HK1263291B - An improved assay for the diagnosis of peanut allergy - Google Patents

Description

Improved assay for diagnosing peanut allergy

The present invention relates to diagnostically useful carriers comprising means for specifically capturing antibodies against Ara h 7 isoform 7.0201 in a sample from a subject; methods comprising the step of detecting the presence or absence of antibodies against Ara h 7 isoform 7.0201 in a sample from a subject; and pharmaceutical compositions comprising Ara h 7 isoform 7.0201 or variants thereof.

Among food allergies, those related to peanut (Arachis hypogea) have received the most research attention because they are common, often permanent, and often severe. The U.S. Food Anaphylaxis Mortality Registry implicates peanut as a trigger in 59% of 63 deaths, with adolescents and young adults at highest risk, with additional risk factors being delayed administration of epinephrine during anaphylaxis and asthma. Peanut is a ubiquitous food, and affected patients (who often react to small doses) face numerous obstacles to avoid ingestion and experience negative impacts on their quality of life.

Peanut allergy is primarily suspected in two situations: when a patient exhibits a severe allergic reaction or in the context of monitoring allergic conditions to other food or aeroallergens. When peanut sensitization is detected by a positive skin prick test (SPT) or peanut-specific IgE assay (whose diagnostic reliability relative to clinical disease is currently insufficient), the patient is referred to a specialized center that provides diagnosis based on a double-blind, placebo-controlled food challenge (DBPCFC). This procedure is the gold standard for food allergy diagnosis, but its use requires hospitalization and is expensive. Furthermore, DBPCFC with peanut can result in severe or even life-threatening reactions, even under the control of a trained physician.

Therefore, there is great interest in improved assays for diagnosing peanut allergy based on the detection of specific antibodies in samples from subjects suspected of having peanut allergy.

Assays described in the prior art rely on the use of natural or recombinant allergens to detect major peanut allergens such as Arah 1, Ara h 2, Ara h 3 and Ara h 6 (Flinterman, A.E., van Hoffen, E., den Hartog, Jager C.F., Koppelman, S., Pasmans, S.G., Hoekstra, M.O., Bruijnzeel-Koomen, C.A., Knulst, A.C., Knol, E.F. (2007): Children with peanut allergy recognize predominantly Arah 2 and Ara h 6, which remains stable over time, Clin Exp Allergy 2007;37;1221-1228;de Leon, M.P., Drew, A.C., Glaspole, I.N., Suphioglu, C., O’Hehir, R.E., Rolland, J.M. (2007): IgE cross-reactivity between the major peanut allergen Arah 2and tree nut allergens, Mol Immunol 2007;44:463-471).

Ara h 2 and Ara h 6 are 2S storage proteins of the conglutin type and belong to the prolamin superfamily. Members of this family are characterized by the presence of a conserved pattern of six or eight cysteine residues within a sequence of approximately 100 amino acid residues that form three or four intramolecular disulfide bonds. They are stable to heat treatment and proteolysis.

Ara h 2 is a major peanut allergen and exists as two isoforms with different molecular weights (MW), resulting from a 12-amino acid insertion with a MW of 1,414 Da. Ara h 6 shares 53% identity with Ara h 2 in its primary protein sequence and, compared to Ara h 2, has 10 cysteines instead of 8 and no N-glycosylation sites. Like Ara h 2, Ara h 6 is resistant to digestion and is stable to heat, particularly during baking. The tertiary structure of Ara h 6 has been elucidated, and the positional parameters of at least four α-helices and disulfide bonds have been identified. In physiological digestion experiments, Ara h 2 and Ara h 6 form stable fragments, even under reducing conditions. This supports the view that the cross-reactivity between Ara h 2 and Ara h 6 is mediated by an IgE-reactive conformational epitope (Schmidt, H., Krause, S., Gelhaus, C., Petersen, A., Janssen, O., and Becker, W. M. (2010): Detection and structural characterization of natural Ara h 7, the third peanut allergen of the 2S albumin family, J Proteome Res 2010; 9: 3701–709.).

Ara h 7 is another peanut allergen, and its isoform Ara h 7.0101 was originally identified and cloned by a phage display system, but no natural counterpart was initially found in peanut extracts, which is one of the reasons why its allergenic impact was described as low in the prior art.

Schmidt et al. (2008, Detection and Structural Characterization of Natural Ara h 7, the Third Peanut Allgergen of the 2S Albumin Family) discovered the existence of another isoform (Ara h 7.0201). Ara h 7.0 is an isoform published in the UNIPROT database in addition to 7.0101. However, Schmidt et al. did not mention diagnostic applications; their work was purely proteomic research.

More importantly, only five of the six patient sera used by Schmidt et al. (preselected for Ara h 7.0101 reactivity) showed positive IgE binding to Ara h 7.0201, indicating that Ara h 7.0201 is actually less reactive and therefore has even lower diagnostic value than Ara h 7.0101. Thus, to the best of the inventors' knowledge, Ara h 7.0201 had not been used for routine diagnosis until the present invention.

In addition, some studies have used purified peanut proteins (Ara h 1-3, Ara h 6) to assess a range of immunological parameters and found that clinical severity correlated with recognition of Ara h 2 and 6 at low concentrations and Ara h 1 and 3 at higher concentrations, indicating a clear increased potency of Ara h 2 (Peeters, K.A., Koppelman, S.J., van Hoffen, E., van der Tas, C.W., den Hartog, Jager C.F., Penninks, A.H., Hefle, S.L., Bruijnzeel-Koomen, C.A., Knol, E.F., and Knulst, A.C. (2007): Does skin prick test reactivity to purified allergens correlate with clinical severity of peanut allergy? Clin Exp Allergy. 2007 Jan;37(1):108-15.).

IgE antibody reactivity to the peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6, and Ara h 7 has also been studied in peanut-allergic patients using recombinant allergens. Codreanu et al. evaluated the performance of the proteins for diagnosing peanut allergy in two large groups of peanut-allergic patients. Specific IgE was measured using the UniCAP platform using the ImmunoCAP test. They reported that Ara h 2 was the most sensitive allergen in peanut-allergic patients, with detectable IgE antibodies present in 77-100% of cases, followed by Ara h 6 (38-80% of cases). In contrast, their data indicated that no patients were monosensitized to Ara h 7 (Codreanu, F., Collignon, O., Roitel, O., Thouvenot, B., Sauvage, C., Vilain, A.C., Cousin, M.O., Decoster, A., Renaudin, JM., Astier, C., Monnez, JM., Vallois, P., Morisset, M., Moneret-Vautrin, D.A., Brulliard, M., Ogier, V., Castelain, M.C., Kanny, G., Bihain, B.E., and Jacquenet, S. (2011): A novel immunoassay using recombinant allergens simplifies peanut allergy diagnosis, Int Arch Allergy Immunol, 2011; 154(3): 216-26.). The finding that no patients were monosensitized to Ara h 7 is another reason not to use any Ara h 7 isoform for routine diagnosis, as it is not believed to increase the sensitivity of diagnostic testing for identifying patients with peanut allergy.

Currently, many patients with peanut allergy are not diagnosed with the allergy based on the results of conventional tests. However, given the severity of symptoms associated with peanut allergy, there is an increasing need for the most sensitive diagnostics.

A problem of the present invention is to overcome any disadvantages associated with prior art diagnostic assays based on the detection of antibodies against peanut allergens.

Another problem of the present invention is to provide novel antigens, novel assays, novel reagents and novel methods which allow the diagnosis of allergies in patients who would not be diagnosed or would be misdiagnosed using conventional assay systems.

Another problem of the present invention was to provide improved antigens for the detection of IgE antibodies in patients suffering from tree nut allergy, preferably peanut allergy.

Another problem of the present invention is to provide alternative methods and alternative reagents for diagnosing tree nut allergy, preferably peanut allergy.

Another problem of the present invention is to provide methods and reagents which, when used in combination with known methods and reagents for diagnosing nut allergy, preferably peanut allergy, increase the overall diagnostic reliability, in particular the sensitivity.

Another problem of the present invention is to provide methods and reagents which allow the diagnosis of nut allergy, preferably peanut allergy, with maximum overall diagnostic reliability, in particular sensitivity, with minimum investment of resources, in particular time and number of antigens used.

The problem of the invention is solved by the subject-matter of the attached independent and dependent claims.

In a first aspect, the problem of the present invention is solved by a diagnostically useful vector comprising means for specifically capturing antibodies against Ara h 7 isoform 7.0201, preferably SEQ ID NO 6, more preferably against a sequence selected from the group consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably against SEQ ID NO 8 in a sample from a subject.

In another preferred embodiment of the first aspect, the carrier further comprises means for specifically capturing antibodies directed against one or more other antigens selected from the group consisting of Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, Ara h 8 and Ara h 5, more preferably selected from the group consisting of Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, and most preferably antibodies directed against Ara h 2 and/or antibodies directed against Ara h 6.

In another preferred embodiment of the first aspect, the diagnostically useful carrier is selected from beads, test strips, microtiter plates, microarrays, solid polymers derived from cellulose, blots, preferably selected from protein blots, line blots and dot blots, glass surfaces, biochips and membranes, and most preferably is a microtiter plate or a line blot.

In a second aspect, the problem of the present invention is solved by a kit comprising a diagnostically useful carrier comprising means for specifically capturing antibodies against the antigen Ara h 7 isoform 7.0201, preferably SEQ ID NO 6, in a sample from a subject, preferably further comprising means for specifically capturing antibodies against one or more other antigens, more preferably selected from the group consisting of Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, Ara h 8 and Ara h 5, more preferably selected from the group consisting of Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, most preferably antibodies against Ara h 2 and/or antibodies against Ara h 6, optionally further comprising means for specifically detecting the captured antibodies.

In another preferred embodiment of the second aspect, the diagnostically useful carrier further comprises means for specifically capturing antibodies against one or more other antigens selected from the group consisting of Ara h 2, Ara h 6, Ara h 1, Ara h 3, Arah 9, Ara h 8 and Ara h 5, wherein the means for specifically capturing antibodies against Ara h 7 isoform 7.0201, preferably SEQ ID NO 6, more preferably against a sequence selected from the group consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably against SEQ ID NO 8 and the means for specifically capturing antibodies against one or more other antigens are on separate carriers.

In a preferred embodiment of the second aspect, the diagnostically useful carrier further comprises means for specifically capturing antibodies against one or more other antigens selected from the group consisting of Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, Ara h 8 and Ara h 5, wherein the means for specifically capturing antibodies against Ara h 7 isoform 7.0201, preferably SEQ ID NO 6, more preferably against a sequence selected from the group consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably against SEQ ID NO 8 and the means for specifically capturing antibodies against one or more other antigens are on one carrier, preferably covalently linked to one carrier.

In a third aspect, the problem of the present invention is solved by a method preferably for diagnosing or aiding the diagnosis of nut allergy, more preferably peanut allergy, comprising the step of detecting in a sample from a subject the presence of antibodies against Ara h 7 isoform 7.0201, preferably SEQ ID NO 6, more preferably against a sequence selected from the group consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably against SEQ ID NO 8.

In a preferred embodiment of the third aspect, the method further comprises detecting in a sample from the subject the presence of antibodies to one or more additional antigens, preferably selected from Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, Ara h 8 and Ara h 5, more preferably selected from Ara h 2, Ara h 6, Ara h 1, Ara h 3 and Ara h 9, most preferably Ara h 2 and/or Ara h 6.

In another preferred embodiment of the third aspect, the presence of antibodies against Ara h 7 isoform 7.0201, preferably SEQ ID NO 6, more preferably against a sequence selected from SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably against SEQ ID NO 8, and the presence of antibodies against one or more other antigens, preferably Ara h 2 and/or Ara h 6, are detected simultaneously.

In another preferred embodiment of the third aspect, the presence of antibodies against Ara h 7 isoform 7.0201, preferably SEQ ID NO 6, more preferably against a sequence selected from SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably against SEQ ID NO 8, and the presence of antibodies against one or more other antigens, preferably Ara h 2 and/or Ara h 6, are detected in spatially separated binding reactions.

