1 endochitinase 32 Z78202 Prunus armeniaca apricot

Pru ar 1 hom: Bet v 1 093165

Pru ar 3 lipid transfer protein Prunus avium sweet cherry

Pru av 1 hom: Bet v 1 C U66076

Pru av 2 hom: thaumatin C U32440

Pru av 3 lipid transfer protein 10 C AF221501 Pru av 4 profilin 15 C AF129425

Prunus doinestica

European plum

Pru d 3 lipid transfer protein 9 P 119C

Prunus persica peach Pru p 3 lipid transfer protein 10 P P81402 pru p 4 profilin 14 C see isoallergen list

Asparagus officinalis

Asparagus

Aspa o 1 lipid transfer protein 9 P 119D

Crocus sativus saffron crocus Cro s 1 21 Varasteh A-R p. c .

Lactuca sativa lettuce

Lac s 1 lipid transfer protein 9 Vieths p . c .

Vitis vinifera grape Vit v 1 lipid transfer protein 9 P80274

Musa x paradisiaca banana Mus xp 1 profilin 15 AF377948

Ananas corαosus pineapple

Ana c 1 profilin 15 C AF377949

Ana c 2 bromelain 22.8* C 119E-G, D14059

Citrus liraon lemon Cit 1 3 lipid transfer protein 9 P Torrej on p . c .

Citrus sinensis sweet orange

Cit s 1 germin-like protein 23 P Torrejon p . c . Cit s 2 profilin 14 P Torrej on p . c .

Cit s 3 lipid transfer protein 9 P Torrejon p . c .

Litchi chinensis litchi lit c 1 profilin 15 C AY049013

Sinapis alba yellow mustard

Sin a 1 2S albumin 14 C 120 Glycine max soybean GIy m 1 HPS 7 P 120A GIy m 2 ε A57106 GIy m 3 profilin 14 see list of isoaller- gens

GIy m 4 (SAM22) PR-IO prot . 17 X60043, 120B

Vigna radiata mung bean

Vig r 1 PR-IO protein 15 AY792956

Arachis hypogaea peanut Ara h 1 vicilin 63.5 C L34402

Ara h 2 conglutin 17 C L77197

Ara h 3 glycinin 60 C AF093541

Ara h 4 glycinin 37 C AF086821

Ara h 5 profilin 15 C AF059616

Ara h 6 hom: conglutin 15 C AF092846

Ara h 7 hom: conglutin 15 C AF091737

Ara h 8 PR-IO protein 17 C AY328088 Lens culinaris lentil Len c 1 vicilin 47 see list of isoaller- gens

Len c 2 seed biotinylated prot . 66 120C Pisum savitum pea Pis s 1 vicilin 44 see list of isoaller- gens

Pis s 2 convicilin 63 pending Actinidia chinensis kiwi Act c 1 cysteine protease 30 P00785

Act c 2 thaumatin-like protein 24 SW-.P81370 , 121 Capsicum annuum bell pepper

Cap a Iw osmotin-like protein 23 C AJ297410

Cap a 2 profilin 14 C AJ417552 Lycopersicon esculentum tomato Lye e 1 profilin 14 C AJ417553

Lye e 2 b-fructofuranosidase 50 C see isoallergen list

Lye e 3 lipid transfer prot . 6 C U81996 Solanum tuberosum potato Sola t 1 patatin 43 P P15476

Sola t 2 cathepsin D inhibitor 21 P P16348

Sola t 3 cysteine protease inhibitor 21 P P20347

Sola t 4 aspartic protease inhibitor 16+4 P P30941 Bertholletia excelsa Brazil nut

Ber e 1 2S albumin 9 C P04403, M17146

Ber e 2 IIS globulin seed storage protein 29 C AY221641

Juglans nigra black walnut

Jug n 1 2S albumin 19* C AY102930

Jug n 2 vicilin-like prot . 56* C AY102931

Juglans regia

English walnut

Jug r 1 2S albumin C U66866

Jug r 2 vicilin 44 C AF066055

Jug r 3 lipid transfer protein 9 P Pastorello

Anacardium occidentale

Cashew Ana o 1 vicilin-like protein 50 C see isoallergen list

Ana o 2 legumin-like protein 55 C AF453947

Ana o 3 2S albumin 14 C AY081853

Ricinus communis

Castor bean

Ric e 1 2S albumin C P01089

Sesamum indicum sesame Ses i 1 2S albumin 9 C 121A, AF240005

Ses i 2 2S albumin 7 C AF091841 ses i 3 7S vicilin-like globulin 45 C AF240006

Ses i 4 oleosin 17 C AAG23840

Ses i 5 oleosin 15 C AAD42942

Cucumis meIo muskmelon

Cue m 1 serine protease 66 C D32206

Cue m 2 profilin 14 C AY271295

Cue m 3 pathogenesis-rel p . PR-I 16* P P83834

I . Others

Anisakis simplex nematode

Ani S 1 24 P 121B, A590 S9

Ani S 2 paramyosin 97 C AF173004

Ani S 3 tropomyosin 41 C 121C, Y19221

Ani S 4 9 P P83885

Argas reflexus pigeon tick

Arg r 1 17 AJ697694

Ascaris suum worm As c s 1 10 122

Carica papaya papaya Car p 3w papain 23.4* 122A, M15203

Dendronephthya nipponica soft coral

Den n 1 53 122B

Hevea brasiliensis rubber (latex)

