A method for quantification of allergens

TECHNICAL FIELD [0001] The present invention relates to the quantification of allergens in the field.

BACKGROUND OF THE INVENTION [0002] Allergens are antigenic molecules that induce allergic responses and the production of IgE antibodies in humans. They are used for both diagnosis and treatment of allergies, i.e. immunotherapy, in which they are used in the form of allergen vaccines. Sources of allergenic materials to which humans are exposed, such as food, pollen or mite excrement particles, naturally occur as complex mixtures of primary and secondary allergens. The main allergens are allergens to which most patients who are allergic to the source react. However, it seems that any protein is a potential allergen, as more and more minor allergens are identified as knowledge progresses.

[0003] Due to the complexity of allergen sources, the amino acid sequences of several allergens were first deduced from the nucleotide sequence derived from cDNA. Cloning of genes encoding allergens has shown that most allergens are heterogeneous and that they exist as mixtures of isoallergens and variants. Alignment of the amino acid sequences of homologous allergens and isoallergens has shown that they can be identified by and / or divided into constant and variable region sequences. The amino acid sequences of the constant regions are unique for the species, however the amino acid sequences of the variable regions are unique for each of the isoallergens.

[0004] Conventional immunotherapy and allergen-specific diagnostics are currently carried out by using standardized natural allergen extracts, which are further formulated as allergen vaccines. These aqueous vaccines are based on allergenic natural source materials, such as tree and grass pollen, cultures of mites and hair particles and dandruff. It is known that the composition of these natural source materials varies considerably depending on when and where these allergenic source materials are collected. Commercial allergen vaccines with mixtures of allergens can also be formulated using various species.

[0005] Knowledge of the composition of the extracts and the content of relevant allergens is essential for the repeatability, safety and efficacy of the final product. A major challenge in the production of allergen vaccines is standardization, i.e. obtaining constant potency from batch to batch of product. Since the raw material has a natural origin, there is considerable variability that must be controlled by science-based measures. The composition of the extract should ideally be a reflection of the water-soluble components of the source of the allergenic material, as it is extracted on the surface of the mucous membranes of the respiratory tract and is presented to the human immune system. However, all extracts contain several allergens involved in total IgE binding in various combinations for individual patients. Ideally, all ingredients must be controlled both qualitatively and quantitatively, but this is not practically possible with current technology.

[0006] Standardization is currently carried out in different ways because each manufacturer has specific standardization procedures for the company. Standardization is carried out using techniques such as SDSPAGE, isoelectric focusing in addition to various immunoelectrophoretic (QIE) and ELISA techniques using mono and / or polyclonal antibodies and radioallergosorption techniques (RAST) or related techniques. Optimal batch-to-batch standardization, such as the SQ standardization procedure is essentially a three-step procedure: 1) ensuring optimal composition and constant proportions between all

EP 1 931 998 B1 to the components by semi-quantitative immuno-electrophoretic techniques, 2) determination of major allergen components by quantitative immunoelectrophoresis, and 3) adjustment of the overall IgE binding power as determined by Magic Lite® assays. In Europe, all standardization is currently carried out according to the company's own reference preparations, while in the US, the FDA sets standards that all manufacturers should apply. All quantitative steps in these currently used techniques depend on the antibodies as reagents and as such are sensitive to changes over time.

[0007] The absolute quantification of specific vaccine components in complex mixtures is not simple and has not yet been established as a sensitive, routine high-throughput technique.

[0008] Routine and high throughput techniques for reliable detection and quantification of food allergens are also needed in the food industry. Nuts can be found as a hidden part of food due to accidental cross-contamination during production. Companies that produce similar foods with and without e.g. nuts may have difficulty cleaning food production equipment between producing different types of food. Traces of previously produced foods such as nuts may remain on the equipment. The first batches of food produced without nuts that pass through the same equipment will probably contain trace amounts of nuts. Foods that may cause allergic reactions due to cross-contamination with nuts or peanuts are e.g. chocolate, sweets, cakes, desserts, sweets, donuts, cereals, milkshakes, granola bars, granola, pancakes, muffins, ice cream, barbecue sauce. Cow's milk may cause an allergic reaction to small amounts of milk protein from dairy products, cow's milk, baby formula based on cow's milk, or baby food containing milk protein. To avoid milk protein contamination during the production of baby foods or baby formula for baby allergic to milk, there is a need for a method for detecting and quantifying milk allergens. Reliable detection and quantification methods for food allergens are needed to ensure compliance with food labeling and to improve consumer protection. Physicochemical methods, e.g. mass spectrometry and immunological methods have been described. The usual criteria for sensitivity, specificity, repeatability, precision and accuracy must be met. However, problems of cross-reactivity, matrix effects and food processing still remain. Biological activity may remain when the protein is denatured.

[0009] Biological mass spectrometry (MS) was used for the first time to assess molecular weight and the identity of proteins and peptides. In recent years, advances in mass spectrometry have resulted in techniques that can be used to quantify various biomolecules from complex mixtures, such as plasma, cell and tissue samples. Earlier quantification techniques only established relative quantification of proteins, while newer techniques assess the absolute amounts of molecules of interest. The rapid development of quantification techniques is mainly the result of advances in the field of proteomics, particularly in applications that distinguish between health and disease states, and in the identification of marker molecules for several diseases such as cancer, rheumatoid arthritis and Alzheimer's disease. The main advantage of these techniques for quantification by MS is the high sensitivity of the techniques in the range from 300 amol to 300 fmol samples.

[0010] WO 2004/070352 discloses a method for quantifying peptides against an internal standard using isobaric labeling reagents or sets of isobaric labeling reagents. [0011] US 6,872,575 discloses a method for determining one or more proteins in complex sample mixtures without purifying the protein or obtaining its composite peptide signature.

[0012] US 6,864,089 discloses a quantitative assay method employing differential isotope labeling of peptide or protein samples.

[0013] Other methods for quantifying proteins using MS techniques are e.g. the AQUA technique using internal calibration peptides synthesized with incorporated stable isotopes (¹³C ¹⁵N) to mimic native peptides created by enzymatic digestion using e.g. trypsin (Stemmann O et al. Cell 2001; 107 (6): 715-26, Gerber SA et al. Proc Natl Acad Sci USA 2003; 100 (12): 6940-5).

[0014] Another method is the ICPL (Isotope Coded Protein Labeling) method described by Kellermann et al., Proteomics 5, 4-15, using e.g. ¹²C /¹³Nicotinic acid C6-succinnamide as an ICPL tag.

[0015] Mass spectrometry was first introduced in allergy research to characterize natural allergens, including post-translational modifications such as glycosylation patterns. It was further used to characterize recombinant isoallergens and / or variants, many of which were expressed in various expression systems, such as Escherichia coli, Pichia pastoris and Baculovirus expression systems.

[0016] Helsper et al., J Alergia Clin Immunol, vol. 110, No. 1 (2002), pages 131-138 describes the use of MS to study the actual expression of allergen isoforms identified by PCR cloning, and in

Swoboda et al., J. Biol Chem, vol. 270, No. 6 (1995), pp. 2607-2613, liquid chromatography, MS and cDNA cloning is used to analyze the isoforms of the main birch pollen allergen, Bet v 1.

[0017] In Kristiansson et al., RAPID COMMUNICATIONS IN MASS SPECTROMETRY, vol. 18, No. 14 (2004), pp. 1592-1598, mass spectrometry is used to measure the amount of tryptic peptides with HHPA adducts of human serum albumin in nasal washes.

[0018] None of these references describe mass spectrometry to quantify a group of allergen or homologous allergen isoforms in a sample, and none of these references describe the use of calibration standard peptides with an amino acid sequence that is identical to the constant amino acid sequence found in an allergen group to be quantified.

[0019] Thus, there is still a need for a sensitive method by which active ingredients such as allergens from the same species or different species, and / or isoallergens, e.g. in a vaccine, can be quantified. The method using MS techniques and species and allergen specific sequences is a very sensitive method by which the content of allergen groups (isoallergens or homologous allergens) can be quantified. The method according to the invention is useful e.g. in the release assay to ensure a safe and accurate amount of allergen during vaccine manufacture in the final product, as well as during various stages of storage of active ingredients and / or products. This method will also be beneficial in developing second-generation allergen vaccines, e.g. using recombinant allergens as active ingredients. This method will allow optimization of the active ingredients in second-generation allergen vaccines based on the knowledge and / or composition of current vaccines.

SUMMARY OF THE INVENTION [0020] In accordance with the present invention, a method for absolute quantification of allergens from a number of sources is provided.