In another preferred embodiment of the third aspect, the presence of antibodies to Ara h 7 isoform 7.0201 and the presence of antibodies to one or more other antigens, preferably Ara h 2 and/or Ara h 6, are detected in a one-pot reaction.

In another preferred embodiment of the third aspect, the method comprises the step of contacting a diagnostically useful carrier according to the invention with a sample from a subject.

In another preferred embodiment of any aspect, the subject suffers from or is suspected of suffering from an allergy, preferably an allergy to tree nuts, more preferably peanuts.

In another preferred embodiment of any aspect, the antibody against Ara h 7 isoform 7.0201, preferably SEQ ID NO 6, is a monospecific antibody against Ara h 7 isoform 7.0201, more preferably against a sequence selected from SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably against SEQ ID NO 8.

In a fourth aspect, the problem of the present invention is solved by a pharmaceutical composition comprising SEQ ID NO 8, more preferably a sequence selected from SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, more preferably SEQ ID NO 6, most preferably Ara h 7 isoform 7.0201, or a variant thereof, and preferably comprising one or more further antigens selected from Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, Ara h 8 and Ara h 5 and variants thereof, more preferably selected from Ara h 2, Ara h 6, Ara h 1, Ara h 3 and Ara h 9 and variants thereof, most preferably Ara h 2 and/or Ara h 6.

In a fifth aspect, the problem of the present invention is solved by comprising a polypeptide comprising SEQ ID NO 8, more preferably a sequence selected from SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, more preferably SEQ ID NO 6, most preferably Ara h 7 isoform 7.0201, or a variant thereof.

In a preferred embodiment, the polypeptide according to the present invention is immobilized, purified or a fusion protein, preferably purified, more preferably immobilized and purified, more preferably purified, recombinant and immobilized.

In a sixth aspect, the problem is solved by the use of a polypeptide, a vector or a kit according to the invention for diagnosing peanut allergy, wherein preferably the sensitivity is increased.

In a seventh aspect, the problem is solved by the use of a polypeptide according to the invention for the preparation of a kit for diagnosing peanut allergy, wherein preferably the sensitivity of the diagnosis is increased.

Preferably, the polypeptide comprising SEQ ID NO 8, more preferably a sequence selected from SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, more preferably SEQ ID NO 6, most preferably Ara h 7 isoform 7.0201, or a variant thereof is a composition comprising said polypeptide and Ara h 2 and/or Ara h 6 or a variant thereof.

In an eighth aspect, the problem is solved by an antibody, preferably an IgE class antibody, that specifically binds to Ara h 7 isoform 7.0201, preferably SEQ ID NO 6, more preferably a sequence selected from SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably SEQ ID NO 8. The antibody is preferably an isolated antibody. The antibody can be isolated from a patient sample by affinity chromatography using the antigen as an affinity ligand using standard protocols.

In a ninth aspect, the problem is solved by the use of an antibody according to the present invention for diagnosing peanut allergy or for preparing a kit for diagnosing peanut allergy.

The present invention revolves around the detection of antibodies directed against Ara h 7 isoform 7.0201, more particularly its surprisingly immunoreactive C-terminus, in particular an epitope comprising SEQ ID NO 8, as part of a diagnostic method performed on samples from patients suspected of suffering from nut allergy. Surprisingly, some allergic patients possess antibodies that are monospecific only for Ara h 7 isoform 7.0201 comprising SEQ ID NO 8, and do not bind to other Ara h 7 isoforms, more particularly 7.0101 and Ara h 7.0, let alone to Ara h 2 or Ara hh 6.

This is in stark contrast to previously published studies, such as those by Codreanu et al., which showed that detection of antibodies against any Arah 7 isoform had limited diagnostic value compared to, or in particular, exceeded the diagnostic value of data from detection of Ara h 2 and/or Ara h 6. In particular, these studies demonstrated that no patients had antibodies monospecific for any Ara h 7 isoform.

The present invention relates to a diagnostically useful carrier, which is preferably a solid phase carrier for contacting a tool for specific capture of antibodies (the tool is bound to the carrier) with a body fluid sample from a subject, preferably a mammalian subject, more preferably a human subject. In a preferred embodiment, the solid phase carrier is a diagnostic device, more preferably selected from beads, test strips, microtiter plates, blots, glass surfaces, biochips and membranes, more preferably selected from blots, test strips and membranes. In a most preferred embodiment, the diagnostically useful carrier is a microtiter plate or a linear blot (Raoult, D., and Dasch, G.A. (1989), The line blot: an immunoassay for monoclonal and other antibodies. Its application to the serotyping of gram-negative bacteria. J. Immunol. Methods, 125 (1-2), 57-65; WO2013041540). In a preferred embodiment, the term "line blot" as used herein refers to a test strip coated with one or more tools for capturing antibodies, preferably respective polypeptides, more preferably a membrane-based test strip. If two or more means are used, they are preferably spatially separated on the carrier. Preferably, the width of the band is at least 30%, more preferably 40, 50, 60, 70 or 80% of the width of the test strip. The test strip may include one or more control bands to confirm that it has been in contact with the sample for a sufficient period of time under appropriate conditions (particularly in the presence of human serum, antibody conjugate, or both). A large number of line blots are commercially available, for example from EUROIMMUN, Lübeck, Germany.

The sample from experimenter that is used for implementing the present invention comprises antibody, is also referred to as immunoglobulin.Usually, sample is the body fluid of the representative group of immunoglobulin that comprises whole experimenter.Yet, once provide, sample just can be further processed, and it can comprise whole immunoglobulins or any immunoglobulin classification of fractionation, centrifugation, enrichment or separation experimenter, preferably IgE, and it can affect the relative distribution of the immunoglobulin of various classifications.Sample can be selected from whole blood, serum, cerebrospinal fluid and saliva, and is preferably serum.In the most preferred embodiment, sample comprises IgE class antibody.In a more preferred embodiment, sample comprises from classification IgA, IgG and IgE, preferably IgG and IgE, more preferably the representative group of experimenter's antibody of IgG1, IgG4 and IgE, wherein most preferably antibody quantity and the ratio of different antigens are compared with the ratio in the sample obtained from experimenter and do not change basically.

Diagnostically useful carriers include means for the specific capture of antibodies directed against Ara h 7 isoform 7.0201, preferably SEQ ID NO 8, optionally in combination with one or more additional antigens such as Ara h 2 and/or Ara h 6. In a preferred embodiment, as used herein, the terms "Ara h 7 isoform 7.0201", "Ara h 2", "Ara h 6", "Ara h 1", "Ara h 3", "Ara h 5", "Ara h 8" and "Ara h 9" refer to the sequences encoded by the database B4XID4 (Ara h 7 isoform 7.0201, expressed sequence without a signal peptide: SEQ ID NO 2) and Q6PSU2 (Ara h 2, expressed sequence without a signal peptide: SEQ ID NO 4), Q647G9 (Ara h 6, expressed sequence without a signal peptide: SEQ ID NO 5), P43238 (Ara h 1), O82580 (Ara h 3), Q9SQI9 (Ara h 5), B0YIU5 or Q6VT83 (two isoforms of Ara h 8) and B6CG41 or B6CEX8 (Ara h 9), respectively. Preferably, the term "Ara h 7" as used herein refers to all three isoforms of Ara h 7, the term "Ara h 7.0101" refers to the isoform having the sequence SEQ ID NO: 1, and the term "Ara h 7.0" refers to the isoform having the sequence SEQ ID NO: 3. Any database code cited throughout this application refers to the polypeptide sequence available through the online NCBI database on the priority date of this application. Preferably, antibodies that bind to Ara h 7.0201 according to the present invention, but do not bind to any other Ara h 7 isoform, more particularly Ara h 7.0101 and Ara h 7.0, are detected in the sample, and the means for specifically capturing antibodies against Ara h 7 isoform 7.0201 is configured for this purpose and is preferably a polypeptide comprising a sequence selected from SEQ ID NO 8 or a variant thereof, preferably selected from SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 6, SEQ ID NO 9 and SEQ ID NO 10, more preferably SEQ ID NO 6, most preferably Ara h 7 isoform 7.0201 or a variant thereof.

Preferably, the antibody against Ara h 7 isoform 7.0201 is an antibody against SEQ ID NO 6, more preferably a sequence selected from SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably SEQ ID NO 8. More preferably, it does not bind to any epitope common to the three Ara h 7 isoforms.

In a preferred embodiment, as used herein, the term "means for specifically capturing antibodies against (antigen) X and antibodies against (antigen) Y" refers to the sum of a means for specifically capturing antibodies against (antigen) X but not antibodies against (antigen) Y and a means for specifically capturing antibodies against (antigen) Y but not antibodies against (antigen) X.

For example, X can be Ara h 2 and Y can be Ara h 6. In this case, the means for specifically capturing antibodies against Ara h 2 and Ara h 6 will include means for specifically capturing antibodies against Ara h 2 and further means for specifically capturing antibodies against Ara h 6. For example, a line blot coated with Ara h 2 and Ara h 6 (which are spatially separated from each other) is a means for specifically capturing antibodies against Ara h 2 and Ara h 6.

According to the present invention, the carrier comprises one or more means for specifically capturing antibodies, preferably one or more, more preferably two or more, more preferably three or more, more preferably four or more such means, each of which is capable of specifically capturing a different antibody. In a most preferred embodiment, the carrier comprises means for specifically detecting antibodies against Ara h 2, Ara h 7 isoform 7.0201 and Ara h 6. The means are preferably immobilized on the carrier. In a preferred embodiment, the means for specifically capturing antibodies is a polypeptide comprising or consisting of an antigen or variant thereof to which the antibody to be captured or detected binds (e.g., selected from Ara h 2, Ara h 7 isoform 7.0201 and Ara h 6, preferably Ara h 7 isoform 7.0201 or a variant thereof). Preferably, the polypeptide, when used to detect antibodies against Ara h 7 isoform 7.0201, comprises a sequence comprising SEQ ID NO: 8, more preferably a sequence comprising SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, more preferably SEQ ID NO: 6, and most preferably Ara h 7 isoform 7.0201, or a variant thereof. The polypeptide may be a linear peptide or a folded polypeptide, the latter preferably being a variant that adopts a fold substantially identical to Ara h 7 isoform 7.0201 as determined by CD spectroscopy. In a preferred embodiment, the peptide or polypeptide comprises an epitope of at least 7, preferably 10, and more preferably 15 amino acids of the antibody to be captured or detected. The antigen, along with the insoluble support to which it is attached, can be directly separated from the reaction mixture upon contact with the sample, for example by filtration, centrifugation, or decantation. The antigen can be immobilized in a reversible or irreversible manner. For example, if the molecule interacts with the support through ionic interactions (which can be masked by the addition of high concentrations of salt), or if the molecule is bound by a cleavable covalent bond, then the immobilization is reversible. In contrast, if the molecule is attached to the support by a covalent bond that cannot be cleaved in aqueous solution, the immobilization is irreversible.Polypeptides can be immobilized indirectly, for example by immobilizing an antibody or other entity that has affinity for the polypeptide, then adding the polypeptide and forming a polypeptide-antibody complex.

In a preferred embodiment, the carrier is selected from beads, test strips, microtiter plates, microarrays, solid polymers derived from cellulose, blots (preferably selected from protein blots, line blots, and dot blots), glass surfaces, slides, biochips, and membranes, and most preferably is a microtiter plate or a line blot. In another preferred embodiment, the carrier is selected from beads, test strips, microtiter plates, microarrays, solid polymers derived from cellulose, blots selected from line blots and dot blots, glass surfaces, slides, and biochips, and most preferably is a microtiter plate or a line blot.