Hev b 1 elongation factor 58 P 123 , 124 Hev b 2 1 , 3-glucanase 34/36 C 125 Hev b 3 24 P 126, 127 Hev b 4 component of 100- P 128 microhelix complex 115 Hev b 5 16 C D42640

Hev b 6.01 hevein precursor 20 C M36986, pO2877 Hev b 6.02 hevein 5 C M36986, pO2877 Hev b 6.03 C-terminal fragment 14 C M36986, pO2877 Hev b 7.01 hom: patatin from B-serum 42 C O80598 Hev b 7.02 hom: patatin from C-serum 44 C AJ223038 Hev b 8 profilin 14 C see list of isoaller- gens

Hev b 9 enolase 51 C AJ132580

Hev b 10 Mn superoxide dismut . 26 C see list of isoaller- gens

Hev b 11 class 1 chitinase see list of isoaller- gens

Hev b 12 lipid transfer protein 9.3 AY057860

Hev b 13 esterase 42 P83269 Homo sapiens human autoallergens

Hom s 1 73* C Y14314

Hom s 2 10.3* C X80909

Hom s 3 20.1* C X89985

Hom s 4 36* C Y17711

Hom s 5 42.6* C P02538

Triplochxton scleroxylon obeche Trip s 1 class 1 chitinase 38.5 Kespohl p . c . References

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The knowledge of the nucleic acid sequences encoding these allergens allows their recombinant production. Therefore especially these allergens are preferably used in immunotherapies and in methods according to the present invention. Another aspect of the present invention relates to a method for evaluating the allergen sensitivity of an individual and/or the clinical efficacy of an allergen immunotherapy comprising the steps :

- providing cells capable of releasing mediators in response to an IgE-allergen complex,

- contacting said cells with serum and/or plasma of said individual spiked with at least one pure allergen or derivative thereof, and

- determining the amounts of mediators released from said sample and evaluating the allergen sensitivity of the individual prior to therapy and/or the clinical efficacy of the immunotherapy by comparing said amounts .

The cells which are capable of releasing mediators comprise normally IgE molecules bound thereto . Such cells can be isolated from samples which are obtained from the individual subj ected to the method according to the present invention or from other individuals . Of course, it is also possible to use cell lines capable of binding IgE in a method according to the present invention .

The method according to the present invention is especially suited for the determination of the allergen sensitivity of an individual because it allows to determine the ratio between the allergen specific IgE and IgG molecules in the plasma and serum of said individual . Since only IgE-allergen complexes and not free IgE are able to induce the release of mediators from mediator-releasing cells like leukozytes the level of released mediator correlates with the amount of IgE-complex present in the sample . In turn the amount of IgE-complex in said sample correlates with the amount of allergen specific IgE, allergen and allergen specific antibodies other than IgE such as IgG, IgA or IgM which compete with IgE for the free allergen and consequently inhibits the formation of an IgE-allergen complex . This means that a low level of allergen specific IgE or a high level of allergen specific IgG leads to the formation of a low number of IgE complex and thus to a reduced mediator release .

The concentration of allergen in said serum and/or plasma is preferably within 1 ng/ml to 100 μg/ml, more preferably within 1 pg/ml to 10 μg/ml .

Another aspect of the present invention relates to a kit for evaluating the allergen sensitivity of an individual and/or the clinical efficacy of an allergen iinmunotherapy for at least one allergy comprising

- at least one allergen for inducing a mediator release of cells capable of releasing the mediator in response to an allergen,

- means for detecting mediator, and

- optionally at least one mediator standard

The kit provided herein comprises at least one allergen, which can be used to induce the release of a mediator from mediator releasing cells contained in a sample . The released mediator is then detected directly or preferably - after the removal of solid parts of the sample - in the supernatant of the reaction mixture . Optionally also means for the detection of IgE molecules binding said allergen are enclosed in the kit according to the present invention . IgE is able to bind a distinct allergen and to mediate, when bound to a mediator releasing cell and the allergen, the release of mediator from said cells . However, IgE specific for an allergen is not normally detected in the blood and is only produced when a person becomes sensitised to an allergen . In order to accurately determine the amount of mediator in the sample (for the provision of a standard curve) a mediator standard may be optionally part of the kit .