[0021] According to one aspect, the invention provides a method for quantifying the absolute amount of an allergen in an allergen sample, wherein the allergen consists of more than one isoallergen (isoallergens) or homologous allergen (allergens), comprising the following steps:

a) providing a known amount of one or more reference peptide (s) for calibration of allergens having an amino acid sequence which is identical to the sequence that can be found in the allergen to be quantified by identifying a constant amino acid sequence within the allergen, by comparing the amino acid sequence of isoallergens or homologous allergens and preparing a synthetic calibration standard peptide having this constant sequence and labeling this calibration peptide (s) for allergen calibration by introducing mass modifying functional groups,

b) degradation of the allergen sample to obtain a mixture of peptides, and optionally labeling these peptides with one or more labeling agents by introducing mass modifying functional groups, wherein if both the peptides in the degraded allergen sample and the allergen calibration standard peptide (s) are labeled, the factor (s) used to label the allergen calibration standard peptide is different from the factor (s) used to label the degraded allergen sample,

c) quantifying the absolute amount of allergen by correlating the amount of allergen calibration standard peptide (s) with the amount of corresponding peptide (s) of the degraded allergen sample by mass spectrometry.

[0022] In one embodiment of the invention, the allergen to be quantified is composed of more than one isoallergen, e.g. members of a group of allergens from the same species with greater than> 67% amino acid sequence identity.

[0023] In another embodiment, the allergen to be quantified is composed of more than one homologous allergen.

[0024] According to the invention, the use of an amino acid sequence which is identical to the sequence that can be found in isoallergens (isoallergens) or homologous allergen (allergens) to be quantified is provided as an allergen calibration standard peptide to determine the absolute amount of allergen (allergens) and possibly identification. Preferably, the degradation step bb) results in a mixture of peptides, wherein one of the peptides contains the same amino acid sequence as the allergen calibration standard peptide.

[0025] According to the invention there is provided a method of obtaining an allergen calibration standard peptide for use in the quantification of isoallergens or homologous allergens, wherein the allergen calibration standard peptide is obtained by:

identifying an amino acid sequence that is constant in isoallergens or homologous allergens to be quantified by comparing with the isoallergen or homologous allergen sequences and preparing a synthetic reference peptide for calibrating the allergen having this constant sequence.

BRIEF DESCRIPTION OF THE FIGURES [0026]

Fig.1. Alignment of the amino acid sequence of the mite group 2 allergen (fig 1a) and Bet v 1 (fig 1b), the major birch allergen using Vector NTI software (Invitrogen). Presumed sequences

The amino acids that can be used as internal calibration standard peptides (useful for quantifying isoallergens) are in bold.

Fig.2. Theoretical enzymatic cleavage by trypsin of house dust mite allergens, a) Der f 2 and b) Der p 2; c) Phl p 1, d) Phlp 5a e) Phl p 5b and f) Bet v 1 (GPMAW, Lighthouse data). Species specific peptides selected for quantification are bold and gray in color.

Figure 3. a) MALDI-TOF MS fingerprint type analysis of a mixture of purified and trypsin digested natural Der f 2 and Der p 2 (1: 1) and b) MALDI-TOF MS fingerprint analysis of a mixture of recombinant Der f purified and digested trypsin 2 and Der p 2 (1: 1).

Fig. 4. SDS-PAGE analysis of the HDM (House Dust Mite) allergen extract separated using hydrophobic interaction chromatography. The HDM protein fractions were divided into two main pools of protein (I and II) and then quantified.

Fig. 5. Strategy for sample labeling using ITRAQ ™ chemistry (Applied Biosystems, Foster City, CA, USA) in allergen quantification.

Fig. 6. MS analyzes of iTRAQ labeled a) Peptide 1, m / z 2353.44 (Der p 2, 32-48) b) Peptide 2, m / z 2326.35 (Der f 2, 32-48) c) trypsin digested and labeled iTRAQ nDer p 2 peptides and d) nDer f 2 peptides.

Figure 7. MS / MS fragmentation of the mixture nDer f 2, nDer p 2, Peptide 1 and Peptide 2. The amount of isoallergens, nDer p 2 (114.10) and Der f 2 (115.10), in the sample mixture was calculated as the ratio of signal surface m / z 114 to surface m / z 116 (Peptide 1) and as the ratio of the surface m / z 115 to the surface m / z 117 (Peptide 2). Fig.8. Reverse phase analysis of a mixture of nDer f 2, nDer p 2, Peptide 1 and Peptide 2 from SCX chromatography. MS and MS / MS analysis was used to identify the peaks detected for the MALDITOF target.

Figure 9. MS analysis of the mixture of nBet v 1 trypsin digested and internal calibration standard (AQUA peptide). The 6 Da mass difference between the native peptide and the calibration standard is shown in the upper corner of the figure.

DETAILED DESCRIPTION OF THE INVENTION [0027] In the present context, the term "allergen" refers to any naturally occurring protein, modified protein, recombinant protein, recombinant mutant protein, or any protein fragment or protein mixtures thereof that has been reported to be subject to secondary exposure induce allergic reactions, i.e. with IgE.

[0028] Examples of naturally occurring allergens include pollen allergens (pollen allergens from trees, weeds, herbs and grasses), mite allergens (from e.g. house dust mites and storage mites), insect allergens (derived from inhalants, saliva and venom), animal allergens from e.g. saliva, hair and dandruff e.g. dog, cat, horse, rat, mouse etc., fungal allergens and food allergens.

[0029] Significant pollen allergens from trees, grasses and herbs are those derived from the taxonomic orders of Fagales, Oleales, Pinales and platanaceae including, inter alia, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus), olive tree (Olea), cedar (Cryptomeria and Juniperus), plane tree (Platanus), the Poales range including grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum and the rows of Asterales and Urticales including, among others herbs of the orders Ambrosia, Artemisia and Parletaria. Other important inhalant allergens are those from house dust mites of the order Dermatophagoides and Euroglyphus, storage mites e.g. Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, flies and fleas e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and those from mammals such dog and horse, venom allergens

EP 1 931 998 B1 including those derived from stinging or biting insects such as those of the Hymenoptera taxonomic order including bees (superfamily Apidae), wasps (superfamily Vespidea) and ants (superfamily Formicoldae). Important inhalation allergens from fungi include those derived from the orders Alternaria, Cladosporium, Aspergillus and Penicillium.

[0030] Examples of food allergens are wheat allergens (e.g. Tri a 18-19), crustaceans including shrimp (e.g. Met e 1, Pen a 1, Pen I 1, Pen m 1 and Pen m 2), king prawn, crab and lobster, fish (e.g. Gad c 1 and Sal s 1), chicken egg (e.g. Gal d 1, Gal d 2), peanuts (e.g. Ara h 18), soybean (Gly m 1-4), cow's milk (Bos d 4-8), nuts such as almond (Pru du 4), brazil nut (Ber e 1, Ber e 2), cashew (Ana o 1-3), hazelnut (e.g. Cor a 1.04, Cor a 2, Cor a 8) and walnut (e.g. Jug n 1-2, Jug r 1-3), celery (Api g 1, Api g 4, Api g 5), mustard (Sin a 1 and Bra j 1) and sesame seeds (sets and 1-6), and in particular allergens from wheat (e.g. Tri a 18-19), chicken egg (e.g. Gal d 1, Gal d 2), peanuts (e.g. Ara h 1-8), soy (Gly m 1-4), cow's milk (Bos d 4-8).

[0031] Examples of recombinant allergens include, but are not limited to, protein / peptide from plant pollen, grass pollen, tree pollen, weed pollen, insect venom, dust mite proteins and storage, animal dandruff, saliva, mushroom ingestion and food allergens ( i.e. peanut, milk, gluten and egg) prepared using recombination techniques. Recombinant allergens can be obtained e.g. on a large scale using microorganism expression systems that can be grown in fermenters, produced by recombinant DNA techniques, or chemical precursors or other chemical compounds when chemically synthesized. In one embodiment of the invention, the allergen is rBet v 1, rAin g 1, rCor a 1, rCar b 1, rCry j 1, rCry j 2, rOle e 1, rAmb a 1, rArt v 1, rCyn d 1, rDac g 1 , rLol p 1, rLol p 5, rPhl p 1, rPhl p 5, rPoa p 1, rPoa p 5, rSor h 1, rDer f 1, rDer f 2, rDer p 1, rDer p 2, rEur m 1, rEur m 2, rGly d 1, rLep d 2, rBla g 1, rBla g 2, rFel d 1, rCan f 1, rCan f 2, rBos d 2, rEqu c 1, rEqu c 2, rMus m 1, rApis m 1 , rApi m 2, rVes v 1, rVes v 2, rVes v 5, rDol m 1, rDol m 2, rDol m 5, rPol a 1, rPol a 2, rPol a 5, rAlt a 1 or rCla h 1 (r is recombinant).