If the diagnostically useful carrier is a bead, a mixture of beads each carrying a type of means for specifically capturing antibodies can be used. The mixture comprises at least one bead carrying means for specifically capturing antibodies against Ara h 7 isoform 7.0201 (preferably against a sequence selected from the group comprising SEQ ID NO: 6, more preferably against a sequence from the group comprising SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and most preferably against SEQ ID NO: 8 or a variant thereof). In addition, the mixture of beads may comprise at least one additional bead, each carrying means for specifically capturing antibodies against one or more of the group comprising Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, Ara h 8, and Ara h 5, and variants thereof, more preferably against one or more of the group comprising Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, and variants thereof, and most preferably against Ara h 2 and/or Ara h 6.

If the diagnostically useful carrier is a bead, the bead may alternatively comprise, in addition to means for specifically capturing antibodies against Arah 7 isoform 7.0201, preferably against a sequence from the group comprising SEQ ID NO: 6, more preferably against a sequence from the group comprising SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, most preferably against SEQ ID NO: 8 or against a variant thereof, at least one or more further means for specifically capturing antibodies, which may be antibodies against one or more of the group comprising Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, Ara h 8 and Ara h 5 and variants thereof, more preferably against one or more of the group comprising Ara h 2, Ara h 6, Ara h 1, Arah 3, Ara h 9 and variants thereof, most preferably against Ara h 2 and/or Ara h 6. Most preferably, such beads comprise means for specifically capturing antibodies against Ara h 7 isoform 7.0201, preferably against a sequence from the group comprising SEQ ID NO 6, more preferably against a sequence from the group comprising SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably against SEQ ID NO 8, means for specifically capturing antibodies against Ara h 2 and means for specifically capturing antibodies against Ara h 6.

However, the teachings of the present invention can be performed not only using polypeptides such as Ara h 7 isoform 7.0201 (having the exact sequence mentioned in this application, e.g., explicitly or implicitly by function, name, sequence or accession number), optionally in combination with one or more additional antigens such as Ara h 2 and/or Ara h 6, but also using variants of these polypeptides.

In a preferred embodiment, the term "variant" as used herein may refer to at least one fragment of the full-length sequence mentioned, more particularly to one or more amino acid or nucleic acid sequences that are truncated at one or both ends by one or more amino acids relative to the full-length sequence. Such fragments comprise or encode a peptide having at least 10, 15, 25, 50, 75, 100, 150 or 200 consecutive amino acids of the original sequence or a variant thereof. The total length of the variant may be 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids.

In another preferred embodiment, the term "variant" relates not only to at least one fragment, but also to a polypeptide comprising an amino acid sequence or a fragment thereof, preferably a fragment comprising at least 25, more preferably 50, more preferably 200 consecutive amino acids that are at least 40, 50, 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% identical to a reference amino acid sequence or a fragment thereof, wherein amino acids other than those essential for biological activity (e.g., the ability to specifically bind to an antibody of interest, or the folding or structure of the polypeptide) are deleted or substituted and/or one or more such essential amino acids are replaced in a conservative manner and/or amino acids are added or deleted such that the biological activity of the polypeptide is at least partially retained. The state of the art includes various methods that can be used to align two given nucleic acid or amino acid sequences and calculate the degree of identity, see, for example, Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3rd edition. In a preferred embodiment, Clustal W software (Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007): Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948) is used using default settings.

In a preferred embodiment, the variant may also include chemical modifications, such as isotope labeling or covalent modifications, such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation, etc. Those skilled in the art are familiar with methods for modifying polypeptides. In addition, variants can also be generated by fusion with other known polypeptides or variants thereof.

The polypeptide variant has biological activity. In a preferred embodiment, such biological activity is the ability to bind to a corresponding antibody. For example, a variant of Ara h 7 isoform 7.0201 has the ability to specifically bind to an antibody against Ara h 7 isoform 7.0201 in a sample obtained from a subject allergic to peanut, preferably an antibody of the IgA, IgE, or IgG class, more preferably an antibody of the IgE, IgG1, or IgG4 class, because it comprises the epitope bound by the antibody. Preferably, it comprises the epitope recognized by an antibody in a sample from an allergic patient, said antibody binding to Ara h 7 isoform 7.0201, more preferably to an epitope comprising SEQ ID NO: 8, most preferably to SEQ ID NO: 8, and more preferably not to isoforms 7.0101 or 7.0201. Taking into account the significance of SEQ ID NO 8, one skilled in the art is able to design biologically active variants starting from the original Ara h 7 isoform 7.0201 sequence, introduce modifications such as point mutations, truncations, etc., and then confirm that the variants are still biologically active by testing whether they bind to IgE antibodies against Ara h 7 isoform 7.0201 in samples obtained from subjects allergic to peanuts, preferably using the ELISA described in detail in Example 1.

If a polypeptide is used as a tool for specifically capturing an antibody (e.g., an antibody against Ara h 7 isoform 7.0201), the polypeptide can be provided as a purified and/or isolated polypeptide that can be substantially pure in any form and at any degree of purification from tissues, fruits, or cells containing the polypeptide in endogenous form, more preferably cells that overexpress the polypeptide, or a crude or enriched lysate of such cells when used to implement the teachings of the present invention. In a preferred embodiment, the polypeptide is a native polypeptide, wherein the term "native polypeptide" as used herein refers to a folded polypeptide, more preferably a folded polypeptide purified from a tissue or cell, more preferably from a mammalian cell or tissue, optionally from a non-recombinant tissue or cell. If a native polypeptide is used, it is preferably enriched compared to its native state.

According to the present invention, the polypeptide may be a recombinant protein, wherein the term "recombinant" as used herein refers to a polypeptide produced using genetic engineering methods at any stage of the production process, for example by fusing a nucleic acid encoding the polypeptide to a strong promoter for overexpression in a cell or tissue or by modifying the sequence of the polypeptide itself. Those skilled in the art are familiar with methods for modifying nucleic acids and encoded polypeptides (e.g., as described in Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989), Molecular Cloning, CSH or in Brown T.A. (1986), Gene Cloning—an introduction, Chapman & Hall) and for producing and purifying natural or recombinant polypeptides (e.g., as described in the Handbook "Strategies for Protein Purification", "Antibody Purification" and Burgess, R.R., Deutscher, M.P. (2009): Guide to Protein Purification, published by GE Healthcare Life Sciences). In another preferred embodiment, the polypeptide is an isolated polypeptide, wherein the term "isolated" means that the polypeptide has been enriched compared to the state in which it was produced using biotechnological or synthetic methods, and is preferably pure, i.e., at least 60, 70, 80, 90, 95 or 99% of the polypeptide in a corresponding sample consists of said polypeptide, as judged by SDS polyacrylamide gel electrophoresis followed by Coomassie blue staining and visual inspection. Any polypeptide on a support used as a means for capturing antibodies is preferably pure.

In a preferred embodiment, according to the present invention, there is provided a method for providing antibodies, preferably against Arah 7 isoform 7.0201, comprising the steps of: a) providing a sample comprising antibodies against Ara h 7 isoform 7.0201, preferably against a sequence from the group comprising SEQ ID NO 6, more preferably against a sequence from the group comprising SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably against SEQ ID NO 8, b) contacting the sample with means for specifically capturing antibodies against Ara h 7 isoform 7.0201, preferably against a sequence from the group comprising SEQ ID NO 6, more preferably against a sequence from the group comprising SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably against SEQ ID NO 8, under conditions compatible with formation of a complex comprising the antibody and the means, and c) isolating the complex, optionally followed by releasing and/or detecting the antibody from the complex.

The subject of the present invention providing the sample is an organism that produces antibodies, more preferably a mammal, most preferably a human, and the antibodies are preferably IgA, IgE or IgG, more preferably IgE, IgG1 or IgG4 class or equivalent allergy-related antibodies.

Within the scope of the present invention are diagnostically useful carriers comprising means for the specific capture of antibodies directed against an antigen such as Ara h 7 isoform 7.0201. In preferred embodiments, the term "specific capture antibody" as used herein refers to the ability to specifically bind to the antibody of interest so that it is bound and removed from a sample, while other antibodies, preferably antibodies of the same class, do not substantially bind and remain in the sample.

The diagnostically useful carrier according to the present invention serves as a scaffold for one or more tools for specifically capturing antibodies, preferably diagnostically relevant antibodies. The carrier is suitable for performing a diagnostic method. By using a carrier rather than a free soluble tool to specifically capture the antibody, it is more direct to separate and separate the complex comprising the tool and the antibody from the sample and to wash the complex, for example in order to remove any molecules that are non-specifically bound to the tool, the complex or the carrier. In a preferred embodiment, the diagnostically useful carrier is a diagnostic device, preferably selected from the group comprising beads, test strips, microtiter plates, blots and membranes, and preferably a microtiter plate or a line blot.

According to the present invention, means for specifically detecting captured antibodies are provided, optionally as part of a kit. In preferred embodiments, the term "specifically detecting captured antibodies" as used herein refers to detecting antibodies that specifically bind to the means for specifically capturing antibodies, preferably Ara h 7 isoform 7.0201, after capture, rather than detecting any other antibodies present in the sample. In preferred embodiments, the term "specific binding" as used herein refers to binding that is stronger than a binding reaction characterized by a dissociation constant of 1 x ^10-5 M, more preferably 1 x ^10-7 M, more preferably 1 ^{x 10-8 M} , more preferably 1 x ^10-9 M, more preferably 1 x ^10-10 M, more preferably 1 x 10-11 M, more preferably 1 x ^10-12 M, as measured by surface plasmon resonance using a Biacore instrument at 25°C in PBS buffer at pH 7.

In a preferred embodiment, the means for specifically capturing antibodies against Ara h 7 isoform 7.0201 and the means for specifically capturing antibodies against one or more additional antigens are on separate supports. This means that the means are not attached to a single support, but rather to one or more separate supports that are separate and/or can be separated without damaging them. For example, the means for specifically capturing antibodies against Ara h 7 isoform 7.0201 can be attached to a first test strip, and the means for specifically capturing antibodies against Ara h 2 can be attached to another test strip that is separate from the first test strip.

In another preferred embodiment, the means for specifically capturing antibodies against Ara h 7 isoform 7.0201 and the means for specifically capturing antibodies against one or more additional antigens are on one support, preferably covalently linked to one support. This means that the means are attached to a support that cannot be removed without damaging the support so that the means are on separate supports. For example, the device can be entirely coated on a test strip (particularly in the form of a line blot).

The teachings of the present invention provide a kit, preferably for diagnosing allergies, more preferably for diagnosing peanut allergies. Such a kit is a container containing the specific reagents required for carrying out the method of the invention, in particular a diagnostically useful carrier according to the invention, and optionally further containing one or more solutions required for carrying out the method of the invention, preferably selected from or all selected from a sample dilution buffer, a wash buffer, and a buffer containing means for detecting any specifically captured antibody (e.g., a secondary antibody) and optionally a means for detecting the latter. In addition, it may include instructions detailing how to use the kit and the diagnostically useful carrier of the invention (e.g., a line blot, wherein the antibody of the invention for specifically capturing Ara h 7 isoform 7.0201 is immobilized on the line blot) to contact the polypeptide of the invention with a body fluid sample from a subject, preferably a human subject. In addition, the kit may contain a positive control, such as a recombinant antibody known to bind Ara h 7 isoform 7.0201, and a negative control, such as a protein with no detectable affinity for Ara h 7 isoform 7.0201, such as bovine serum albumin. Finally, such a kit may contain a standard solution containing an antibody binding to Ara h 7 isoform 7.0201 to prepare a calibration curve. In a preferred embodiment, the kit comprises a device, preferably a blot-based device, such as a line blot coated with means for specifically capturing antibodies to Ara h 7 isoform 7.0201 and optionally antibodies to one or more other antigens such as Ara h 2 and/or Ara h 6.

According to the present invention, a tool for detecting one or more captured antibodies is needed and can be included in a kit. Those skilled in the art are aware of many methods that can be used, which are also described in the prior art, such as Zane, H.D. (2001), Immunology-Theoretical & Practical Concepts in Laboratory Medicine, W.B. Saunders Company, particularly in Chapter 14. In a preferred embodiment, a second antibody is used that is bound to the constant region of one or more captured antibodies (which are the corresponding primary antibodies), and the second antibody can be bound to a directly detected label such as a fluorescent, radioactive or enzymatically active label (the latter can catalyze a chemiluminescent reaction or produce a molecule that can be detected by colorimetry or spectroscopy or other analytical methods). In a more preferred embodiment, the second antibody is bound to a label selected from a fluorescent label, a radioactive label and a chemiluminescent label.