Preferably the cells are mast and/or basophilic and/or eosinophilic cells .

According to another preferred embodiment of the present invention the allergen is selected from the group consisting of maj or birch pollen allergens , in particular Bet v 1 and Bet v 4 , maj or timothy grass pollen allergens , in particular PhI p 1 , PhI p 2 , PhI p 5, PhI p 6 and PhI p 7 , major house dust mite allergens , in particular Der p 1 and Der p 2 , maj or cat allergen FeI d 1, maj or bee allergens, maj or wasp allergens , profilins, especially PhI p 12 , and storage mite allergens , especially Lep d 2 and the allergens listed in table 1.

The means for detecting mediators are preferably antibodies .

A mediator, as outlined above, is preferably detected by immunological methods . Therefore the kit may provide at least one antibody which is able to bind specifically mediator . Preferably enzyme linked immuno sorbent assays (ELISA) , radio immuno assays (RIA) or lateral flow devices are employed. Another aspect of the present invention relates to a kit for evaluating the allergen sensitivity of an individual or the clinical efficiency of an allergen immunotherapy for at least one allergy comprising at least two of the following components :

- at least one allergen for inducing a mediator release of cells capable of releasing mediators in response to an allergen,

- means for detecting the mediator,

- at least one mediator standard, and

- cells capable of releasing mediators in response to an IgE-allergen complex .

The present invention is further illustrated by the following figures and example, without being restricted thereto .

Fig . 1 shows the association of results from intradermal end-point titration (x-axis : Allergen concentration giving the first positive reaction) and rBet v 1- specific serum IgE (y- axis : kU/L CAP System) .

Fig . 2 shows the association of results from basophil histamine release (x-axis : Allergen concentration giving 30% histamine release) and rBet v 1-specific serum IgE (y-axis : kU/L CAP System) .

Fig. 3 shows the association of results from intradermal end-point titration (x-axis : Allergen concentration giving the first positive reaction) and results from basophil histamine release (y-axis : Allergen concentration giving 30% histamine release) .

Fig . 4 shows the association of Bet v 1-specific IgE determined by CAP (x-axis : kϋ/ L) and of rBet v 1-specific IgE determined with labelled a-chain (y-axis : counts per minute (c . p .m. ) ; 1 : 5 serum dilution) .

Fig . 5 shows the association of results from basophil histamine release (x-axis : maximal histamine release (% ) ) and results from skin prick testing (y-axis : weal reaction (mm²) induced by skin prick testing with 2 μg/ml of recombinant Bet v m) .

EXAMPLES : Example 1 :

The cross-linking of effector cell (mast cells and basophils) - bound IgE antibodies by allergens is a crucial event for the induction of the immediate symptoms of type I allergy (Ka- wakami T, et al . , Nat Rev Immunol (2002 ) 2 : 773-86) . As described in the classical experiments by Prausnitz and Kϋstner (Prausnitz C, at al . , Centralbe F Bact 1 Abt Orig ( 1921) 86 : 160-8 ) , this event depends on three major factors , i . e . allergen-specific IgE antibodies, effector cells and allergens . Because the characterisation of IgE antibodies and the development of diagnostic tests capable of measuring the precise amount of allergen-specific IgE antibodies, several studies have investigated the association of allergen-specific serum IgE levels and biological sensitivity to allergens in allergic patients ( Stenius B, et al . , Clin Allergy ( 1971 ) 1 : 37-55 ; Bryant DH, et al . , Clin Allergy (1975 ) 5 : 145-57 ; Pauli G, et al . , Clin Allergy ( 1977 ) 7 : 337- 46; Bousquet J, et al . , Clin Allergy ( 1987 ) 17 : 529-36; Witteman AM, et al . , J Allergy Clin Immunol ( 1996) 97 : 16-25 ; Niederberger V, et al . J Invest Dermatol (2001) 117 : 848-51 ; Norman PS , et al . , J Allergy Clin Immunol ( 1973 ) 52 : 210-24 ; Lichtenstein LM, et al . J Allergy Clin Immunol ( 1971 ) 47 : 103 (A37 ) ) . It is well established that the presence of allergen-specific serum IgE is a pre-requisite for the occurrence of an immediate type of reaction, but whether the amount of allergen-specific IgE correlates with immediate type sensitivity to the given allergen has been a matter of great debate . To address the problem almost all of the investigations carried out in the past have used allergen extracts , i . e . mixtures of allergens and non-allergenic molecules (Stenius B, et al . , Clin Allergy ( 1971 ) 1 : 37-55 ; Bousquet J, et al . , Clin Allergy ( 1987 ) 17 : 529-36; Norman PS, et al . , J Allergy Clin Immunol (1973 ) 52 : 210-24 ; Lichtenstein LM, et al . J Allergy Clin Immunol ( 1971 ) 47 : 103 (A37 ) ) . This is the reason why these studies could not analyse the association between allergen-specific IgE levels and biological activities at molecular levels . Recent studies using purified natural and recombinant allergens to re-investigate the relation between skin sensitivity and allergen-specific IgE levels report considerable discrepancies between these parameters (Witteman AM, et al . , J Allergy Clin Immunol ( 1996) 97 : 16-25 ; Niederberger V, et al . J Invest Dermatol (2001 ) 117 : 848-51) . In this example, purified recombinant Bet v 1 , the maj or birch pollen allergen, was used as a paradigmatic tool to further investigate the association between allergen-specific IgE levels , effector cell responses and in vivo sensitivity. In a population of 18 birch pollen-allergic patients , selected on well-defined clinical criteria, and out of the pollen season, skin sensitivity and basophil degranulation in response to defined amounts of structurally folded recombinant Bet v 1 was quantified. The results of the biological and of the serological tests were compared. For the measurement of Bet v 1-specific IgE antibody levels two different assays were used: one to detect any Bet v 1-specific IgE, and the other to detect Bet v 1-specific IgE able to bind to effector cells .