[0032] The recombinant mutant allergen differs from the wild type in that the genes for the allergens have been modified by genetic manipulation methods such that the polypeptides they encode exhibit substitution, deletion and / or addition of single or several amino acids compared to the wild type. Examples of the recombinant mutant allergen include substituted allergen variants, addition variants, oligomers, fragments, deletion variants, hybrid molecules and other variants.

[0033] Examples of the modified allergen include allergens that in naturally occurring forms are associated with allergic disease conditions in sensitive individuals, wherein the modified recombinant allergen is altered compared to the naturally occurring allergen. Allergen variants containing several amino acid substitutions, allergen mutants, oligomers, fragments, deletion variants, hybrid molecules, myristylated, glycosylated, palmitylated and phosphorylated allergens and other variants are included. The modified allergen can be produced by any suitable method such as site-directed mutagenesis, PCR method, chemical synthesis and combinations of these methods.

[0034] In one embodiment of the invention, the allergen to be quantified is selected from one or more from the group Bet v 1, Aln g 1, Cor a 1 and Car b 1, Que a 1, Cry j 1, Cry j 2 , Cup a 1, Cup s 1, Jun a 1, Jun a 2, Jun a 3, Ole e 1, Lig v 1, Syr v 1, Pla I 1, Pla a 1, Pla a 2, Amb a 1, Amb a 2, Amb t 5, Art v 1, Art v 2, Art v 3, Par j 1, Par j 2, Par j 3, Sal k 1, Ave e 1, Cyn d 1, Cyn d 7, Dac g 1 , Fes p 1, Hol I 1, Lol p 1 and 5, Pha a 1, Pas n 1, Phl p 1, Phl p 2, Phl p 3, Phl p 4, Phl p 5, Phl p 6, Poa p 1, Poa p 5, Sec c 1, Sec c 5, Sor h

EP 1 931 998 B1

1, Der f 1, Der f 2, Der f 3, Der f 7, Der p 1, Der p 2, Der p 3, Der p 7, Der m 1, Eur m 1, Eur m 2, Gly d 1, Gly d 2, Lep d 1, Lep d 2, Blo t 1, Tyr p 2, Bla g 1, Bla g 2, Per a 1, Per a 3, Per a 7, Fel d 1, Fel d 2, Fel d 3, Fel d 4, Can f 1, Can f 2, Bos d 2, Equ c 1, Equ c 2, Equ c 3, Mus m 1, Rat n 1, Apis m 1, Apl m 1, Apl m 2, Ves v 1, Ves v 2, Ves v 5, Ves f 5, Ves g 5, Ves m 1, Ves m 2, Ves m 5, Ves p 5, Ves s 5, Ves vi 5, Dol m 1, Dol m 2, Dol m 5, Dol a 5, Pol a 1, Pol a 2, Pol a 5, Sol I 1, Sol i 2, Sol f 3 and Sol i 4, Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, Cla h 1, Cla h 2, Cla h 6 Asp f 1, Bos d 4, Mal d 1, Mal d 3, Gly m 1, Gly m 2, Gly m 3, Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5 or hybrid any of them.

[0035] In another embodiment of the invention, the allergen to be quantified is one or more isoallergens selected from the group of grass pollen allergens such as Phl p 1, Phl p 5, Phl p 6, Poa p 1, Poa p 5, Dac g 1, Fes p 1, Lol p 1 and Lol p 5, dust mite allergens such as Der f 1, Der f 2, Der p 1 and Der p 2, venom allergens such as Api m 1, Apl m 2, Ves v 1 , Ves v 2, Ves v 5, Dol m 1, Dol m 2, Dol m 5, Dol a 5, Pol a 1, Pol a 2, and Pol a 5, weed allergens such as Amb a 1, Amb a 2, Par j 1, Par o 1 and Par m 1, birch allergens such as Bet v 1, Japanese cedar allergens such as Cry j 1 and Cry ji 2, cockroach allergens such as Per a 1, olive pollen allergens such like Ole e 1, cat allergens such as Fel d 1, dog allergens such as Can f 1 and Can f 2, horse allergens such as Equ c 1 and Equ c 2, mugwort allergens like Art v 1, Art v 2, Art v 3, mold allergens such as Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, Cla h 1, Cla h 2 and Cla h 6 and Solenopsis ant allergens such as Sol 2, Sol I 3 and Sol i 4.

[0036] In yet a further embodiment of the invention, the allergen to be quantified is one or more isoallergens selected from the group of grass pollen allergens such as Phl p 1, Phl p 5 and Phl p 6, olive pollen allergens such as Ole e 1 , dust mite allergens such as Der f 1, Der f 2, Der p 1 and Der p 2, venom allergens such as Ves v 1, Ves v 2 and Ves v 5, weed allergens such as Amb a 1, Amb a 2, Par j 1, Par o 1 and Par m 1 and tree allergens such as Bet v 1, Cry j 1 and Cry j 2.

[0037] In yet a further embodiment, the allergen to be quantified is one or more isoallergens selected from the group consisting of Der f 1, Der p 1, Der f 2 and Der p 2.

[0038] In yet a further embodiment, the allergen to be quantified is one or more isoallergens selected from the group consisting of Phl p 1, Phl p 5, Phl p 6, Poa p 1, Poa p 5, Dac g 1, Fes p 1, Lol p 1, Lol p 5.

[0039] In yet a further embodiment, the allergen to be quantified is one or more isoallergens selected from the group consisting of Amb a 1 and Amb a 2.

[0040] An allergen from a single species may be composed of several very similar molecules. These similar molecules are referred to as isoallergens when they share the following biochemical characteristics in common: a. Similar molecular size; b. identical biological function, if known, e.g. enzymatic activity; and c.> 67% amino acid sequence identity. In the present context, members of the allergen group who have> 67% amino acid sequence identity and are of the same species are referred to as isoallergens. Each isoallergen can have multiple forms of very similar sequences with only a few amino acids difference; these are referred to as variants, and fall under the term "isoallergen" in the present context.

[0041] In the present context, the term "homologous allergens" refers to allergens from various species known to have similar three-dimensional structures, molecular size, identical biological function, if known, e.g. enzymatic activity and may have common structural epitopes for antibodies IgE. In a further embodiment of the invention, the homologous allergens have> 20% identity

And a common amino acid sequence, preferably a sequence of at least 2-20 amino acids, more preferably 4-15 and most preferably 6-10.

[0042] As an example of homologous allergens, for example, Amp m 2 and Ves v 2, and Der f 2 and Der p 2 can be mentioned. [0043] In the present context, the expression "allergen extract" refers to any extract obtained by extraction of biological source material, as generally described in "Allergenic extracts", H. Ipsen et al., chapter 20 in Allergy, principle and practice (ed. S. Manning) 1993; Mosby-Year Book, St. Louis. Such an extract can be obtained by aqueous extraction of water-soluble material, followed by purification steps, such as filtration, to obtain a solution, i.e. an extract. The extract may then be subjected to further purification and / or treatment, such as freeze-drying, substantially removing all water. Usually, the allergen extract contains a mixture of proteins and other molecules. Allergen proteins are often classified as the main allergen or indirect allergen, minor or non-allergenic. Allergen extracts generally contain both major and minor allergens. Main allergens will generally constitute about 5-15% of the average allergen extract, more often about 10%. Allergen classification is based on an assessment of the clinical significance of an allergen and is given below. Examples of important major allergens found in the extract include group 1 and 5 and 6 grass allergens (e.g. Phl p 1, 5, and 6), dust mite allergens group 1 and 2 (e.g. Der p 1, Der p 2), pollen allergen grasses 1 (Bet v 1), cedar pollen allergen 1 and 2 (e.g. Cry j 1, Cry j 2), ragweed pollen 1 and 2 (Amb a 1, Amb a 2), cat allergen 1 (i.e. Fel d1) .

[0044] The expression "biological allergen source material" as used herein refers to any biological material containing one or more allergens. Examples of such materials are PMB (Pure Mite Body) mites or WMC (Whole Mite Culture), degreased or non-degreased pollen from e.g. grasses, herbs, weeds and trees, animal hair and dandruff, fur, mycelium and spores of fungi, insects venom or saliva and foods.

[0045] Biological allergen source materials may include contaminating materials such as residual foreign pollen and plants and flowers from the allergen source material. The maximum level of pollution accepted from pollen from other species is 1%. They should also not contain flower and plant remains with a 5% by weight limit.

[0046] The term "allergen vaccine" as used in the present context includes at least one allergen either from the same allergic source or from other allergic sources e.g. group 1 and group 5 grass allergens or group 1 and group 2 mite allergens from different mite and grass species, respectively, weed antigens such as short and giant ragweed allergens, allergens of various fungi such as alternaria and cladosporium, tree allergens such as birch, hazel, hornbeam allergens, oak and alder, food allergens like peanut, soy and milk allergens.