Alternatively, a biofunctional assay can be used as a tool for detecting one or more captured antibodies, provided that the antibodies are IgE class antibodies, preferably an assay based on IgE antibody basophil activation. Such assays have been described in the prior art, for example, Hausmann, O.V., Gentinetta, T., Bridts, C.H., and Ebo, E.G. (2009): The Basophil Activation Test in Immediate-Type Drug Allergy, Immunol. Allergy Clin. N.Am. 29, 555-566.

In a preferred embodiment, the term "diagnosis", as used herein, refers to any type of procedure aimed at obtaining useful information that helps to assess whether a patient has had, is likely to have, or is more likely than the average or comparison subjects (the latter preferably having similar symptoms) a certain disease or condition in the past, at the time of diagnosis or in the future, to understand how a disease has progressed or is likely to progress in the future or to assess a patient's responsiveness to a certain treatment (e.g., the administration of a suitable medication, such as a medication for desensitization of allergy patients). In other words, the term "diagnosis" includes not only diagnosis, but also prediction and/or monitoring of the course of a disease or condition. In a more preferred embodiment, the term "diagnosis", as used herein, means that it is unknown whether the subject (from whom the sample is analyzed according to the present invention) is allergic to nuts, preferably peanuts, as demonstrated by the presence in their blood of antibodies against peanut-specific allergens (e.g., selected from the group comprising Ara h 2, Ara h 6 and Ara h 7, preferably Ara h 7).

Therefore, the term "diagnosis" preferably does not mean that the diagnostic method or reagent according to the present invention will be definitive and sufficient to complete the diagnosis based on a single test, let alone a parameter, but may refer to a contribution to the so-called "differential diagnosis", that is, a systematic diagnostic procedure that considers the possibility of a series of possible conditions based on a series of diagnostic parameters. The term "diagnosis" may also refer to a method or reagent for selecting the most promising treatment regimen for a patient. In other words, the method or agent may involve selecting a treatment regimen for a subject.

The present invention relates to a method comprising detecting in a sample from a subject the presence or absence of antibodies directed against Ara h 7 isoform 7.0201, preferably against a sequence from the group comprising SEQ ID NO 6, more preferably against a sequence from the group comprising SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, and most preferably against SEQ ID NO 8. Such a method may comprise the steps of: a) providing a sample from a subject, b) contacting the sample with a diagnostically useful carrier according to the invention under conditions compatible with the formation of a complex comprising the diagnostically useful carrier and an antibody (more particularly a means for specifically capturing the antibody and an antibody), c) isolating any said complex, for example by removing the sample, d) optionally washing the complex, and e) detecting the complex. The method is preferably an in vitro method. Detection of the complex, method or test kit for prognosis or diagnosis according to the present invention comprises the use of a method selected from the group consisting of immunodiffusion techniques, basophil activation by IgE antibodies, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, and labeled immunoassays, for example, labeled immunoassays selected from the group consisting of radiolabeled immunoassays, enzyme immunoassays such as colorimetric assays, chemiluminescent immunoassays, and immunofluorescent techniques. In a preferred embodiment, the complex is detected using a method selected from the group consisting of immunodiffusion techniques, basophil activation by IgE antibodies, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, and labeled immunoassays selected from the group consisting of radiolabeled immunoassays, chemiluminescent immunoassays, and immunofluorescent techniques. Those skilled in the art are familiar with these methods, which are also described in the prior art, for example, Zane, H.D. (2001): Immunology—Theoretical & Practical Concepts in Laboratory Medicine, W.B. Saunders Company, in particular in Chapter 14.

In many cases, the presence or absence of the antibody in the test sample (optionally meaning to determine whether the concentration of the antibody exceeds a certain threshold (usually indicated by the detection limit) in the implicit detection limit of the present method, preferably as set by measurement using an ELISA (preferably as described in Example 1)) is sufficient for diagnosis. If the antibody can be detected, this is useful information for the clinician's diagnosis and indicates that the patient has an increased likelihood of having the disease. In a preferred embodiment, the relative concentration of the antibody in the serum compared to the level that can be found in an average healthy subject can be determined. In a preferred embodiment, the term "detection presence" as used herein means being sufficient to verify whether a signal sufficient to exceed any background level is detected using a composite detection method indicating that the antibody of interest exists or that there is more of the antibody of interest than in a healthy subject. In a more preferred embodiment, this may involve determining whether the concentration is at least 0.1, preferably 0.2, 0.5, 1, 2, 5, 10, 20, 25, 50, 100, 200, 500, 1000, 10000 or 100000 times the concentration of the antibody of interest found in an average healthy subject.

In a preferred embodiment, the presence or absence of at least one antibody, such as an antibody against Ara h 7 isoform 7.0201 and an antibody against Ara h 2 and/or an antibody against Arah 6, is detected simultaneously (i.e., at the same time). This is convenient for efficient diagnostic procedures because maximum diagnostic information is obtained within a given time period. A prerequisite, of course, is sufficient capacity to run all reactions.

In a preferred embodiment, the absence or presence of at least two antibodies (e.g., an antibody against Ara h 7 isoform 7.0201 and an antibody against Ara h 2 and/or an antibody against Ara h 6) is detected in spatially separated reactions. This means that these reactions are run in separate vessels in different reaction mixtures.

If more than one antibody is to be detected, the method can be performed in a one-pot reaction. Preferably, the term "one-pot reaction" as used herein refers to two or more, preferably all, reactions carried out in order to detect the presence or absence of an antibody, being carried out in the same reaction mixture in one reaction vessel, with no physical barriers between the reactions, as opposed to experimental setups where at least two reactions are envisaged to be carried out in separate solutions and reaction vessels.

The antibody to be detected can be a monospecific antibody. In a preferred embodiment, the term "monospecific antibody" as used herein refers to an antibody that binds only to one antigen, preferably only to one diagnostically relevant antigen, more preferably only to one diagnostically relevant antigen from the group consisting of Ara h 7, Ara h 2, and Ara h 6. In a more preferred embodiment, the monospecific antibody to be detected can bind to Ara h 7 isoform 7.0201, preferably to SEQ ID NO 6, more preferably to a sequence from the group consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10, most preferably to SEQ ID NO 8, but does not bind to any other allergens, such as isoform Ara h 70101, Ara h 7.0, or, for example, Ara h 2 and/or Ara h 6. If a patient has only monospecific antibodies, their allergy can only be detected serologically using a tool that specifically captures and detects the monospecific antibody. If a diagnostic assay based on a tool for detecting antibodies against an Ara h 7 isoform different from 7.0201 that does not comprise SEQ ID NO 8 is used, the assay result may be false negative because antibodies monospecific for Ara h 7 isoform 7.0201, more preferably SEQ ID NO 8, cannot be detected.

The present invention provides the use of a means for specifically capturing antibodies against Ara h 7 isoform 7.0201, preferably a polypeptide comprising Ara h 7 isoform 7.0201 or a variant thereof, for increasing the sensitivity of a diagnostically useful carrier, for detecting antibodies against Ara h 7, preferably monospecific antibodies against Ara h 7, for preparing a kit for diagnosing peanut allergy or a method for diagnosing nut allergy, preferably peanut allergy, optionally in combination with a means for specifically capturing antibodies against one or more additional antigens, such as Ara h 2 and/or Ara h 6.

The present invention provides for the use of an antibody that specifically binds to Ara h 7 isoform 7.0201, preferably SEQ ID NO 6, more preferably to a sequence from the group consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10, and most preferably to SEQ ID NO 8. Preferably, the antibody is a monospecific antibody. More preferably, the antibody is detected in or isolated from a patient sample and is of the IgA, IgE, or IgG class, more preferably of the IgE, IgG1, or IgG4 class, and most preferably of the IgE class.

The present invention provides antibodies or fragments thereof that specifically bind to Ara h 7 isoform 7.0201, preferably SEQ ID NO: 6, more preferably to a sequence from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and most preferably to SEQ ID NO: 8. The antibody is preferably a monospecific antibody. The antibody may be a monoclonal antibody. The antibody may be a polyclonal antibody. The antibody may be used in a variety of diagnostic applications, for example, to measure the affinity of an antibody from a sample. Alternatively, the antibody may be used in competitive assays, such as competitive ELISAs. Alternatively, the antibody may be used to immobilize a polypeptide, preferably comprising SEQ ID NO: 6, more preferably comprising a sequence from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and most preferably SEQ ID NO: 8. Alternatively, the antibody may be used as a positive control. Those skilled in the art will appreciate how to obtain such antibodies, for example, using recombinant methods. Production may involve steps of purifying and/or isolating the antibody.

The present invention provides a pharmaceutical composition, preferably a vaccine, comprising Ara h 7 isoform 7.0201, preferably a polypeptide comprising SEQ ID NO: 6, more preferably a sequence from the group comprising SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, most preferably SEQ ID NO: 8 or a variant thereof (optionally in combination with one or more additional antigens, such as Ara h 2 and/or Ara h 6 or a variant thereof), the composition preferably being suitable for administration to a subject, preferably a mammalian subject, more preferably a human. Such a pharmaceutical composition may comprise a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered, for example, orally, parenterally, by inhalation spray, topically, by eye drops, rectally, nasally, orally, vaginally or via an implanted reservoir, wherein the term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition may be provided in a suitable dosage form, such as capsules, tablets and aqueous suspensions and solutions, preferably in sterile form. It can be used in a method for treating a disease, preferably an allergy, comprising administering to a subject an effective amount of a polypeptide of the invention. Hypoallergenic variants of Ara h 7 and optionally one or more additional antigens such as Ara h 2 and/or Ara h 6 can be used. Those skilled in the art are familiar with methods for generating hypoallergenic variants of known allergens.

The present invention provides a vaccine comprising Ara h 7 isoform 7.0201, more preferably comprising a polypeptide of SEQ ID NO 6, more preferably a sequence from the group comprising SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably SEQ ID NO 8.

The present invention provides a method for treating, preventing or ameliorating allergy, preferably nut allergy, more preferably peanut allergy, by administering the pharmaceutical composition of the present invention to a subject.

The present invention provides the use of Ara h 7 isoform 7.0201, preferably in combination with Ara h 2 and/or Ara h 6 or variants thereof, more preferably a polypeptide comprising SEQ ID NO 8, more preferably a sequence from the group comprising SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, more preferably SEQ ID NO 6, and most preferably Ara h 7 isoform 7.0201 or variants thereof, for the preparation of a kit for diagnosing peanut allergy, wherein a diagnostic assay with increased sensitivity is provided. Such preparation may involve a method comprising the step of immobilizing Ara h 7 isoform 7.0201, preferably in combination with Ara h 2 and/or Ara h 6 or variants thereof, more preferably a polypeptide comprising SEQ ID NO 6, more preferably a sequence from the group comprising SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, most preferably SEQ ID NO 8, on a diagnostically useful carrier.

The present invention provides the use of Ara h 7 isoform 7.0201, more preferably a polypeptide comprising SEQ ID NO 8, more preferably a sequence from the group comprising SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, more preferably SEQ ID NO 6, most preferably Ara h 7 isoform 7.0201 or a variant thereof, preferably in combination with Ara h 2 and/or Ara h 6 or variants thereof, for the preparation of a medicament, preferably a vaccine, preferably for preventing, ameliorating or treating allergic conditions, particularly nut allergy, more particularly peanut allergy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows IgE binding to the Ara h 7 isoform in peanut-sensitized patients as measured by ELISA as described in Example 1.