Material and methods

Study population

The examination of the patients was performed between January and April before the beginning of the birch pollen season . Eighteen patients , eight women and 10 men aged between 28 and 58 years (mean age : 45.6 years ) , were included in the study on the basis of clinical history of birch pollinosis and positive skin prick tests to birch pollen extract . All patients had moderate to severe rhino-conjunctivitis first diagnosed at least 3 years before . Five patients had mild asthma during birch pollen season and 12 patients had oral allergy syndrome with fruits of the Rosaceae family (apple, peach, apricot and almonds ) and vegetables from Solanaceae (potato, tomato) and Apiaceae family (celery, carrot) . Skin prick tests with a standard panel of respiratory allergens (Stallergenes, France) consisting of house dust mites, mixtures of fungal allergens, dog and cat dander, cockroach, grass, trees (including birch, olive and ash) and weed pollens were performed to identify the sensitisation profile . Patients ' characteristics are displayed in- the following Table 1.

Study design

To analyse the possible association between allergen-specific IgE levels, skin sensitivity, and basophil degranulation, patients were bled and their skin was tested on the same day . The analyses were carried out strictly out of the birch pollen season to exclude effects because of seasonal allergen contact . Patients were not allowed to take anti-allergic or anti-inflammatory medication at least 1 week before the study was performed. None of the patients had received allergenspecific immunotherapy over the last' 5 years . After informed consent was given, blood was collected for basophil histamine release and for serum sampling. Immediately thereafter, intradermal skin tests were performed using the end-point titration method (Grammer LC, et al . , J Allergy Clin Immunol ( 1985) 76 : 123-7 ) .

Detection and quantification of allergen-specific antibodies

Allergen-specific IgGl to IgG4 subclass levels as well as allergen-specific IgM and IgA levels were measured by ELISA using isotype-specific monoclonal antibodies as described (Vrtala S, et al . , J Allergy Clin Immunol ( 1996) 97 : 781-7 ) . Results represent means of duplicate determinations and are shown as OD values corresponding to the amount of bound antibodies .

Basophil histamine release test

The challenge of whole blood with rBet v 1 and anti-IgE as a positive control was performed in a dose response fashion according to the method described by Tanisaki et al . (Tanisaki Y, et al . , Int Arch Allergy Appl Immunol (1984 ) 73 : 141-5) . Ten mil- lilitres of venous blood was drawn into a plastic syringe containing ImI of heparin . 250μl of different concentrations of rBet v 1 (from 10^-4 to 10 mg/ml) or anti-IgE (from ICT⁴ to 10^-3; e- specific, Dako, Glostrup, Denmark) were added to the test tubes containing 500 ml of whole blood diluted 1 : 4 in Tris buffer ( 10 mmol/1 Tris, 136 mmol/1 NaCl, 2.7 mmol/1 KCl, 0.23 mmol/1 MgCl₂, 1.8 mmol/L CaCl₂, 5.5 mmol/1 glucose; pH 7.3) . The mixed solution was incubated for 30 min at 37⁰C . The reaction was stopped, and the cells were separated by cold centrifugation (4⁰C) at 375 x g

for 5 min . 200μl of the cell-free supernatant was used for histamine quantification in a radioimmunoassay with acylated histamine monoclonal antibodies ( Immunotech, Marseille, France) as described previously (Morel AM, et al . J Allergy Clin Immunol ( 1988 ) 82 : 646-54 ) . Total histamine was measured after cell lysis by repeated thawing and freezing . All experiments were performed in duplicate . The parameter used to describe the degree of basophil sensitivity was the lowest allergen concentration inducing 30% of total histamine release .