[0047] Preparation of vaccines is generally well known in the art. Vaccines are typically prepared for injection either as liquid solutions or suspensions. Such a vaccine may also be emulsified or formulated to allow nasal as well as oral administration, including buccal and sublingual administration. The immunogenic component of interest may be suitably mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Examples of suitable excipients are water, saline, dextrose, glycerol and the like as well as combinations thereof. The vaccine may additionally contain other substances such as wetting agents, emulsifying agents, buffering agents or adjuvants that increase the effectiveness of the vaccine.

[0048] According to an aspect of the invention, a method for quantifying the absolute amount of an allergen in an allergen sample, which allergen consists of more than one isoallergen (isoallergens) or homologous allergen (allergens) comprises the following steps:

a) providing a known amount of one or more peptide (s) for calibration of the allergen having an amino acid sequence which is identical to the sequence to be found within the allergen to be quantified by identifying a constant amino acid sequence within the allergen, which is to be quantified by comparing the amino acid sequence of isoallergens or homologous allergens and preparing a synthetic allergen calibration standard peptide having this constant sequence and labeling this allergen (allergen) calibration standard peptide by introducing mass modifying functional groups,

b) degrading the allergen sample to obtain a mixture of peptides, and optionally labeling these peptides with one or more labeling agent (s) by introducing mass-modifying functional groups, wherein if both peptides in the degraded allergen sample and the allergen calibration standard peptide (s) are marked, the labeling factor (s) used to label the peptide (s) for calibration of the allergen is different from the labeling factor (s) used to label the peptides of a degraded allergen sample,

c) determination of the absolute amount of allergen by correlating the amount of peptide (s) to calibrate the allergen with the amount of the corresponding peptide (s) of the degraded allergen sample by mass spectrometry.

[0049] In one particular embodiment of the invention, both the calibration standard peptide (s) and the peptides of the degraded sample are labeled, but with other labeling agents.

[0050] In another embodiment of the invention, the calibration standard peptide (s) is labeled and the peptides of the degraded sample are not labeled.

[0051] In a preferred embodiment of the invention as described above, one allergen calibration standard peptide is provided in step a). Thus, preferably, only one allergen calibration standard peptide is used for each allergen sample.

[0052] According to another preferred embodiment of the invention, the degraded sample in step b) is labeled with only one labeling agent if it is labeled.

[0053] Thus, preferably, only one allergen calibration standard peptide is provided in step a) and if labeled, the degraded sample in step b) is only labeled with one labeling agent.

[0054] Furthermore, the calibration standard peptide is preferably labeled with only one labeling agent.

[0055] Mass analysis, e.g. MS as such, can be carried out on mixtures of several pairs of allergen samples and the allergen calibration standard peptide provided according to the invention for this particular allergen sample.

[0056] In the present context, the term "allergen calibration standard peptide (s) having an amino acid sequence, which sequence is identical to the one found in the allergen (s) to be quantified" refers to the region of the amino acid sequence that it is constant, i.e. identical in the group of allergen isoallergens or in homologous allergens to be quantified. According to a preferred embodiment of the invention, the allergen calibration standard peptide (s) is selected such that degradation at stage b) of the allergen (isoallergens or homologous

(Allergens) to be quantified gave a mixture of peptides, wherein one of the peptides in the mixture contains the same amino acid sequence as the allergen calibration standard peptide.

[0057] Mass analysis, e.g. MS as such, can be performed on mixtures of several pairs of allergen samples and the allergen calibration standard peptide provided according to the invention, for this particular allergen sample.

[0058] The number of amino acids in the allergen calibration standard peptide is preferably in the range of 2-20 amino acids, more preferably in the range of 4-15 and most preferably in the range of 6-15. The number depends on the optimal enzymatic cleavage site that was found as the sequence corresponding to the amino acid sequence within the sample, i.e. the constant or variable region sequence when the sample is enzyme cleaved. Furthermore, the allergen calibration pattern to be used according to the invention depends on the label and quantification method to be used to obtain detectable signal and fragmentation when analyzed in an MS instrument.

[0059] In the present context, the term "allergen sample" refers to a sample containing one or more allergens.

[0060] In one embodiment of the invention, the allergen sample comprises an allergen extract, a naturally occurring purified allergen, a modified allergen, a recombinant allergen, a recombinant mutant allergen, any allergen fragment, a mixture of isoallergens, or a mixture of homologous allergens, or a combination thereof, and an allergen extract containing a synthetic addition of purified natural or recombinant allergens.

[0061] The allergen sample may be in the form of a final product, such as an allergen vaccine in the form of a tablet or solution, or a product / intermediate taken during manufacture, for example after extraction of the biological source material of the allergen or raw material.

[0062] In a preferred embodiment of the invention, the allergen sample is in the form of an allergen extract, a final tablet product or an intermediate product.

[0063] In one aspect of the invention, an allergen extract is provided, which allergen extract is composed of a natural allergen and a recombinant allergen and is obtained by quantifying the amount of allergen in the natural extract and adding the recombinant allergen or natural purified allergen to the extract the amount of allergen in the final extract, such as a natural extract to which a purified natural or recombinant allergen has been artificially added.

[0064] The method of the invention makes it possible to quantify an allergen (s) present as isoallergens or homologous allergens and having a common constant sequence of amino acids from one or more species in an allergen sample simultaneously or in one operation.

[0065] It is possible to quantify species isoallergens in an allergen sample simultaneously or in one procedure using the method of the invention.

[0066] Depending on the sample, it may be necessary to use denaturing agents and buffer solutions to obtain a suitable solution.

[0067] If the sample contains substances such as thiols eg DTT or mercaptoethanol, high concentrations of detergent and / or denaturing agents such as SDS, octyl BD-glucopyranoside and Triton^® X-100 and / or active proteases or primary amines (other than the allergen of interest) that may interfere with the method of the invention, sample preparation may involve various treatments e.g. acetone precipitation. Recommended buffers and alternative detergents and / or denaturing agents and substances that may interact with the method of the invention are listed e.g. in Applied

EP 1 931 998 B1

Biosystems iTRAQ ™ Reagents Amine-Modifying Labeling Reagents for Multiplexed Relative and Absolute Protein Quantification Protocol from Applied Biosystems, Foster City, CA, USA.

[0068] Depending on the complexity of the sample, it may be advantageous to pre-fractionate the sample before degradation, e.g. if there are molecules present in the sample that interfere with the detection of the allergen (s) of interest. It may also be necessary to separate / elute the sample from its formula and / or any adjuvant e.g. aluminum hydroxide or calcium phosphate. To obtain a less complex mixture, the sample can be fractionated by using various chromatography techniques such as hydrophobic interaction chromatography, ion exchange chromatography and / or immunoaffinity.

[0069] In one embodiment of the invention, the allergen sample is a fraction resulting from the pre-fractionation of e.g. an allergen extract fraction containing one or more isoallergens.

[0070] In one embodiment of the invention, the allergen sample is a fraction from a preliminary fractionation step where the sample was fractionated according to size, solubility, electric charge and / or ligand specificity. In a further embodiment of the invention, the pre-fractionation is carried out by means of chromatography, such as by means of hydrophobic interaction chromatography, reverse phase chromatography, ion exchange chromatography, size exclusion chromatography or affinity chromatography e.g. by means of hydrophobic interaction chromatography.

[0071] An example of a pre-fractionation of an intermediate containing HDM allergens of groups 1 and 2 by the use of hydrophobic interaction chromatography is shown in Fig. 4. Fractions containing HDM allergens of groups 1 and 2, respectively, are separated based on their physicochemical properties and identified by immunoprecipitation. Both fractions can then be subjected to quantitative testing.

[0072] In one embodiment of the invention, the allergen sample is desalted after chromatography.

[0073] In one embodiment of the invention, the allergen sample is reduced and any cysteine ​​residue is blocked from degradation, for example by alkylation.

[0074] According to the invention, the sample is degraded by treatment with one or more enzymes to obtain a mixture of peptides. The enzyme can be selected to have a very predictable degradation pattern, resulting in peptides that can be identified and quantified by comparison with an allergen calibration standard peptide. The enzyme may be one or more protease (s) such as e.g. two proteases or one or more other enzyme (s). Examples of proteolytic enzymes include trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin and carboxypeptidase C. For example, the proteolytic enzyme trypsin is a serine protease that cleaves peptide bonds between lysine or arginine and non-specific thus producing peptides that have an amino terminus (N-terminus) and as the carboxyl terminal amino acid (C-terminus) lysine or arginine. In this way, peptides from protein cleavage are predictable and their presence and / or amount, in the sample after digestion with trypsin, indicates the presence and / or amount of protein from which they are derived. Furthermore, the free amino terminus of the peptide may be a good nucleophile that facilitates its labeling. Because enzyme activity is predictable, peptide sequences that are produced by degradation of a protein of a given sequence can be predicted. With this information, you can generate "theoretical" peptide information. The determination of "theoretical" peptide fragments in e.g. computer-assisted analysis of the resulting ion fragments from the mass spectrometry of the actual sample can therefore be used to identify one or more peptides.