FIG2 shows IgE binding to the Ara h 7 isotype as well as Ara h 2 and Ara h 6 from peanut-sensitized patients as detected by line blot as described in Example 2. FIG2 shows IgE binding to the Ara h 7 isotype as well as Ara h 2 and Ara h 6 from peanut-sensitized patients as described in Example 2.

Figure 3 shows the inhibition experiment described in Example 3. Maximum inhibition was observed for the five peanut components in patients sensitized to the immobilized allergen. The horizontal line represents the median. Gray, open markers indicate inhibition using the same allergen as the immobilized allergen.

Figure 4 shows the results of epitope mapping of serum IgE against a linear peptide at the C-terminus of Ara h 7.0201. Normalized signal-to-noise ratios (z-scores) are presented for each peptide on the microarray. Positive binding requires at least 6 subsequent peptides to produce a z-score higher than 3.

sequence:

The present invention includes a series of novel polypeptides, more specifically

SEQ ID NO 1 (expression sequence of Ara h 7.0101 without signal sequence)

TRWDPDRGSRGSRWDAPSRGDDQCQRQLQRANLRPCEEHMRRRVEQEQEQEQDEYPYSRRGSRGRQPGESDENQEQRCCNELNRFQNNQRCMCQALQQILQNQSFWVPAGQEPVASDGEGAQELAPELRVQVTKPLRPL

SEQ ID NO 2 (expression sequence of Ara h 7.0201 without signal sequence)

TRWDPDRGSRGSRWDAPSRGDDQCQRQLQRANLRPCEEHIRQRVEKEQEQEQDEYPYIQRGSRGQRPGESDEDQEQRCCNELNRFQNNQRCMCQALQQILQNQSFRFQQDRSQLHQMERELRNLPQNCGFRSPSRCDLSSRTPY

SEQ ID NO 3 (expression sequence of Ara h 7.0 without signal sequence)

TRWDPDRGSRGLRWDAPSRGDDQCQRQLQRANLRPCEEHIRQRVEQEQEQEQDEYPYSQRGSRGRRPGESDEDQEQRCCNELNRFQNNQRCMCQALQQILQNQSFRFQQDRSQLHQNGEGAQELAPELRVQVTKPLRP

SEQ ID NO 4 (expression sequence of Ara h 2.0201 without signal sequence)

RQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQSDRLQGRQQEQQFKRELRNLPQQCGLRAPQRCDLEVESGGRDRY

SEQ ID NO 5 (expression sequence of Ara h 6.0101 without signal sequence)

MRRERGRQGDSSSCERQVDRVNLKPCEQHIMQRIMGEQEQYDSYDIRSTRSSDQQQRCCDELNEMENTQRCMCEALQQIMENQCDRLQDRQMVQQFKRELMNLPQQCNFRAPQRCDLDVSGGRCSEQ ID NO6 (C-terminus of Ara h 7 isoform 7.0201)

HQMERELRNLPQNCGFRSPSRCDLSSRTPY

SEQ ID NO 7 (C-terminus of Ara h 7 isoform 7.0201)

NCGFRSPSRC

SEQ ID NO 8 (reactive epitope from the C-terminus of Ara h 7 isoform 7.0201)

GFRSPS

SEQ ID NO 9 (C-terminus of Ara h 7 isoform 7.0201)

CGFRSPSRCD

SEQ ID NO 10 (C-terminus of Ara h 7 isoform 7.0201)

QNCGFRSPSRCDL

SEQ ID NO 11 (Ara h 7 isoform 7.0101, as expressed in Example 1)

MSHHHHHHHHLEVLFQGPSMTRWDPDRGSRGSRWDAPSRGDDQCQRQLQRANLRPCEEHMRRRVEQEQEQEQDEYPYSRRGSRGRQPGESDENQEQRCCNELNRFQNNQRCMCQALQQILQNQSFWVPAGQEPVASDGEGAQELAPELRVQVTKPLRPL

SEQ ID NO 12 (Ara h 7 isoform 7.0201, as expressed in Example 1)

MSHHHHHHHHLEVLFQGPSMTRWDPDRGSRGSRWDAPSRGDDQCQRQLQRANLRPCEEHIRQRVEKEQEQEQDEYPYIQRGSRGQRPGESDEDQEQRCCNELNRFQNNQRCMCQALQQILQNQSFRFQQDRSQLHQMERELRNLPQNCGFRSPSRCDLSSRTPY

SEQ ID NO 13 (Ara h 7 isoform 7.0, as expressed in Example 1)

MSHHHHHHHHLEVLFQGPSMTRWDPDRGSRGLRWDAPSRGDDQCQRQLQRANLRPCEEHIRQRVEQEQEQEQDEYPYSQRGSRGRRPGESDEDQEQRCCNELNRFQNNQRCMCQALQQILQNQSFRFQQDRSQLHQNGEGAQELAPELRVQVTKPLRP

SEQ ID NO 14 (Ara h 2 isoform 2.0201, as expressed in Example 1)

MSHHHHHHIEGRTMRQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQSDRLQGRQQEQQFKRELRNLPQQCGLRAPQRCDLEVESGGRDRYSEQ ID NO 15 (Ara h 6 isoform 6.0101, as expressed in Example 1)

MSHHHHHHHHLEVLFQGPSMRRERGRQGDSSSCERQVDRVNLKPCEQHIMQRIMGEQEQYDSYDIRSTRSSDQQQRCCDELNEMENTQRCMCEALQQIMENQCDRLQDRQMVQQFKRELMNLPQQCNFRAPQRCDLDVSGGRC

SEQ ID NO 16: Ara h 1.01.01 as used in Example 3

MSHHHHHHIEGRTMKSSPYQKKTENPCAQRCLQSCQQEPDDLKQKACESRCTKLEYDPRCVYDPRGHTGTTNQRSPPGERTRGRQPGDYDDDRRQPRREEGGRWGPAGPREREREEDWRQPREDWRRPSHQQPRKIRPEGREGEQEWGTPGSH VREETSRNNPFYFPSRRFSTRYGNQNGRIRVLQRFDQRSRQFQNLQNHRIVQIEAKPNTLVLPKHADADNILVIQQGQATVTVANGNNRKSFNLDEGHALRIPSGFISYILNRHDNQNLRVAKISMPVNTPGQFEDFFPASSRDQSSYLQGFSR NTLEAAFNAEFNEIRRVLLEENAGGEQEERGQRRWSTRSSENNEGVIVKVSKEHVEELTKHAKSVSKKGSEEEGDITNPINLREGEPDLSNNFGKLFEVKPDKKNPQLQDLDMMLTCVEIKEGALMLPHFNSKAMVIVVVNKGTGNLELVAVRK EQQQRGRREEEEDEDEEEEGSNREVRRYTARLKEGDVFIMPAAHPVAINASSELHLLGFGINAENNHRIFLAGDKDNVIDQIEKQAKDLAFPGSGEQVEKLIKNQKESHFVSARPQSQSQSPSSPEKESPEKEDQEEENQGGKGPLLSILKAFN

SEQ ID NO 17: Ara h 3.01.01 as used in Example 3

MSHHHHHHHHLEVLFQGPSMRQQPEENACQFQRLNAQRPDNRIESEGGYIETWNPNNQEFECAGVALSRLVLRRNALRRPFYSNAPQEIFIQQGRGYFGLIFPGCPRHYEEPHTQGRRSQSQRPPRRLQGE DQSQQQRDSHQKVHRFDEGDLIAVPTGVAFWLYNDHDTDVVAVSLTDTNNNDNQLDQFPRRFNLAGNTEQEFLRYQQQSRQSRRRSLPYSPYSPQSQPRQEEREFSPRGQHSRRERAGQEEENEGGNIFSGF TPEFLEQAFQVDDRQIVQNLRGETESEEEGAIVTVRGGLRILSPDRKRRADEEEEYDEDEYEYDEEDRRRGRGSRGRGNGIEETICTASAKKNIGRNRSPDIYNPQAGSLKTANDLNLLILRWLGPSAEYGN LYRNALFVAHYNTNAHSIIYRLRGRAHVQVVDSNGNRVYDEELQEGHVLVVPQNFAVAGKSQSENFEYVAFKTDSRPSIANLAGENSVIDNLPEEVVANSYGLQREQARQLKNNNPFKFFVPPSQQSPRAVA

The present invention is further illustrated by the following non-limiting examples, from which further features, embodiments, aspects and advantages of the present invention can be derived.

Example

Example 1: Detection of IgE antibodies against the Ara h 7 isotype in sera from peanut-sensitized patients by ELISA

Recombinant proteins

Heterologous expression and purification of recombinant allergens

Ara h 7.0101 (SEQ ID NO 11), Ara h 7.0201 (SEQ ID NO 12), Ara h 7.0 (SEQ ID NO 13), Ara h 6 (SEQ ID NO 15), and Ara h 2 (SEQ ID NO 14) were expressed and purified in E. coli as His6 fusion proteins having the sequences shown in the corresponding SEQ ID NOs as described in Sitaru C, C, Probst C, Komorowski L, I, Schmidt E, Schlumberger W, Rose C, W, Zillikens D. Enzyme-linked immunosorbent assay using multimers of the 16th non-collagenous domain of the BP180 antigen for sensitive and specific detection of pemphigoid autoantibodies. Exp Dermatol. 2007 Sep; 16(9): 770-7. Purification was performed by immobilized metal ion chromatography under denaturing conditions.

1.2. Detection of IgE binding by ELISA

The IgE binding capacity of the three Ara h 7 isoforms was analyzed by enzyme-linked immunosorbent assay (ELISA) using sera from 33 patients who showed IgE positivity to peanut extract (Figure 1). In addition, 10 sera from atopic patients without peanut allergy and 23 healthy blood donors were incubated as negative controls (all reactions were negative, data not shown).

Microtiter plates were coated with recombinant peanut allergen (9 μg/ml in PBS, pH 7.5) for 2 hours at 4° C. as described by Sitaru et al. Sera were diluted 1/10 (v/v) in blocking buffer (PBS-0.1% bovine serum albumin) and 100 μl per well were applied to the plates in duplicate and allowed to react overnight at 4° C.

Bound antibodies were detected using anti-human IgE peroxidase conjugate and stained with tetramethylbenzidine (Total IgE ELISA EV 3840-9601E, Euroimmun, Lubeck, Germany). Optical density (OD) was read at 450 nm with a reference at 620 nm using an automated spectrophotometer (Spectra Mini, Tecan, Germany).

result

IgE reactivity to Ara h 7 isoform 7.0101, Ara h 7 isoform 7.0201, and Ara h 7.0 was measured in 66 human sera. None of the 23 blood donors and 10 sera from atopic patients who did not have IgE to peanut extract were positive. Of the 23 peanut extract-positive sera, 11 showed positive results for Ara h 7.0201, while only 6 and 10 showed positive results for Ara 7.0101 and Ara h 7, respectively.

Among the positive sera, the average OD of Ara h 7.0201 was 1.0, while the average OD of Ara h 7.0101 and Ara h 7.0 was much lower (both 0.4).

These results suggest that, on the one hand, Ara h 7.0201 reacts more strongly with patient IgE, and on the other hand, more patients can be correctly diagnosed by using Ara h 7.0201 as a target antigen to detect IgE antibodies compared to the other two isotypes (Figure 1). In fact, some patients can only be identified and diagnosed as allergic if antibodies to Ara h 7.0201 are detected.

Example 2: Detection of IgE antibodies by line blotting

The IgE binding capacity of 2S albumin Ara h 2, Ara h 6, and the three Arah 7 isoforms was investigated by immunoblotting analysis. Serum samples from 34 peanut-sensitized subjects (Figure 2) were incubated with line blot-immunoblotting and the intensity was assessed using EUROLINEScan software. In addition, 20 sera from atopic subjects without peanut allergy and 17 healthy blood donors were incubated as negative controls (all negative, data not shown).

For line blot immunoblotting, recombinant allergens are coated onto a nylon-based membrane using standard methods. The membrane is then blocked, dried, and mounted on foil. The foil is cut into strips, which are incubated with serum.