Intradermal testing

Threshold intradermal skin tests were performed by inj ection of 0.03ml of 10-fold dilutions of rBet v 1 on the lateral part of the arm. Serial dilutions were prepared from a solution of 1000 mg/ml and the first dilution tested was 10 mg/ml . The tests were read 15 min after inj ection. The area of weal and erythema was recorded. The test was considered positive when the induced weal area exceeded that of the weal induced by inj ection and the lowest concentration of allergen inducing a positive test result was used for comparison with other parameters (Grammer LC, et al . , J Allergy Clin Immunol (1985) 76 : 123-7 ) .

Statistical analysis of the data

Correlation between different parameters was tested by Spearman s non-parametric tests using VisualStats Professional software (version 2003) .

Results

Poor association between rBet v 1-specific immunoglobulin E levels and skin sensitivity to rBet v 1

To compare rBet v 1-specific IgE levels and skin sensitivity, rBet v 1-specific IgE levels were measured by CAP and correlated with the threshold concentration of rBet v 1 inducing a positive intradermal test weal reaction . Fig . 1 shows that there is no association between allergen-specific IgE levels and skin sensitivity (r=-0.007 , P=O .977 ) . In individual patients a strong discrepancy between allergen-specific IgE and skin sensitivity was observed. For example, patient 8 exhibited high Bet v 1-specific IgE level ( 79.9 kU/1) but showed a positive ID reaction only at 10 mg/ml of rBet v 1 (Table 1) . On the other hand, patient 10 had low rBet v 1- specific IgE ( 4.5 kU/1) , yet with a 1000-fold greater skin sensitivity to Bet v 1 (positive ID test reaction at 1 ng/ml of rBet v 1 ) than patient eight . Seven pa- tients (2 , 5 , 7 , 9 , 11 , 12 and 17 ) with similar rBet v 1-specif- ic IgE levels (17.1 26.6kU/l) exhibited an extremely broad range of skin sensitivity to rBet v 1 ( from 3 to 10 5 mg/ml) (Table 1 ) .

Poor association between rBet v 1-specific immunoglobulin E levels and rBet v 1-related basophil sensi tivity

Fig. 2 ( IgE vs . 30% histamine release) shows that there is also a lack of association between rBet v 1-specific IgE levels and Bet v 1-induced basophil sensitivity (Fig . 2 : r=-0.113 , P=O .656) . The concentration of rBet v 1 required to induce 30% histamine release varied from 10^-3 to 1 mg/ml . For given levels of rBet v 1-specific IgE (RAST class 3 : 4.51 17.1kU/l) , the concentration of rBet v 1 inducing 30% histamine release varied 1000-fold ( 1 10^-3 mg/ml) .

Association between rBet v 1-induced basophil histamine release and skin sensitivity

Fig. 3 shows that the results of intradermal testing and basophil histamine release tests are better associated than the results of serological and biological tests . There is a significant trend between the concentrations of rBet v 1 eliciting 30% histamine release and intradermal weal reactions (r=0.614 ; P=O .007 ) . Patients with extremely bad association between rBet v 1-specific IgE levels and results of biological testing (e . g . patients 8 and 10 ) showed better association when intradermal testing results were compared with basophil histamine release (Table 1) . Results of other tests performed in order to explain the discrepancies between serological and biological tests are given below .

Measurements of rBet v 1-specific immunoglobulin G subclasses, immunoglobulin A and immunoglobulin M

It has been described that Bet v 1-allergic patients ' sera contain Bet v 1-specific IgG antibodies that may interfere with IgE binding to Bet v 1 or recognise epitopes on the Bet v 1 molecule other than IgE and hence have no effect on IgE binding to Bet v 1 (Visco V, et al . J Immunol ( 1996) 157 : 956-62 ; Denepoux S, et al . FEBS Lett (2000 ) 465 : 39-46) . Therefore the levels of rBet v 1-specific IgG were determined (IgGl IgG4 ; Table 1) . The patients exhibited varying rBet v 1-specific IgGl IgG4 subclass responses with most pronounced responses in the IgGl and IgG4 subclasses . No significant levels of rBet v 1-specific IgA and IgM antibodies were detected in the sera, excluding the possibility that these antibody classes may influence IgE recognition of Bet v 1.