[0075] In one embodiment of the invention, the allergen sample is degraded prior to labeling by digesting the sample with at least one proteolytic enzyme to degrade the sample partially or completely. In a further embodiment of the invention, the proteolytic enzyme is selected from the group consisting of trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, prase, chymotrypsin or carboxypeptidase C or a combination thereof such as selected from the group ArgC, LysC and trypsin or their combination. In yet a further embodiment of the invention, the enzyme is trypsin.

[0076] The digested sample can be prepared before labeling by any of several methods if desired.

[0077] It will be apparent to those skilled in the art that there are numerous options for labeling the sample and allergen calibration standard peptide to introduce, in a predetermined manner, various mass modifying functions, which allows quantification of allergen peptides. Labeling can e.g. be carried out as described in WO 2004/070352, US 6,864,089, Stemmann O et al. Cell 2001; 107 (6): 71526, and Gerber SA et al. Proc Natl Acad Sci USA 2003; 100 (12): 6940-5.

[0078] In one embodiment of the invention, labeling is carried out using ITRAQ chemistry^™ (Applied Biosystems, Foster City, CA, USA).

[0079] According to this embodiment of the invention, labeling of a degraded allergen sample and / or calibration standard peptide is performed by a set of isomeric or isobaric labeling reagents such as iTRAQ ™ Reagents (Applied Biosystems, Foster City, CA, USA). Each of these reagents contains a reactive group (RG) that reacts with the analyte and a unique reporter group (RP) that generates a unique 'signature ion' signature ion) in MS / MS analysis. These two groups are further linked together by a linker residue (LK) using X and Y bonds. Labeling of the degraded allergen sample thus gives the analyte referred to as RP-X-LK-Y sample. Analyte analysis is performed by setting the mass spectrometer such that both X and Y bonds break up. The breakdown of the X bond releases the reporter group from the analyte and the reporter group can then be determined independently of the analyte. The breakdown of the Y bond releases the RP-LK combination from the analyte. Thus, based on fragmentation, the presence and / or amount of a reporter group can be correlated with the presence and / or amount of an analyte in a sample.

[0080] Labeling, e.g. with 4 iTRAQ ™ reagents, allows absolute quantification of different allergen samples simultaneously (the allergen samples are degraded and each peptide mixture is labeled with different iTRAQ ™ reagents. The ability to simultaneously analyze different allergen samples allows comparison of labeled peptides, sample (s) with a known amount of calibration peptide (s) and thus enables quantification and identification by using MS / MS in one step.

[0081] Labeling of samples using ITRAQ ™ and / or other labeling reagents can be performed according to the manufacturer's procedure as shown in Fig. 5.

[0082] Another labeling method in connection with the quantification of proteins using MS techniques is e.g. the AQUA technique using internal calibration peptides synthesized with stable isotopes incorporated (¹³C ¹⁵N) to mimic native peptides formed by enzymatic digestion using e.g. trypsin (Stemmann O et al. Cell 2001; 107 (6): 715-26, Gerber SA et al. Proc Natl Acad Sci USA 2003; 100 (12): 6940-5).

[0083] Another method is the ICPL (Isotope Coded Protein Labeling) method described by Kellermann et al., Proteomics 5, 4-15, using e.g. ¹²C /¹³Nicotinic acid C6-succinimide as an ICPL tag.

[0084] In one embodiment of the invention, differently labeled peptide (s) and allergen calibration standard peptide (s) are labeled separately and mixed after labeling prior to quantification. [0085] Depending on how labeling of the allergen and calibration standard peptide is carried out, the appropriate identification method can be chosen.

[0086] In one embodiment of the invention, the allergen is further positively identified by comparing the labeled allergen peptide (s) and the allergen calibration standard peptide (s) by peptide identification analysis.

[0087] Cation exchange chromatography can also be used to separate peptides in combination with reverse phase chromatography as two-dimensional chromatography and to reduce, if necessary, any salts and organic compounds prior to MS analysis.

[0088] In one embodiment of the invention, the quantification is carried out by mass spectrometry.

[0089] In a further embodiment of the invention, the identification of the allergen and / or isoallergens can be carried out using tandem mass spectrometers and other mass spectrometers that are able to select and fragment ion molecules. This is particularly suitable when iTRAQ ™ reagents are used for labeling.

[0090] Tandem mass spectrometers (and to a lesser extent single-stage mass spectrometers) have the ability to select and fragment molecular ions depending on their mass-to-charge ratio (m / z), and then record the ion spectra of the resulting (daughter) ion fragments. More specifically, it is possible to generate ion spectra of daughter fragments by subjecting selected ions to dissociative energy levels (e.g. CID - collision-results in dissociation). For example, ions corresponding to labeled peptides with a given m / z ratio can be selected in the first mass analysis, fragmented and re-analyzed in the second mass analysis. Representative instruments that can perform such tandem mass analyzes include, but are not limited to, four-sector magnetic mass spectrometers, tandem time-of-flight, triple, quadruple, ion traps, and hybrid four-field time-of-flight (Q-TOF) quadrupole time-offlight.

[0091] These types of mass spectrometers can be used in conjunction with a number of ionization sources, including but not limited to electrospray ionization (ESI ) and matrix assisted laser desorption ionization (MALDI). Ionization sources can be used to generate charged forms for the first analysis when the analyzes are no longer charged. Additional mass spectrometry instruments and fragmentation methods include post-source decay in MALDI-MS instruments and high energy CID using MALDI-TOF (Time of Flight) - TOF MS. For a recent review of tandem mass spectrometers, see R. Aebersold and D. Goodlett, Mass Spectrometry in Proteomics. Chem. Rev. 101: 269-295 (2001). See also US Patent No. 6,319,476 for discussion of TOF TOF mass analysis techniques.

[0092] The allergen calibration standard peptide (s) (constant or variable sequence (s)) is selected depending on whether the quantification to be carried out is the determination of the allergen or homologous allergens or specific allergens or isoallergens.

[0093] In one embodiment of the invention, an absolute allergen quantification (absolute amount of allergen isoallergens) can be performed.

[0094] When iTRAQ ™ reagents are used, the selected allergen calibration standard peptide (s) is labeled with an isomeric or isobaric tag from a set of tags, e.g., iTRAQ-114, iTRAQ115, iTRAQ-116 or iTRAQ-117, used to label allergen peptides .

[0095] Thus, in an embodiment of the invention, the marking is carried out with ITRAQ-114, ITRAQ-115, ITRAQ116 and / or ITRAQ-117.

[0096] Once the relative amount of the reporter group for the reference peptide or calibration peptides has been established, relative to the relative number of reporter groups for the differently labeled peptides, it is possible to calculate the absolute amount (often expressed as concentration and / or amount) of all differently labeled peptides in the sample mixture and thus calculating the amount of allergen (e.g., absolute amount of isoallergen (isoallergens) from the species when the sample is an extract). Obtaining MS and MS / MS from ITRAQ-labeled samples^™ can be performed e.g. using 4700 Explorer software^™. In addition, GPS Explorer software can be used to perform a database search that will ultimately identify peptides from MS / MS. The data obtained can then be used for quantification based on a known amount of allergen calibration standard peptide, i.e. the ratio between the sample and the calibration standard peptide.

[0097] In one embodiment of the invention (i) the allergen to be quantified is Der f 2 and the allergen calibration standard peptide contains amino acids 32-48 Der f 2, or (ii) the allergen to be quantified is Der p 2 and the allergen calibration standard peptide contain amino acids 32-48 Der p 2.

[0098] The method of the invention is useful, e.g. in a release assay, to provide a safe and predictable amount of allergen during vaccine manufacture, and in the final product, and also during the various stages of storage of ingredients and / or products and raw extract. The method of the invention is useful in the development of second generation allergen vaccines e.g. using the recombinant allergen as the active ingredient, by optimizing the active ingredients in the second generation allergen vaccine based on the knowledge and / or composition of the current vaccine. Current vaccines are often formulated using allergens from a number of types of allergens, and the method would also be beneficial in determining the composition of these allergen mixtures. The method of the invention can be used to check purification, where trace amounts of allergen (s) are measured, in assay releases and analysis of intermediates and end products.

[0099] Peptides that can be used as calibration calibration peptides can be prepared using protein and / or nucleotide databases and progam (programs) cleavage analysis and / or in vitro mass fingerprinting experiment (s).