Manual incubation of serum samples was performed according to EUROIMMUN immunoblotting instructions (e.g., in DPA-Dx pollen 1, DP 3210-1601-1E). All incubation and washing steps were completed on a rocking shaker at room temperature (+18°C to +25°C). Reagents were removed from the test kit (DPA-Dx pollen 1, DP 3210-1601-1E) provided by EUROIMMUN. The immunoblotting strips were pre-incubated with working strength universal buffer (WSUB) for 5 minutes. After removing all liquids, the strips were incubated overnight (12 to 24 hours) with 100 μl of each serum sample diluted with 1.0 ml WSUB in the first step. In the second step, the strips were incubated for 60 minutes with 1.0 ml enzyme conjugate (alkaline phosphatase-conjugated anti-human IgE). After the first and second steps, the liquid was aspirated and the strips were washed 3 × 5 minutes with 1.0 ml WSUB. In the third step, the strips are incubated with 1.0 ml of chromogen/substrate solution for 10 minutes. The liquid is then aspirated again, and the enzyme reaction is stopped by washing each strip three times for 1 minute with deionized or distilled water. Finally, the test strips are placed on the evaluation platform, air-dried, and evaluated using EUROLINEScan software.

result

A total of 71 samples were analyzed by immunoblotting. No sIgE to Ara h 2, Ara h 6, or all three Ara h 7 isotypes was detected in 20 sera from atopic subjects without specific IgE (sIgE) to peanut extract and in 17 healthy blood donors. Of the 34 samples from subjects with sIgE to peanut extract, 17 samples reacted positively to Ara h 2 and Ara h 6, 15 samples reacted positively to Ara h 7.0201, 14 samples reacted positively to Ara h 7.0101, and 11 samples reacted positively to Ara h 7.0, respectively.

Among the positively reacting sera, the average immunoblot intensities from Ara h 2, Ara h 6, and Ara h 7.0201 were 84, 59, and 74, respectively, and the average intensities from Ara h 7.0101 and Ara h 7 were considerably lower (20 and 28) (Fig. 2).

The results showed that subjects with sIgE to peanut extract had higher mean IgE levels for Ara h 7.0201 than for Ara h 7.0101 and Ara h 7.0. Furthermore, subjects positive for peanut extract IgE had very similar mean levels of Ara h 2 and Ara h 7.0201 IgE, suggesting that Ara h 7.0201 IgE, like Ara h 2, is another major allergen component of peanut allergy.

Example 3: Studies based on a larger patient cohort using line blot and ELISA

Patient selection

To evaluate the diagnostic value of different peanut storage proteins, adults suspected of peanut allergy based on history and/or sensitization who had undergone a diagnostic open or double-blind placebo-controlled food challenge (DBPCFC) with peanut at the University Medical Center Utrecht between 2003 and September 2014 (n=127) were selected. Residual serum from routine blood collection one year before and after the food challenge date was available for 95 subjects. From this cohort, 40 peanut-allergic patients and 40 tolerant patients (using positive and negative challenges, respectively) were selected based on the availability of ImmunoCAP ISAC sensitization data. DBPCFC was performed according to the international consensus protocol as described above (Taylor SL, Hefle SL, Bindslev-Jensen C, Atkins FM, Andre C, Bruijnzeel-Koomen C et al. A consensus protocol for the determination of the threshold doses for allergenic foods: How much is too much? Clin Exp Allergy 2004; 34: 689–95. doi: 10.1111/j.1365-2222.2004.1886.x.). All positive challenges were DBPCFC. Negative challenges also included two open peanut challenges. For the inhibition experiments, serum was collected from 10 allergic adults sensitized to peanut extract and using active DBPCFC (randomly selected from a cohort previously characterized by Peeters KABM, Koppelman SJ, van Hoffen E, van der Tas CWH, den Hartog Jager CF, Penninks AH, et al. Does skin prick test reactivity to purified allergens correlate with clinical severity of peanut allergy? Clin Exp Allergy 2007;37:108–15. doi:10.1111/j.1365-2222.2006.02628.x).

Line Blot

Sensitization to recombinant peanut storage proteins Ara h 1.0101, 2.0201, 3.0101, 6.0101, and Ara h 7 isoform 7.0201 (Table 1) was assessed using line blots as described in Example 2. Briefly, the test strips were incubated with 100 μl of patient serum diluted 1:11 in dilution buffer at room temperature on an orbital shaker overnight. After washing, the strips were incubated with enzyme-labeled anti-human IgE antibodies and then with the substrates nitro blue tetrazolium/5-bromo-4-chloro-3'-indolyl phosphate, and the reactions were assessed using the "EUROLineScan" software. The intensity of the bands was reported as intensity level and category, corresponding to the enzyme-allergy-adsorbent test classification (category 0-6) (Williams PB, Barnes JH, Szeinbach SL, Sullivan TJ. Analytical precision and accuracy of commercial immunoassays for specific IgE: Establishing a standard. J Allergy Clin Immunol 2000; 105: 1221–30. doi: 10.1067/mai.2000.105219). Categories of 1 or higher (equivalent to intensity levels of 3 or higher) were considered positive.

Heterologous expression and purification of recombinant allergens

Ara h 7.0101 (SEQ ID NO 11), Arah 7.0201 (SEQ ID NO 12), Ara h 7 (SEQ ID NO 13), Ara h 6 (SEQ ID NO 15), and Ara h 2 (SEQ ID NO 14) were expressed in E. coli without a signal sequence as fusion proteins with an N-terminal His (6x) residue, represented by the corresponding SEQ ID NOs described in Sitaru et al. (2007). Purification was performed by immobilized metal ion chromatography under denaturing conditions. For inhibition experiments, the recombinant proteins were dialyzed against TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 3% sucrose, 1.2 mM glutathione (non-reducing), 3.8 mM glutathione (reducing)).

ELISA and inhibition assays

Microtiter plates were coated with immobilized recombinant peanut allergen (9 μg/ml in PBS pH 7.5) for 2 hours at 4°C as described by Sitaru et al.

For dose-dependent cross-inhibition experiments, the optimally diluted serum in blocking buffer described in Example 1 (see Table 3) was pre-incubated with competing allergens at final concentrations of 0.1, 1, and 10 μg/ml at room temperature with shaking for 30 minutes before application to the plate. Inhibition values were given as the percentage of OD reduction compared to the control to which blocking buffer alone was added.

Data analysis and statistics

Correlations between sIgE test results were analyzed using Spearman correlation. Differences in positive tests between tolerant and allergic patients were assessed using the chi-square test. The area under the receiver operating characteristic (ROC) curve (AUC) was determined to assess the ability of the test to discriminate between allergy and tolerance established by food challenge. Analyses were performed using SPSS Statistics 21 (IBM Corporation, Armonk, NY, USA).

result

Evaluation of peanut storage protein sensitization

Table 2 lists the patient characteristics of the 80 peanut-challenged patients evaluated for diagnostic testing. The median age was 25 years (range: 16-77), and 36% were male. Sensitization to all peanut storage proteins was significantly more frequent in the peanut-allergic group. In the peanut-allergic group, 73% were sensitized to at least one of the five storage proteins, compared with 15% in the peanut-tolerant group. Peanut 2S albumin Ara h 2 was the most common sensitizer in peanut-allergic patients (65%), followed by two other 2S albumins, Ara h 6 and 7 (both 60%).

The highest discrimination value was found for Ara h 6 (AUC 0.85), followed by Ara h 7 and 2 (AUC 0.83 and 0.81). EUROLINE intensity values showed strong to very strong correlation with the ISAC ISU results for peanut storage proteins ( _rs values Ara h 1: 0.73, Ara h 2: 0.87, Ara h 3: 0.80, Ara h 6: 0.84, all p < 0.001). Ara h 7 intensity values were strongly correlated with the intensity values of Ara h 2 and 6 on EUROLINE ( _rs = 0.81, both p < 0.001). Table 2 lists the sensitization results and the discriminatory power of the components on EUROLINE and ImmunoCAP ISAC.

Cosensitization to peanut storage proteins was the most common. Of all patients sensitized to peanut storage proteins Ara h 1, 2, 3, 6, or 7, almost half were sensitized to all five (46%; Figure E1 in the online repository), followed by cosensitization to Ara h 2, 6, and 7 alone (14%). When focusing specifically on patients sensitized to 2S albumin Ara h 2, 6, or 7, the majority were cosensitized to all three (n=24, 68%; Figure 1). Monosensitization was observed for Ara h 2 (n=6), Ara h 6 (n=2), and Ara h 7 (n=2), with positive results in 4 of 6, 1 of 2, and 1 of 2 patients, respectively.

ELISA inhibition using Ara h 2, 6, and 7 isoforms

For the 10 patients included in the inhibition study, the average age was 25 years, of whom four were male. Sensitization to peanut extract was detected in all subjects, with ImmunoCAP titers ranging from 1.7 to >100 kU/L. First, IgE reactivity against the three Ara h 7 isotypes as well as Ara h 2 and 6 was assessed by ELISA in 10 sera. Four sera showed reactivity to all five components. In addition, two sera reacted with Ara h 7.0201, Ara h 2 and 6, and one serum reacted only with Ara h 2 and 6. The OD values of the remaining three patients were too low for inhibition experiments. The optimal dilution of each serum was determined (data not shown). Cross-inhibition experiments showed that there was variability in the maximum inhibition obtained between different 2S albumins for each patient serum (Figure 3). In some patients, no inhibition was observed, while other patients showed partial or almost complete inhibition using different immobilized allergens and inhibitors. For the Ara h 7.0101 and 7 isoforms, the other two isoforms were able to achieve strong inhibition, similar to inhibition with the same allergens. For Ara h 7.0201, inhibition using the other two epitopes resulted in very limited inhibition.

The variability in inhibition between the different 2S albumins was even more pronounced (Figure 4). For example, for immobilized Ara h 7.0201, inhibition with Ara h 6 resulted in 8 to 86% inhibition (median 22%), while inhibition with Ara h 2 resulted in 4 to 71% inhibition (median 16%). On the other hand, binding to Ara h 6 and Ara h 2 was inhibited by the Ara h 7 isoform, but Ara h 2 inhibited sIgE directed against Ara h 6 by 12% to 70% (median 43%), while inhibition of sIgE directed against Ara h 6 was as high as 77% (median 24%). In summary, Ara h 2 and 6 were able to inhibit binding to Ara h 7.0201 in some patients but not in others. Although Ara h 2 and 6 were able to inhibit each other's binding to varying degrees, the Ara h 7 isoform had difficulty inhibiting binding to Ara h 2 and 6.

discuss

In our cohort of 80 peanut-challenged patients, we demonstrated that Ara h 7.0201 had a discriminatory capacity very similar to that of the major allergens Ara h 2 and 6, which can be explained by their frequent co-sensitization and the strong correlation between their results. Overall, we found that Ara h 6 had the best discriminatory capacity on both the ImmunoCAP ISAC and EUROLINE systems, slightly higher than Ara h 2.

In addition to common co-sensitization, we also observed monosensitization to Ara h 2, 6, or 7.0201. Although monosensitization to Ara h 2 was the most common (n=6), it is important to recognize the existence of monosensitization to Ara h 6 and 7.0201, which would have been missed when testing only sensitization to Ara h 2. This is the first study to demonstrate monosensitization to Ara h 7.0201 in two subjects.

Previous studies have investigated sensitization to Ara h 7, but only the Ara h 701.01 isoform. After the initial cloning of Ara h 7.0101, Kleber-Janke et al. detected sensitization to rAra h 7.0101 in 17 of 40 (43%) peanut-allergic subjects with a history of established peanut allergy, compared with 85% for Ara h 2. Codreanu et al. investigated the effects of several recombinant peanuts in immunoassays and showed that rAra h 7.0101 had poorer sensitivity than rAra h 2 and 6.