Evaluation of Bet v 1-specific immunoglobulin E as a percentage of total immunoglobulin E

If Bet v 1-specific IgE only accounts for a low percentage of total IgE, poor histamine release and skin reactivity might be explained by the fact that basophils and mast cells are primarily occupied by IgE directed against other allergens . Therefore the total IgE values were determined and the percentage of Bet v 1-specific IgE was calculated . The patients in this example had relatively low total IgE values (<168kU/L) and no association between a low percentage of Bet v 1-specific IgE and poor biological responses was found. For example, in patient 11 , who showed high sensitivity. Bet v 1-specific IgE only accounted for 20% of the total IgE . On the other hand, patient 13 was less sensitive, although 62.6% of the total IgE was directed against Bet v 1 (Table 1) .

Discussion

The question of whether allergen-specific IgE antibody levels , effector cell sensitivities , and clinical sensitivity correlate remains a matter of controversy. Several studies have shown a significant correlation of allergen-specific serum IgE antibodies with allergen-induced immediate type reactions even when using a complex mixture of various allergenic and non-al- lergenic components , which may make it difficult to compare skin tests and RAST (Stenius B, et al . , Clin Allergy (1971 ) 1 : 37-55 ; Bousquet J, et al . , Clin Allergy ( 1987 ) 17 : 529-36; Norman PS, et al . , J Allergy Clin Immunol (1973) 52 : 210-24 ; Lichtenstein LM, et al . J Allergy Clin Immunol ( 1971 ) 47 : 103 (A37 ) ) . Recently, other studies using purified allergens (Witteman AM, et al . , J Allergy Clin Immunol (1996) 97 : 16-25 ) and recombinant allergens (Niederberger V, et al . J Invest Dermatol (2001) 117 : 848-51) have demonstrated considerable discrepancies between antibody levels and biological sensitivity.

A clinical study using a defined purified and structurally folded allergen (i . e . the major birch pollen allergen, Bet v l ) to investigate the relation between specific IgE, basophil de- granulation, and skin sensitivity at a molecular level was per- formed. Good agreement between the three methodologies and clinical relevance of birch sensitivity was found; however, strong discrepancies were noted between the levels of allergen-specific IgE, the basophil sensitivity and in vivo sensitivity (i . e . skin sensitivity as determined by end-point titration) . In certain patients, very low specific IgE levels but high sensitivity in basophil degranulation and skin tests and vice versa was observed. A review of the literature reveals the scarcity of studies comparing skin tests , basophil histamine release and specific IgE levels . The few available studies showed greatly varying results and were performed with crude allergen extracts . For example, Norman et al . (Norman PS, et al . , J Allergy Clin Immunol (1973) 52 : 210-24 ) found that the three tests were in good agreement with each other in the diagnosis of ragweed hayfever . Lich- tenstein et al . (Lichtenstein LM, et al . J Allergy Clin Immunol

(1971) 47 : 103 (A37 ) ) found a quantitatively significant relationship between skin tests and histamine release . However, no measurement of specific IgE was performed in this example . The response of sensitised leucocytes and mast cells to antigen can depend on a great variety of factors .

One possibility for low sensitivity and poor release of histamine would be that only a small proportion of the total serum IgE accounts for allergen-specific IgE . Therefore the total IgE levels were determined and the percentage of allergen- specific IgE was calculated. However, an association between low percentages of allergen-specific IgE responses and poor biological activity was found. The possibility that a low percentage of specific IgE out of the total IgE may be responsible for poor biological responses towards the given allergen may be of greater importance in polysensitised subj ects (Norman PS, et al . , J Allergy Clin Immunol (1973) 52 : 210-24 ; Conroy MC, et al . J Immunol (1977 ) 118 : 1317-21 ; MacGlashan DW Jr, et al . , J Immunol

(1986) 136 : 2231-9) .

There are several other factors that may be responsible for the discrepancy between allergen-specific IgE levels and biological responses but they cannot be addressed even in a system using purified allergens . They include interindividual differences in basophil and mast cell sensitivities because of variability in IgE-receptor cell surface density, a parameter that is regulated by serum IgE levels (Conroy MC, et al . J Immunol ( 1977 ) 118 : 1317-21 ; Malveaux FJ, et al . , J Clin Invest ( 1978 ) 62 : 176-81 ; Dembo M, et al . , J Immunol ( 1978 ) 121 : 345-53 ; MacGla- shan DW Jr, et al . J Allergy Clin Immunol (1999) 104 : 492-8 ) . Different cell sensitivities have been demonstrated by variable shifts of the dose response curves (measured by 50% or 30% sensitivity) in case of similar total and antigen-specific IgE serum concentrations (Conroy MC, et al . J Immunol ( 1977 ) 118 : 1317-21 ; MacGlashan DW Jr . , J Allergy Clin Immunol ( 1993) 91 : 605-15) .