[0100] In the present context, the term "allergen calibration standard peptide" refers to an allergen calibration standard with an amino acid sequence identical to the constant sequence in the group of isoallergens or homologous allergens. The allergen calibration peptide is preferably prepared by peptide synthesis.

[0101] In some cases, the sequence of the allergen of interest is already known. For example, the official list of allergens can be found on the website (<a href="http://www.alergen.org">www.alergen.org</a>), which is maintained by the IUIS Allergen Nomenclature Sub-committee. Known allergen sequences can be obtained from protein and nucleotide sequence databases, e.g. Uniprot Knowlegdebase. Protein and / or nucleotide sequences can be used

EP 1 931 998 B1 search using e.g. Sequence Retrieval System (SRS), or by using keywords, e.g. entering the introduced name (ID), description (DE), gene (GN), species (OS) and / or organelles (OG). Further analysis of the protein / allergen of interest is carried out using e.g. the Vector NTI software (Invitrogen) and / or by using the ExPASy (Expert Protein Analysis System) Swiss Institute of Bioinformatics (SIB) proteomic server. Sequence alignment of existing allergen isoforms and allergen types is performed using, e.g., Blast screening, which can be used to align homologous protein and / or nucleotide sequences. Sequence alignments provide a way of comparing e.g. new sequences with previously characterized genes and / or protein (s). Sequence alignment of homologous allergens or isoallergens can be used to show identical (constant) and variable sequences within allergen species as shown in Fig. 1.

[0102] To obtain optimal reference peptides for calibration, it is possible to simulate cleavage analysis for the allergen of interest using the cleavage (degradation) analysis program (s). Allergen sequences can be submitted, for example, to the GPMAW program (Ughthouse data, Odense, Denmark), which was created to support MS analyzes. Allergen cleavage (degradation) analysis can be deduced for several known proteases such as trypsin, Asp-N and Lys-C and / or a combination of two or more of them. The resulting theoretical peptides (Fig. 2) are then used to check the optimal enzyme (s) for degradation and further to design synthetic peptides that can be used as calibration calibration peptides.

[0103] On the other hand, calibration calibration peptides can be deduced from an in vitro mass fingerprint experiment (s) in which the allergens are cleaved by the enzyme and mixed. Type-specific peptides can be detected by mass fingerprint analysis and identified by searching databases e.g. using a Mascot Search as described below.

[0104] Purified natural (n) Der f 2 and nDer p 2 and recombinant (r) Der f 2 (A61501) and Der p 2 (BAA01241) can be dissolved in 25 mM Tris-Cl pH 7.5, 1.0 M urea. Samples of recombinant molecules (rDer f 2 and rDer p 2) and natural (nDer f 2 and nDer p 2) molecules can be mixed (e.g. 1: 1) or digested as individual allergens with trypsin and then mixed. The digestion of mixed and / or individual allergens is desalted and evaluated using a mass fingerprint. Peptides that are species specific can be identified by mass fingerprint analysis from a mixture of these two species. Digested single HDM 2 allergens can be mixed after digestion with trypsin and the same species-specific constant sequences can be shown. Synthetic peptides can be designed based on type-specific constant sequences. Quantification and peptide sequence checking can be performed by labeling individual allergens and standard calibration peptides, e.g. with ITRAQ ™ reagents, and analyzing using tandem mass spectrometry.

[0105] Natural allergen extracts, eg the intermediate product of two HDM species Dermatophagoides farinae and Dermatophagoides pteronyssinus (ALK-Abelló, Hrsholm, Denmark) (1.0 mg / ml dry matter) are dissolved. To remove the interfering component, samples can be precipitated with acetone. The precipitate containing proteins can then be dissolved in the selected buffer. For quantification, the sample (s) and selected synthetic peptides from two species used as a calibration calibration peptide can be labeled e.g. ITRAQ ™ reagents and analyze using MS / MS.

[0106] The final product, e.g., a house mite allergen tablet, is dissolved in 20 mM Naphosphate pH 7.0 buffer. To remove interfering components, it may be necessary to pre-fractionate the sample and / or

Its precipitation with acetone. The dissolved tablet and selected calibration standard peptide are labeled with e.g. ITRAQ ™ reagents and analyzed using tandem MS / MS.

[0107] Release study; the final product, e.g. a mixture (mixtures) of 5 grasses containing 5 species of grasses combined with aluminum hydroxide is dissolved in a selected buffer that can leach bound allergens from aluminum hydroxide. Unbound allergens can be further separated and / or desalted to a selected buffer for digestion and selected calibration standard peptides can be labeled with e.g. ITRAQ ™ reagents and quantification can be assessed using MS / MS.

EXAMPLES

Example 1

Identification of unique constant regions for natural Der f 2, Der p 2, Phl p 1, Phl p 5 and Bet v 1 and synthetic calibration standard peptides [0108] Absolute quantification of isoallergens using unique constant region sequences ie characteristic peptides in natural Der f 2, Der p 2, Phl p 1, Phl p 5 and Bet v 1 have been demonstrated by using two different approaches. Two calibration standard peptides were synthesized to evaluate the iTRAQ ™ labeling technique (Applied Blosystems). In addition, four calibration standard peptides were synthesized to evaluate stable isotope labeling techniques such as Protein-AQUA ™ (SigmaAldrich).

[0109] Standard allergen calibration peptides having species-specific sequences corresponding to amino acid sequences 32 - 48 in Der f 2 and in Der p 2, 149 - 158 in Phl p 1, 123 - 135 in Phl p 5a, 115 - 127 in Phl p 5b ai 151-164 at Bet v 1 was designed based on amino acid sequence alignment (Vector NTI) (Fig. 1), GPMAW cleavage analysis (Fig. 2) and Blast database search. In vitro fingerprint analyzes of trypsin digested natural allergens Der f 2, Der p 2, Phl p 1. Phl p 5, Bet v 1 and mixed Der f 2 and Der p 2 were performed to show the occurrence of species-specific peptides. All in vitro digested samples and mixtures were identified by using a Mascot screen scanner (Matrix Science Inc., Boston, MA, USA).

[0110] Synthetic peptides corresponding to amino acid sequence 32-48 in Der f 2 and Der p 2 were obtained from Sigma GENOSYS, Texas, USA. Peptide concentration was determined by amino acid analysis (Sigma GENOSYS, Texas, USA). Synthetic stable isotope peptides (Protein-AQUA ™ peptides) corresponding to amino acid sequence 149-158 in Phl p 1 (Arg¹³C ¹⁵N), 123-135 in Phl p 5a (Arg ¹³C¹⁵N), 115-127 in Phl p 5b a (Arg ¹³C ¹⁵N) and 151 - 164 at Bet v 1 (Val ¹³C ¹⁵N) obtained from Sigma GENOSYS, Texas, USA (Table 1).

Example 2

Purification of natural and recombinant grass, birch and second mite allergens [0111] Natural Der f 2 and Der p 2 were purified from 100 mg extracts of Dermatophagoides farinae and Dermatophagoides pteronyssinus (ALK-Abelló, Horsholm, Denmark). Natural Phl p 1 and 5 were purified from 50 mg Phleum prantense extract (ALK-Abelló) and natural Bet v 1 from 50 mg Betula verrucosa extract (ALKAbelló). Particle purification was performed as described in the literature (Johannessen BR et al. FEBS Lett 2005; 579: 1208-12, Aasmul-Olsen S. et al. New Horizons in Allergy Immunotherapy, edited by Sehon et al. Plenum Press. New York 1996, p. 261-65, Petersen A et al. Clin Exp Allergy 1994 Mar; 24 (3): 250-6, Ipsen H &amp; Lowenstein HJ Allergy Clin Immunol. 1983; 72 (2): 150-59). Recombinant Der f 2 and Der p 2 were expressed in the Pichia pastoris expression system and purified as described in the literature (Johannessen BR et al. FEBS Lett 2005; 579: 1208-12). Proteins were stored as lyophilized samples at -20 ° C.

[0112] The concentration of purified allergens was measured using the extinction coefficient of one of the isoallergens in A280 and using a Lambda 800 UV / VIS Spectrometer (Perkin Elmer Instruments, CA, USA). Example 3

Pre-fractionation of the HDM extract and protein digestion [0113] Hydrophobic interaction chromatography was used to pre-fractionate the extracts of Dermatophagoides farinae (Der f) and Dermatophagoides pteronyssinus (Der p). Fractionation of mite extracts was performed on a 1.0 ml HITrap Phenyl column (GE-Healthcare, Uppsala, Sweden). The column was equilibrated with 50 mM Na-phosphate buffer (Merck, Darmstadt, Germany), pH 7.0, 1.0 M ammonium sulfate (Fluka, Buchs, Switzerland) and the bound sample was eluted with 50 mM Na-phosphate buffer (Merck), pH 7 , 0 in 5 column volumes using a decreasing linear gradient. Chromatography was performed separately for each of the HDM extracts, Der fi Der p. Fractions were analyzed by SDS-PAGE (Invitrogen, Carlsbad, CA, USA) and based on these analyzes HDM protein was divided into two major protein pools (Fig 4). Der f and Der p pools containing HDM 2 allergens were subjected to a dialysis step against 10 mM ammonium bicarbonate (BDH, Poole, England). Der fi Derized dialysis pools Der p lyophilized, aliquoted ~ 5 mg (dry weight) into vials and stored frozen at -20 ° C. Portions of Der fi Der p containing HDM 2 allergens were further tested for absolute quantity determination.