Inhibition experiments in our study showed that Ara h 7.0201 is the most relevant isoform of Ara h 7. Strong inhibition of other isoforms in sera from patients with sIgE to Ara h 7.0101 and 7.0 suggests recognition of epitopes present on all three Ara h 7 isoforms. On the other hand, the lack of inhibition of other isoforms in subjects sensitized to Ara h 7.0201 suggests cosensitization to a unique epitope not present on the other two isoforms. Of the six sera sensitized to any Ara h 7 isoform, four were cosensitized to all three Ara h 7 isoforms. It seems paradoxical that binding to Ara h 7.0201 cannot be inhibited by other isoforms, while, on the other hand, Ara h 7.0201 can inhibit binding to other isoforms. One explanation is the recognition of multiple epitopes (unique and cross-reactive) of Ara h 7.0201 in a single patient, whereby the abundance of sIgE directed against a unique epitope present on one Ara h 7 isoform results in low inhibition of other isoforms lacking that epitope. Another contributing factor may be the difference in accessibility of epitopes to IgE between immobilized coated allergens on a solid phase and dissolved, folded allergens.

Cross-inhibition by 2S albumin showed that IgE against Ara h 2 and 6 could not be inhibited by the Ara h 7 isoform, but to some extent inhibited each other. This could be explained by shared epitopes on Ara h 2 and 6 that are not part of the Ara h 7 isoform.

In conclusion, this study has demonstrated that Ara h 7 isoform 7.0201 is a third clinically relevant peanut 2S albumin with discriminatory power for peanut allergy at the population level comparable to Ara h 2 and 6. Although co-sensitization to peanut storage proteins and more specifically 2S albumin is most common, monosensitization to Ara h 2, 6, or 7 occurs in individual patients, leading to the risk of misdiagnosis when testing a single 2S albumin.

Example 4: Detection of IgE antibodies against specific Ara h 7 epitopes using peptide microarrays

Peptide microarray incubation and visualization

A commercial peptide microarray (PEPperPRINT) containing 15-mer peptides of Ara h 7.0201 (SEQ ID NO 2) with overlapping sequences (offset: 1, each peptide was printed in duplicate) covering the C-terminus of Ara h 7.0201 (SEQ ID NO 6) was obtained. For incubation experiments, the microarray was blocked with working strength universal buffer (WSUB, see Example 2) on an orbital shaker at room temperature for 1 hour. All further described incubations were also performed in WSUB. Sera from 3 peanut-positive and 2 peanut-negative patients were diluted 1:4 and incubated overnight at 4°C. To detect bound IgE antibodies, biotinylated anti-IgE IT-28 (Squarix) was diluted 1:5000 and incubated on the array for 1 hour at room temperature. After washing, the array was incubated with fluorescent Neutravidin 800 (Thermo Fisher) diluted 1:5000 for 1 hour at room temperature. Peptide microarray slides were scanned using a Licor Odyssey imager at a wavelength of 800 nm (intensity: 8.5). The image focus was set to 0.8 mm, and the maximum image resolution (21 μm) was selected to ensure maximum sensitivity.

Evaluate

After scanning, the TIFF images and peptide map files were loaded into the Pepslide Analyzer software (SICASYS) to quantify the signal corresponding to each single peptide. The raw data were exported as

CSV file and calculate the log2 of the signal-to-noise ratio for each single Ara h 7 peptide (S _Ara ) and empty spot (S _Blank ) on the array. For a more robust and normalized evaluation, the z-score (Zi) of each single peptide was calculated according to the following formula:

Based on these calculations, a positive binding epitope was defined as the detection of at least 6 subsequent peptides comprising a z-score of 3.0 or higher (p=0.003).Data visualization was performed using Microsoft Excel (Figure 4).

result

While the two negative sera did not detect the epitope, a previously unknown Ara h 7.0201 epitope containing the sequence GFRSPS (amino acids 129-134) (numbering according to the amino acid residues of SEQ ID NO 2) was detected using serum 3. This epitope was also detectable with serum 1, albeit below the z-score cutoff. Since this sequence is unique to Ara h 7.0201, it can be concluded that this epitope is associated with increased reactivity to Ara h 7.0201 compared to the other two isoforms.

surface

Table 1: Peanut storage proteins present on EUROLINE bands. Adapted from Becker and Jappe [5] and Van Erp et al. [7].

Table 2: Patient characteristics and sensitization data for the 40 peanut-tolerant and 40 peanut-allergic patients in the cohort

IQR: interquartile range. AUC: area under the curve. CI: confidence interval

*The manufacturer's recommended cut-off value for positive tests was used. Difference in the number of positive tests between tolerant and allergic subjects (Chi-square test).

Table 3: Individual dilutions of sera for the dose-dependent inhibition experiment described in Example 3

Sequence Listing

<110> EUROIMMUN Medizinische Labordiagnostika AG

<120> Improved assay for diagnosing peanut allergy

<130> 17PP016

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 1 (expression sequence of Ara h 7.0101 without signal sequence)

<400> 1

Thr Arg Trp Asp Pro Asp Arg Gly Ser Arg Gly Ser Arg Trp Asp Ala

1 5 10 15

Pro Ser Arg Gly Asp Asp Gln Cys Gln Arg Gln Leu Gln Arg Ala Asn

20 25 30

Leu Arg Pro Cys Glu Glu His Met Arg Arg Arg Val Glu Gln Glu Gln

35 40 45

Glu Gln Glu Gln Asp Glu Tyr Pro Tyr Ser Arg Arg Gly Ser Arg Gly

50 55 60

Arg Gln Pro Gly Glu Ser Asp Glu Asn Gln Glu Gln Arg Cys Cys Asn

65 70 75 80

Glu Leu Asn Arg Phe Gln Asn Asn Gln Arg Cys Met Cys Gln Ala Leu

85 90 95

Gln Gln Ile Leu Gln Asn Gln Ser Phe Trp Val Pro Ala Gly Gln Glu

100 105 110

Pro Val Ala Ser Asp Gly Glu Gly Ala Gln Glu Leu Ala Pro Glu Leu

115 120 125

Arg Val Gln Val Thr Lys Pro Leu Arg Pro Leu

130 135

<210> 2

<211> 144

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 2 (expression sequence of Ara h 7.0201 without signal sequence)

<400> 2

Thr Arg Trp Asp Pro Asp Arg Gly Ser Arg Gly Ser Arg Trp Asp Ala

1 5 10 15

Pro Ser Arg Gly Asp Asp Gln Cys Gln Arg Gln Leu Gln Arg Ala Asn

20 25 30

Leu Arg Pro Cys Glu Glu His Ile Arg Gln Arg Val Glu Lys Glu Gln

35 40 45

Glu Gln Glu Gln Asp Glu Tyr Pro Tyr Ile Gln Arg Gly Ser Arg Gly

50 55 60

Gln Arg Pro Gly Glu Ser Asp Glu Asp Gln Glu Gln Arg Cys Cys Asn

65 70 75 80

Glu Leu Asn Arg Phe Gln Asn Asn Gln Arg Cys Met Cys Gln Ala Leu

85 90 95

Gln Gln Ile Leu Gln Asn Gln Ser Phe Arg Phe Gln Gln Asp Arg Ser

100 105 110

Gln Leu His Gln Met Glu Arg Glu Leu Arg Asn Leu Pro Gln Asn Cys

115 120 125

Gly Phe Arg Ser Pro Ser Arg Cys Asp Leu Ser Ser Arg Thr Pro Tyr

130 135 140

<210> 3

<211> 138

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO3 (expression sequence of Ara h 7.0 without signal sequence)

<400> 3

Thr Arg Trp Asp Pro Asp Arg Gly Ser Arg Gly Leu Arg Trp Asp Ala

1 5 10 15

Pro Ser Arg Gly Asp Asp Gln Cys Gln Arg Gln Leu Gln Arg Ala Asn

20 25 30

Leu Arg Pro Cys Glu Glu His Ile Arg Gln Arg Val Glu Gln Glu Gln

35 40 45

Glu Gln Glu Gln Asp Glu Tyr Pro Tyr Ser Gln Arg Gly Ser Arg Gly

50 55 60

Arg Arg Pro Gly Glu Ser Asp Glu Asp Gln Glu Gln Arg Cys Cys Asn

65 70 75 80

Glu Leu Asn Arg Phe Gln Asn Asn Gln Arg Cys Met Cys Gln Ala Leu

85 90 95

Gln Gln Ile Leu Gln Asn Gln Ser Phe Arg Phe Gln Gln Asp Arg Ser

100 105 110

Gln Leu His Gln Asn Gly Glu Gly Ala Gln Glu Leu Ala Pro Glu Leu

115 120 125

Arg Val Gln Val Thr Lys Pro Leu Arg Pro

130 135

<210> 4

<211> 151

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO4 (expression sequence of Ara h 2.0201 without signal sequence)

<400> 4

Arg Gln Gln Trp Glu Leu Gln Gly Asp Arg Arg Cys Gln Ser Gln Leu

1 5 10 15

Glu Arg Ala Asn Leu Arg Pro Cys Glu Gln His Leu Met Gln Lys Ile

20 25 30

Gln Arg Asp Glu Asp Ser Tyr Gly Arg Asp Pro Tyr Ser Pro Ser Gln

35 40 45

Asp Pro Tyr Ser Pro Ser Gln Asp Pro Asp Arg Arg Asp Pro Tyr Ser

50 55 60

Pro Ser Pro Tyr Asp Arg Arg Gly Ala Gly Ser Ser Gln His Gln Glu

65 70 75 80

Arg Cys Cys Asn Glu Leu Asn Glu Phe Glu Asn Asn Gln Arg Cys Met

85 90 95

Cys Glu Ala Leu Gln Gln Ile Met Glu Asn Gln Ser Asp Arg Leu Gln

100 105 110

Gly Arg Gln Gln Glu Gln Gln Phe Lys Arg Glu Leu Arg Asn Leu Pro

115 120 125

Gln Gln Cys Gly Leu Arg Ala Pro Gln Arg Cys Asp Leu Glu Val Glu

130 135 140

Ser Gly Gly Arg Asp Arg Tyr

145 150

<210> 5

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO5 (expression sequence of Ara h 6.0101 without signal sequence)

<400> 5

Met Arg Arg Glu Arg Gly Arg Gln Gly Asp Ser Ser Ser Cys Glu Arg

1 5 10 15

Gln Val Asp Arg Val Asn Leu Lys Pro Cys Glu Gln His Ile Met Gln

20 25 30

Arg Ile Met Gly Glu Gln Glu Gln Tyr Asp Ser Tyr Asp Ile Arg Ser

35 40 45

Thr Arg Ser Ser Asp Gln Gln Gln Arg Cys Cys Asp Glu Leu Asn Glu

50 55 60

Met Glu Asn Thr Gln Arg Cys Met Cys Glu Ala Leu Gln Gln Ile Met

65 70 75 80

Glu Asn Gln Cys Asp Arg Leu Gln Asp Arg Gln Met Val Gln Gln Phe

85 90 95

Lys Arg Glu Leu Met Asn Leu Pro Gln Gln Cys Asn Phe Arg Ala Pro

100 105 110

Gln Arg Cys Asp Leu Asp Val Ser Gly Gly Arg Cys

115 120

<210> 6

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 6 (C-terminus of Ara h 7 isoform 7.0201)

<400> 6

His Gln Met Glu Arg Glu Leu Arg Asn Leu Pro Gln Asn Cys Gly Phe

1 5 10 15

Arg Ser Pro Ser Arg Cys Asp Leu Ser Ser Arg Thr Pro Tyr

20 25 30

<210> 7

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 7 (C-terminus of Ara h 7 isoform 7.0201)

<400> 7

Asn Cys Gly Phe Arg Ser Pro Ser Arg Cys

1 5 10

<210> 8

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 8 (reactive epitope from the C-terminus of Ara h 7 isoform 7.0201)

<400> 8

Gly Phe Arg Ser Pro Ser

1 5

<210> 9

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 9 (C-terminus of Ara h 7 isoform 7.0201)

<400> 9

Cys Gly Phe Arg Ser Pro Ser Arg Cys Asp

1 5 10

<210> 10

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 10 (C-terminus of Ara h 7 isoform 7.0201)