Furthermore, it has been shown that persons with equivalent numbers of IgE molecules on basophils may release 0-100% of their histamine content (Conroy MC, et al . J Immunol ( 1977 ) 118 : 1317-21) . The same has been observed for cutaneous mast cells ( Petersen LJ, et al . , J Allergy Clin Immunol (1996) 97 : 672-9 ; Bordignon V, Pet al . , Invest Allergol Clin Immunol (2000) 10 : 78-82 ) . In addition, it has been shown that early signal events occur involving sykkinase and IP3 products, which are not linked to the level of specific IgE or basophil sensitivity (MacGlashan DW Jr . , J Allergy Clin Immunol ( 1993 ) 91 : 605-15 ; Miura K, et al . , J Immunol (2001) 167 : 7027 ; MacGlashan DW Jr. , J Immunol (2003) 170 : 4914-25) .

Recent evidence indicates that mast cells may also be influenced via Toll-like receptors (Marshall JS, et al . , Int Arch Allergy Immunol ( 2003) 132 : 87- 97 ) . However, the rBet v 1 preparation used for the experiments did not contain endotoxins .

Finally, it is possible that the presence of IgE antibodies with varying affinities or binding specificities for epitopes inducing varying anaphylactic activity may have influenced serological and biological test results .

In conclusion, this study demonstrates on a molecular level that allergen-specific serum IgE levels are not necessarily related to the biological sensitivity as determined by cellular and in vivo tests . A moderate association was , however, found between the cutaneous tests and the basophil histamine release tests .

Example 2 :

To determine the sensitivity of a patient before therapy to allow the choice of the correct dose a whole blood basophil histamine release test is used. Patients with high sensitivity will be inj ected smaller doses than less sensitive patients . Be- fore treatment a dose response curve will be established with purified allergen . In parallel, cells will be stimulated with anti-IgE to determine overall cell sensitivity which may affect sensitivity to the allergen . Success of treatment should be controlled after IgG antibodies against the allergen become detectable which is usually the case after 4-8 weeks of treatment . Since blocking of IgG antibodies may be responsible for the reduction of sensitivity it may be useful to determine in parallel IgG levels to the given allergen. Again a dose response is determined with the purified allergen and anti-IgE . Either the dose giving maximal cell activation (i . e . , maximal histamine release or CD203c upregulation) is compared or the dose giving a certain degree of activation is determined and compared with the test result obtained before treatment . Materials and methods are as described in example 1.

Example 3 :

When basophil histamine release experiments were performed with washed granulocyte preparations as described (Stahl-Skov et al . 1977. J Exp Immunol 27 : 432-439) no correlation between histamine release data and skin sensitivity was found.

Histamine release was done using basophils from allergic patients . They were enriched by Dextran sedimentation, isolated, washed, re-suspended in histamine release buffer, and exposed to different concentrations of recombinant Bet v 1 ( 10^~5, 10^-4, 10^~3, ICT², 10^-1, 1 μg/ml) or anti-IgE mAb E-124-2-8 ( 1 μg/ml) in 96- well microtiter plates (TPP, Trasadingen, Switzerland) for 30 minutes at 37⁰C . After incubation, cells were centrifuged. Cell- free supernatants were recovered and analyzed for histamine content by using a commercial radioimmunoassay (Immunotech, Marseille, France) . Histamine release was expressed as a percentage of total histamine measured in cell lysates (Valent et al . , 1989 , Proc Natl Acad Sci USA 86 : 5542-5546) .

Skin prick tests were performed with serial dilutions (1 : 2) of recombinant Bet v 1 as described (Pauli et al . , 1996, J Allergy Clin Immunol 97 : 1100-1109) .

Maximal histamine released from basophils exposed to recombinant Bet v 1 (HR%-max) did not correlate with skin prick test reactions (mm²) (SPT 2 μg/ml) (r=0.224 , P=O .342 ) ( Figure 5) .

Claims

Claims :

1. Method for evaluating the allergen sensitivity of an individual and/or the clinical efficacy of an allergen immunotherapy comprising the steps :

- providing at least two samples selected from the group consisting of blood or fractions thereof, connective tissue, nasal, bronchial, skin or gut biopsy material from an individual subj ected or intended to be subj ected to an immunotherapy with at least one pure allergen or derivative thereof, wherein the samples contain cells capable of releasing mediators in response to said allergen

- contacting said sample with said allergen or derivative thereof, and

- determining the amounts of mediators released from said sample and evaluating the allergen sensitivity of the individual prior to therapy and/or the clinical efficacy of the immunotherapy by comparing said amounts .

2. Method for evaluating the allergen sensitivity of an individual and/or the clinical efficacy of an allergen immunotherapy comprising the steps :

- providing cells capable of releasing mediators in response to an IgE-allergen complex,

- contacting said cells with serum and/or plasma of said individual spiked with at least one pure allergen or derivative thereof, and

- determining the amounts of mediators released from said sample and evaluating the allergen sensitivity of the individual prior to therapy and/or the clinical efficacy of the immunotherapy by comparing said amounts .