Example 4

Enzymatic cutting and iTRAQ labeling [0114] iTRAQ ™ labeling was used for three sets of samples:

a) 15 μg natural Der f 2 and 15 μg natural Der p 2

b) 15 μg recombinant Der f 2 and 15 μg recombinant Der p 2 i

c) 100 μg of pre-fractionated Der fi Derp extracts.

[0115] Synthetic reference peptides, 15 μg Peptide 1 (Der p 2) and 15 μg Peptide 2 (Der f 2) were dissolved in 100 mM triethylammonium bicarbonate (TEAB) pH 8.5 and labeled for use as internal calibration standards for each from experimental sets as described below.

[0116] Blocking of free cysteine ​​residues, enzymatic digestion using trypsin and labeling peptides with iTRAQ ™ reagents were carried out according to the manufacturer's protocol:

Each of the six protein samples and two internal calibration standards were dissolved in 20 μΐ 100 mM TEAB, pH 8.5. Protein samples were denatured with 1.0 μl 0.05% SDS and reduced with 2.0 μl 4.8 mM TCEP Tris (2-carboxyethyl) phosphine at 60 ° C after blocking cysteine ​​residues with 1.0 μl 10 mM s-methyl methanothiosufonate (MMTS) at room temperature. Protein samples were digested with 10% (v / v) modified sequencing quality trypsin (Promega, Madison, WI, USA) for 18 hours at 37 ° C.

[0117] iTRAQ ™ labeling of trypsin digested samples and internal calibration standard peptides, Peptide 1 and 2 was performed at room temperature. Each iTRAQ labeling reagent from 114 to 117 was dissolved in 70 μl 70% ethanol, which was then applied to the sample (s). The final volumes of the reagent mixtures were 100 μl / sample. Labeled samples were stored at -20 ° C. [0118] Trypsin digested peptides of natural Der p 2, recombinant Der p 2 and Der p extracts were labeled with iTRAQ-114. Trypsin digested natural peptide Der f 2, recombinant Der f 2 and Der f extracts were labeled with iTRAQ-115. Synthetic peptides Peptide 1 (Der p 2) and Peptide 2 (Der f 2) were labeled with iTRAQ-116 (Der p 2) and iTRAQ-117 (Der f 2) (Fig 5). Each labeled sample was analyzed using a Voyager STR proteomic analyzer and / or 4700 Proteomic Analyzer (Applied

EP 1 931 998 B1

Biosystems) to show labeling of peptides. MS / MS fragment analyzes were performed using

4700 Proteomic Analyzer (Applied Biosystems). Labeled samples were diluted 1:10 and 1.0 μΐ samples (samples) were desalted using C18 Micro columns (ZipTips, Millipore) and / or hand-prepared C18 micro columns (Poros R2, Applied Blosystems). The sample was eluted with 1.0 μΐ 70% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) and 1.0 μl alpha-cyano-4-hydroxy-cinnamic acid matrix (CHCA) (Agilent Technologies, Bóblingen , Germany) was added to the top of the sample. The sample was dried and subjected to MS analysis.

[0119] MS analyzes of iTRAQ ™ labeled internal calibration standards showed weights corresponding to Peptide 1 at m / z 2353.44 and Peptide 2 at m / z 2326.35 (Figures 6a and 6b). The results showed that the modification in Der p 2 specific Peptide 1 and Der f 2 specific Peptide 2 corresponded to modification by the iTRAQ ™ reagent when it was bound at the amino terminus and C-terminal lysine. MS analyzes of iTRAQ ™ labeled natural and recombinant Der p 2 peptides showed iTRAQ ™ modified masses at m / z 2353.29. Similarly, MS analyzes of the labeled natural and recombinant Der f 2 peptides showed an iTRAQ ™ modified peptide at m / z 2326.29 (Fig 6c and 6d, respectively). No cleavage sites missed by trypsin were observed. These results show that iTRAQ-labeled Peptides 1 and 2 can be used as internal calibration standards for the absolute quantification of Der p 2 and Der f 2 isoallergens.

Example 5

Absolute quantification from a mixture of two different HDM species [0120] The absolute quantification of isoallergens of Dermatophagoides farinae and Dermatophagoides pteronyssinus was first performed from a direct mixture of purified natural allergens. ITRAQ ™ labeled natural trypsin digested Der p 2, Der f 2 and Peptides 1 and 2 were mixed in a 1: 1: 1: 1 ratio, diluted 1: 5 and 1:10 and desalted on hand-prepared C18 micro columns (Poros R2, Applied Biosystems). The sample was eluted using 1.0 μl 5 μg / μl CHCA (Sigma) in 70% ACN (Sigma), 0.1% TFA (Fluka). MS / MS fragment analyzes were performed with a 4700 Proteomic Analyzer (Applied Biosystems).

[0121] Analysis of the m / z 2353.29 fragments showed signals at m / z 114 m / z at 116 corresponding to reporter ions for natural Der p 2 and Peptide 1. The amount of natural Der p 2 isoallergens in the sample was calculated as the ratio of the signal surface m / z 114 to surface m / z 116, internal calibration standard (Fig 7a). Analysis of MS / MS m / z 2326.29 fragments showed signals at m / z 115 m / z at 117 corresponding to reporter ions for natural Der f 2 and Peptide 2.

[0122] The amount of natural Der f 2 isoallergens in the sample was calculated as the ratio of the signal surface m / z 115 to m / z 117, an internal calibration standard (Fig 7b). In addition to absolute quantification, ionic peak lists m / z 2353.29 m / z 2326.29 were subjected to database analysis using a Mascot search engine (Matrix Science). Analysis of the databases identified the peptides as 32-48 Dermatophagoides farinae and Dermatophagoides pteronyssinus HDM allergens 2.

Example 6

Separation of labeled peptide mixtures by two-dimensional chromatography [0123] The absolute quantification of the mixture of recombinant Der f 2 and Der p 2 and isoallergens in a complex mixture of Der fi Der p extracts was evaluated by two-dimensional chromatography. Cation exchange chromatography (SCX) was evaluated as the first direction and reverse phase chromatography as the separation step in the second direction.

[0124] Recombinant Der p 2, Der f 2 and Peptides 1 and 2 were mixed in a 1: 1: 1: 1 ratio, in a final volume of 50 μΐ of Der fi Der pi extract Peptides 1 and 2 were mixed in a ratio of 4 : 4: 1: 1, in the final volume of 50 pl.

Separation of mixtures of labeled peptides by cation exchange chromatography (both recombinant Der p 2 and Der f 2 and Der p 2 and Der f 2 extracts);

[0125] Sample mixtures were diluted 1:10 to 5% ACN (Sigma) 0.05% formic acid (Merck) and subjected to SCX. SCX was carried out on a 0.8x50 mm Zorbax BIO-SCX (3.5 μm) column (Agilent Technologies) on a SMART ™ system (GE-HealthCare, Uppsala, Sweden). The column was equilibrated with 5% ACN (Sigma) 0.05% formic acid (Merck) and the chromatography was performed with an increasing linear gradient of 5% ACN (Sigma) from 0 to 100% 0.05% formic acid (Merck) 0.5 M NaCl (Merck) ) for 30 min. The flow rate was 50 / min and the chromatography was monitored at 214 nm. 50 µl fractions were collected and 1.0 µl of each fraction was analyzed using Voyager-STR MS instruments (Applied Biosystems) and / or 4700 Proteomic Analyzer (Applied Biosystems). Peptides at m / z 2353.29 and m / z 2326.29 were identified by eluting at the end of the gradient together with some other HDM peptides. Fractions of the Der fi Der p mixture (extracts) containing peptides at m / z 2353.29 and / z 2326.29 were selected for further separation by reverse phase chromatography, see below. However, the quantification of the mixture of rDer f 2 and rDer p 2 (recombinant) and calibration with an internal standard was performed directly after SCX from the target plate.

[0126] MS / MS m / z fragment analysis of 2353.29 showed m / z 114 m / z signals at 116 corresponding to reporter ions for Der p 2 and Peptide 1. The amount of Der p 2 clone in the mixture rDer p 2 / rDer f 2 was calculated as ratio of signal surface m / z 114 to surface m / z 116, internal calibration standard. Analysis of MS / MS m / z 2326.29 fragments showed signals at m / z 115 m / z at 117 corresponding to reporter ions for Der f 2 and Peptide 2. The amount of Der f 2 clone in rDer p 2 / rDer f 2 mixing was calculated as the ratio of signal surface m / z 115 to m / z 117, an internal calibration standard. The list of ion peak fragments m / z 2353.29 and m / z 2326.29 was subjected to database analysis using a Mascot search engine (Matrix Science). Database analyzes have identified the peptides as 32-48 Dermatophagoides farinae and Dermatophagoides pteronyssinus HDM allergens 2.

[0127] Peptides at m / z 2353.29 m / z 2326.29 in both samples; a mixture of recombinant Der f 2 and Der p 2 and a mixture of the Der f Der Der extract was eluted from the SCX column with similar retention times. This experiment has shown that SCX can be used as a fractionation and desalting step for more simple sample mixtures prior to allergen quantification. Experiments have also shown that SCX can be used as a first-step fractionation step for the analysis of more complex allergen mixtures, such as conventional allergen extracts used in immunotherapy.

Separation of labeled peptides (from Der p 2 and Der f 2 extracts) by SOX followed by reverse phase chromatography followed by absolute quantification of MALDI TOF-TOF MS, [0128] Separation of iTRAQ ™ labeled HDM peptides from SCX fractionation as described above was carried out using a C18 PepMap100 (3 pm) column (LC Packings Dionex, Sunny Vale, CA, USA). The column was equilibrated with 0.05% TFA (Fluka), 2% ACN (Sigma). Peptides were eluted with 0.04% TFA (Fluka), 80% ACN in a 0-50% gradient over 80 min, 50-100% over 120 min. Chromatography was carried out using Ultimate3000 (LC Packings, Dionex) 2.0 pl / min and monitored at 210 and 214 nm. 1.0 µl of SCX fraction was injected onto the column. Fractions were collected by spotting them directly onto a MALDI-TOF target plate. The spotting was done with a Probot instrument (LC Packings, Dionex) which was connected

EP 1 931 998 B1 online to the Ultimate3000 instrument. Spot-on was carried out every 30 s by mixing the HCCA matrix (Agilent Technologies) with the sample in a 1: 1 ratio.

[0129] Spotted samples were analyzed by MS and MS / MS using 4700 Proteomic Analyzer (Applied Biosystems). Peptides at m / z 2353.29 and m / z 2326.29 were identified from target spots and were shown to correspond to signals at 214 nm in chromatography (Fig. 8). MS / MS m / z 2353.29 fragment analyzes showed signals at m / z 114 m / z at 116 corresponding to reporter ions for Der p 2 and Peptide 1. The amount of Der p 2 isoallergens in the Der p / f extract mixture was calculated as the ratio of signal surface m / z 114 to surface m / z 116, an internal calibration standard. Analysis of MS / MS fragments at m / z 2326.29 showed signals at m / z 115 m / z at 117 corresponding to reporter ions for Der f 2 and Peptide 2. The amounts of Der f 2 isoallergens in the Der p / f extract mixture were calculated as the surface ratio signal m / z 115 to m / z 117, internal calibration standard. The list of ion peak fragments m / z 2353.29 and m / z 2326.29 was subjected to database analysis using a Mascot search engine (Matrix Science). Database analyzes have identified the peptides as 32-48 Dermatophagoides farinae and Dermatophagoides pteronyssinus HDM 2 allergens.

[0130] This experiment shows that reverse phase chromatography can be used as the second direction of fractionation of a simple and / or complex mixture of allergen extracts to quantify isoallergens.

Example 7

Absolute quantification of isoallergens using the AQUA strategy [0131] In the AQUA technique (Stemmann O et al. Cell 2001; 107 (6): 715-26, Gerber SA et al. Proc Natl Acad Sci USA 2003; 100 (12): 6940- 5.) peptides for internal calibration are synthesized with stable isotopes (¹³C ¹⁵N) to mimic native peptides produced by enzymatic digestion using e.g. trypsin. Incorporation of one isotope labeled amino acid residue typically changes the peptide molecular weight from 6 to 10 Da. Unlike the iTRAQ technique, either the sample or the internal calibration standard (s) must be modified by labeling agents. In experiments with quantification e.g. using LC-MS / MS, the amount of specific ion fragment from both the native peptide sample and the synthesized internal calibration standard can be measured as a function of retention time in reverse phase chromatography. Absolute quantification determined by comparing the amount of known internal standard with the native peptide of the sample.

[0132] The synthetic internal peptide calibration standard for natural Phl p 1, Phl p 5 forma aibi Bet v 1 was designed as described above (Fig. 2). Synthetic peptides are described in more detail in Table 1.

Table 1: Labeling of the synthetic internal calibration standard

<td>Type</td><td>allergen</td><td>Sequence acid</td><td>Mass theoretical</td><td>Modification</td><td>modified mass</td>

<td>Betula verrucosa</td><td>Bet v 1</td><td>AVESYLLAHSDAYN</td><td> 1552,73</td><td>(Val ¹³C¹⁵N)</td><td> 1558,64</td>

<td>Phleum prantense</td><td>Phl p 1</td><td>SAGEVEIQFR</td><td> 1135,57</td><td>(Arg ¹³C¹⁵N)</td><td> 1145,27</td>

<td>Phleum prantense</td><td>Phl p 5a</td><td>YDAYVATLSEALR</td><td> 1471,74</td><td>(Arg ¹³C¹⁵N)</td><td> 1481,66</td>

<td>Phleum prantense</td><td>Phl p 5b</td><td>FDSFVASLTEALR</td><td> 1455,74</td><td>(Arg ¹³C¹⁵N)</td><td> 1465,66</td>

[0133] Natural Phl p 1, Phl p 5 and Bet v 1 were redissolved in 25 mM Tris-Cl (Sigma), 1.0 M urea (Fluka) pH 7.8 at a concentration of 2.5 pmol / μΐ. Internal calibration standards were mixed with the samples in a 1: 1 ratio. Digestion was carried out with 10% (v / v) trypsin quality sequencing (Promega, Madison, WI, USA) at 37 ° C for 18 hours. Samples were stored at -20 ° C.

[0134] Trypsin digested Phl p 1, Phl p 5 and Bet v 1 were analyzed by MS and MS / MS using Voyager STR and / or 4700 Proteomic Analyzer (Applied Biosystems). All samples were diluted 1:10 in 0.1% TFA (Fluka) and 1.0 μl of each sample was desalted using hand-prepared C18 micro columns (Poros R2 Applied Blosystems).

[0135] MS analyzes of natural Phl p 1 digested trypsin showed native peptide at m / z 1135.60 and internal calibration standard peptide for Phl p 1 isoallergens at m / z 1145.61 (data not shown). MS / MS analyzes of internal calibration peptide showed a fragmentation pattern that corresponded to the unique amino acid sequence of natural Phl p 1 isoallergens.

[0136] MS analyzes of natural Phl p 5 digested trypsin showed native Phl p 5a peptide at m / z 1471.81 and native Phl p 5b peptide at m / z 1455.77 (data not shown). Internal calibration standard peptides for Phl p 5a and Phl p 5b isoallergens were detected at m / z 1481.83 and at m / z 1465.78. MS / MS analyzes of peptides for internal calibration showed fragmentation patterns that corresponded to the unique amino acid sequence of the natural isoallergens Phl p 5a and Phl p 5b.

[0137] MS trypsin digested natural Bet v1 digestion showed native peptide at m / z 1552.76 and internal calibration standard peptide for Bet v 1 isoallergens at m / z 1558.77 (Fig. 9) MS / MS analyzes of peptides for internal calibration showed fragmentation patterns, which correspond to the unique amino acid sequence of Bet v 1 natural isoallergens.

[0138] For the absolute quantification of Betv 1 Phl p 1, Phl p 5a and Phl p 5b isoallergens, the samples can be analyzed using e.g. LC connected MS / MS instruments such as LCQ DecaXP (ThermoFinnigan), QSTAR® Hybrid LC / MS / MS system, and 4000 Q TRAP LC / MS / MS system (Applied Blosystems).

[0139] Experiments with AQUA peptides have shown that stable isotope-labeled synthetic peptides imitating native isoallergen sequences can be used for absolute quantification of isoallergens in natural Phl p 1, Phl p 5 and Bet v 1. Furthermore, trypsin digestion database analysis showed no binding not interrupted by trypsin. Two-dimensional chromatography with a combination of SCX and RP-HPLC as described for iTRAQ chemistry can be used to fractionate more complex mixtures of allergens that are chemically identical to the native and synthetic internal calibration standard.