3. Method according to claim 1 or 2 , characterised in that the mediators are selected from the group consisting of histamine, tryptase, prostaglandins, leukotrienes, especially cysteinyl leukotrienes , eosinophil cationic protein, cytokines, like in- terleukins ( IL) , especially IL-2R, CD63 , CD203c and combinations thereof .

4. Method according to any one of claims 1 to 3 , characterised in that said cells are mast and/or basophilic and/or eosinophil- ic cells .

5. Method according to any one of claims 1 to 4 , characterised in that the sample further comprises immunoglobulins ( Ig) , especially immunoglobulin G ( IgG) .

6. Method according to any one of claims 1 to 5 , characterised in that the samples are provided before and after subj ecting said individual to an immunotherapy.

7. Method according to any one of claims 1 to 6, characterised in that the samples are provided after subj ecting said individual to an immunotherapy .

8. Method according to any one of claims 1 to 5 , characterised in that the at least one sample is provided after a maximum of 1 hour, 12 hours, 24 hours, 10 days , 4 weeks , 6 months and 36 months, after subj ecting said individual to an immunotherapy.

9. Method according to any one of claims 1 to 8 , characterised in that said allergen is recombinantly produced.

10. Method according to claim 9, characterised in that said allergen comprises at least one deletion, at least one substitution or at least one insertion.

11. Method according to claim 9 , characterised in that said allergen is modified by reshuffling the fragments of said allergen by genetic engineering .

12. Method according to any one of claims 1 to 11, characterised in that said sample is contacted with varying concentrations of said allergen .

13. Method according to claim 12 , characterised in that the concentration of said allergen is selected within the range of lng/ml to lOOμg/ml, preferably within the range of lpg/ml to lOμg/ml .

14. Method according to any one of claims 1 to 13 , characterised in that further total amount of the mediator of said cells is determined.

15. Method according to claim 14 , characterised in that a degree of cellular sensitisation is defined by determining the concentration of said allergen inducing the release of 10%, preferably 30%, of the total amount of the mediator of said cells .

16. Method according to claim 15, characterised in that the the allergen sensitivity of an individual and/or the clinical efficiency of an allergen immunotherapy is evaluated by observing the degree of cellular sensitisation in the course of said immunotherapy.

17. Method according to any one of claims 1 to 16, characterised in that the mediator in the sample is determined by an immunological and/or a chromatographical method.

18. Method according to claim 17 , , characterised in that the method is selected from the group consisting of radioimmunoassay

(RIA) , enzyme linked immunosorbent assay (ELISA) , high performance liquid chromatography (HPLC) , reverse transcriptase polymerase chain reaction, immunofluorescence flow cytometry and combinations thereof .

19. Method according to any one of claims 1 to 18 , characterised in that said allergen is selected from the group of the maj or birch pollen allergens, in particular Bet v 1 and Bet v 4 , the major timothy grass pollen allergens, in particular PhI p 1 , PhI p 2, PhI p 5, PhI p 6 and PhI p 7 , the major house dust mite allergens, in particular Der p 1 and Der p 2 , the maj or cat allergen FeI d 1, the major bee allergens, the major wasp allergens , profilins, especially PhI p 12 , and storage mite allergens , especially Lep d 2

20. Kit for evaluating the allergen sensitivity of an individual or the clinical efficiency of an allergen immunotherapy for at least one allergy comprising

- at least one allergen for inducing a mediator release of cells capable of releasing mediators in response to an allergen, - means for detecting the mediator, and

- optionally at least one mediator standard.

21. Kit for evaluating the allergen sensitivity of an individual or the clinical efficiancy of an allergen immunotherapy for at least one allergy comprising at least two of the following components :

- at least one allergen for inducing a mediator release of cells capable of releasing mediators in response to an allergen,

- means for detecting the mediator,

- at least one mediator standard, and

- cells capable of releasing mediators in response to an IgE-allergen complex .

22. Kit according to claim 21 , characterised in that said cells are mast and/or basophilic and/or eosinophilic cells .

23. Kit according to any one of claims 20 to 22 , characterised in that said allergen is selected from the group consisting of major birch pollen allergens, in particular Bet v 1 and Bet v 4 , maj or timothy grass pollen allergens, in particular PhI p 1 , PhI p 2, PhI p 5, PhI p 6 and PhI p I ₁ major house dust mite allergens, in particular Der p 1 and Der p 2 , maj or cat allergen FeI d 1, major bee allergens, maj or wasp allergens, profilins, especially PhI p 12 , and storage mite allergens , especially Lep d 2.

24. Kit according to any one of claims 20 to 23, characterised in that the means for detecting the mediator are selected from the group consisting of antibodies .