<400> 10

Gln Asn Cys Gly Phe Arg Ser Pro Ser Arg Cys Asp Leu

1 5 10

<210> 11

<211> 159

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO11 (Ara h 7 isoform 7.0101, as expressed in Example 1)

<400> 11

Met Ser His His His His His His His Leu Glu Val Leu Phe Gln

1 5 10 15

Gly Pro Ser Met Thr Arg Trp Asp Pro Asp Arg Gly Ser Arg Gly Ser

20 25 30

Arg Trp Asp Ala Pro Ser Arg Gly Asp Asp Gln Cys Gln Arg Gln Leu

35 40 45

Gln Arg Ala Asn Leu Arg Pro Cys Glu Glu His Met Arg Arg Arg Val

50 55 60

Glu Gln Glu Gln Glu Gln Glu Gln Asp Glu Tyr Pro Tyr Ser Arg Arg

65 70 75 80

Gly Ser Arg Gly Arg Gln Pro Gly Glu Ser Asp Glu Asn Gln Glu Gln

85 90 95

Arg Cys Cys Asn Glu Leu Asn Arg Phe Gln Asn Asn Gln Arg Cys Met

100 105 110

Cys Gln Ala Leu Gln Gln Ile Leu Gln Asn Gln Ser Phe Trp Val Pro

115 120 125

Ala Gly Gln Glu Pro Val Ala Ser Asp Gly Glu Gly Ala Gln Glu Leu

130 135 140

Ala Pro Glu Leu Arg Val Gln Val Thr Lys Pro Leu Arg Pro Leu

145 150 155

<210> 12

<211> 164

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 12 (Ara h 7 isoform 7.0201, as expressed in Example 1)

<400> 12

Met Ser His His His His His His His Leu Glu Val Leu Phe Gln

1 5 10 15

Gly Pro Ser Met Thr Arg Trp Asp Pro Asp Arg Gly Ser Arg Gly Ser

20 25 30

Arg Trp Asp Ala Pro Ser Arg Gly Asp Asp Gln Cys Gln Arg Gln Leu

35 40 45

Gln Arg Ala Asn Leu Arg Pro Cys Glu Glu His Ile Arg Gln Arg Val

50 55 60

Glu Lys Glu Gln Glu Gln Glu Gln Asp Glu Tyr Pro Tyr Ile Gln Arg

65 70 75 80

Gly Ser Arg Gly Gln Arg Pro Gly Glu Ser Asp Glu Asp Gln Glu Gln

85 90 95

Arg Cys Cys Asn Glu Leu Asn Arg Phe Gln Asn Asn Gln Arg Cys Met

100 105 110

Cys Gln Ala Leu Gln Gln Ile Leu Gln Asn Gln Ser Phe Arg Phe Gln

115 120 125

Gln Asp Arg Ser Gln Leu His Gln Met Glu Arg Glu Leu Arg Asn Leu

130 135 140

Pro Gln Asn Cys Gly Phe Arg Ser Pro Ser Arg Cys Asp Leu Ser Ser

145 150 155 160

Arg Thr Pro Tyr

<210> 13

<211> 158

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 13 (Ara h 7 isoform 7.0, as expressed in Example 1)

<400> 13

Met Ser His His His His His His His Leu Glu Val Leu Phe Gln

1 5 10 15

Gly Pro Ser Met Thr Arg Trp Asp Pro Asp Arg Gly Ser Arg Gly Leu

20 25 30

Arg Trp Asp Ala Pro Ser Arg Gly Asp Asp Gln Cys Gln Arg Gln Leu

35 40 45

Gln Arg Ala Asn Leu Arg Pro Cys Glu Glu His Ile Arg Gln Arg Val

50 55 60

Glu Gln Glu Gln Glu Gln Glu Gln Asp Glu Tyr Pro Tyr Ser Gln Arg

65 70 75 80

Gly Ser Arg Gly Arg Arg Pro Gly Glu Ser Asp Glu Asp Gln Glu Gln

85 90 95

Arg Cys Cys Asn Glu Leu Asn Arg Phe Gln Asn Asn Gln Arg Cys Met

100 105 110

Cys Gln Ala Leu Gln Gln Ile Leu Gln Asn Gln Ser Phe Arg Phe Gln

115 120 125

Gln Asp Arg Ser Gln Leu His Gln Asn Gly Glu Gly Ala Gln Glu Leu

130 135 140

Ala Pro Glu Leu Arg Val Gln Val Thr Lys Pro Leu Arg Pro

145 150 155

<210> 14

<211> 165

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 14 (Ara h 2 isoform 2.0201, as expressed in Example 1)

<400> 14

Met Ser His His His His His Ile Glu Gly Arg Thr Met Arg Gln

1 5 10 15

Gln Trp Glu Leu Gln Gly Asp Arg Arg Cys Gln Ser Gln Leu Glu Arg

20 25 30

Ala Asn Leu Arg Pro Cys Glu Gln His Leu Met Gln Lys Ile Gln Arg

35 40 45

Asp Glu Asp Ser Tyr Gly Arg Asp Pro Tyr Ser Pro Ser Gln Asp Pro

50 55 60

Tyr Ser Pro Ser Gln Asp Pro Asp Arg Arg Asp Pro Tyr Ser Pro Ser

65 70 75 80

Pro Tyr Asp Arg Arg Gly Ala Gly Ser Ser Gln His Gln Glu Arg Cys

85 90 95

Cys Asn Glu Leu Asn Glu Phe Glu Asn Asn Gln Arg Cys Met Cys Glu

100 105 110

Ala Leu Gln Gln Ile Met Glu Asn Gln Ser Asp Arg Leu Gln Gly Arg

115 120 125

Gln Gln Glu Gln Gln Phe Lys Arg Glu Leu Arg Asn Leu Pro Gln Gln

130 135 140

Cys Gly Leu Arg Ala Pro Gln Arg Cys Asp Leu Glu Val Glu Ser Gly

145 150 155 160

Gly Arg Asp Arg Tyr

165

<210> 15

<211> 143

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO 15 (Ara h 6 isoform 6.0101, as expressed in Example 1)

<400> 15

Met Ser His His His His His His His Leu Glu Val Leu Phe Gln

1 5 10 15

Gly Pro Ser Met Arg Arg Glu Arg Gly Arg Gln Gly Asp Ser Ser Ser

20 25 30

Cys Glu Arg Gln Val Asp Arg Val Asn Leu Lys Pro Cys Glu Gln His

35 40 45

Ile Met Gln Arg Ile Met Gly Glu Gln Glu Gln Tyr Asp Ser Tyr Asp

50 55 60

Ile Arg Ser Thr Arg Ser Ser Asp Gln Gln Gln Arg Cys Cys Asp Glu

65 70 75 80

Leu Asn Glu Met Glu Asn Thr Gln Arg Cys Met Cys Glu Ala Leu Gln

85 90 95

Gln Ile Met Glu Asn Gln Cys Asp Arg Leu Gln Asp Arg Gln Met Val

100 105 110

Gln Gln Phe Lys Arg Glu Leu Met Asn Leu Pro Gln Gln Cys Asn Phe

115 120 125

Arg Ala Pro Gln Arg Cys Asp Leu Asp Val Ser Gly Gly Arg Cys

130 135 140

<210> 16

<211> 615

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO16: Ara h 1.01.01 as used in Example 3

<400> 16

Met Ser His His His His His Ile Glu Gly Arg Thr Met Lys Ser

1 5 10 15

Ser Pro Tyr Gln Lys Lys Thr Glu Asn Pro Cys Ala Gln Arg Cys Leu

20 25 30

Gln Ser Cys Gln Gln Glu Pro Asp Asp Leu Lys Gln Lys Ala Cys Glu

35 40 45

Ser Arg Cys Thr Lys Leu Glu Tyr Asp Pro Arg Cys Val Tyr Asp Pro

50 55 60

Arg Gly His Thr Gly Thr Thr Asn Gln Arg Ser Pro Pro Gly Glu Arg

65 70 75 80

Thr Arg Gly Arg Gln Pro Gly Asp Tyr Asp Asp Asp Arg Arg Gln Pro

85 90 95

Arg Arg Glu Glu Gly Gly Arg Trp Gly Pro Ala Gly Pro Arg Glu Arg

100 105 110

Glu Arg Glu Glu Asp Trp Arg Gln Pro Arg Glu Asp Trp Arg Arg Pro

115 120 125

Ser His Gln Gln Pro Arg Lys Ile Arg Pro Glu Gly Arg Glu Gly Glu

130 135 140

Gln Glu Trp Gly Thr Pro Gly Ser His Val Arg Glu Glu Thr Ser Arg

145 150 155 160

Asn Asn Pro Phe Tyr Phe Pro Ser Arg Arg Phe Ser Thr Arg Tyr Gly

165 170 175

Asn Gln Asn Gly Arg Ile Arg Val Leu Gln Arg Phe Asp Gln Arg Ser

180 185 190

Arg Gln Phe Gln Asn Leu Gln Asn His Arg Ile Val Gln Ile Glu Ala

195 200 205

Lys Pro Asn Thr Leu Val Leu Pro Lys His Ala Asp Ala Asp Asn Ile

210 215 220

Leu Val Ile Gln Gln Gly Gln Ala Thr Val Thr Val Ala Asn Gly Asn

225 230 235 240

Asn Arg Lys Ser Phe Asn Leu Asp Glu Gly His Ala Leu Arg Ile Pro

245 250 255

Ser Gly Phe Ile Ser Tyr Ile Leu Asn Arg His Asp Asn Gln Asn Leu

260 265 270

Arg Val Ala Lys Ile Ser Met Pro Val Asn Thr Pro Gly Gln Phe Glu

275 280 285

Asp Phe Phe Pro Ala Ser Ser Arg Asp Gln Ser Ser Tyr Leu Gln Gly

290 295 300

Phe Ser Arg Asn Thr Leu Glu Ala Ala Phe Asn Ala Glu Phe Asn Glu

305 310 315 320

Ile Arg Arg Val Leu Leu Glu Glu Asn Ala Gly Gly Glu Gln Glu Glu

325 330 335

Arg Gly Gln Arg Arg Trp Ser Thr Arg Ser Ser Glu Asn Asn Glu Gly

340 345 350

Val Ile Val Lys Val Ser Lys Glu His Val Glu Glu Leu Thr Lys His

355 360 365

Ala Lys Ser Val Ser Lys Lys Gly Ser Glu Glu Glu Gly Asp Ile Thr

370 375 380

Asn Pro Ile Asn Leu Arg Glu Gly Glu Pro Asp Leu Ser Asn Asn Phe

385 390 395 400

Gly Lys Leu Phe Glu Val Lys Pro Asp Lys Lys Asn Pro Gln Leu Gln

405 410 415

Asp Leu Asp Met Met Leu Thr Cys Val Glu Ile Lys Glu Gly Ala Leu

420 425 430

Met Leu Pro His Phe Asn Ser Lys Ala Met Val Ile Val Val Val Asn

435 440 445

Lys Gly Thr Gly Asn Leu Glu Leu Val Ala Val Arg Lys Glu Gln Gln

450 455 460

Gln Arg Gly Arg Arg Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu Glu

465 470 475 480

Gly Ser Asn Arg Glu Val Arg Arg Tyr Thr Ala Arg Leu Lys Glu Gly

485 490 495

Asp Val Phe Ile Met Pro Ala Ala His Pro Val Ala Ile Asn Ala Ser

500 505 510

Ser Glu Leu His Leu Leu Gly Phe Gly Ile Asn Ala Glu Asn Asn His

515 520 525

Arg Ile Phe Leu Ala Gly Asp Lys Asp Asn Val Ile Asp Gln Ile Glu

530 535 540

Lys Gln Ala Lys Asp Leu Ala Phe Pro Gly Ser Gly Glu Gln Val Glu

545 550 555 560

Lys Leu Ile Lys Asn Gln Lys Glu Ser His Phe Val Ser Ala Arg Pro

565 570 575

Gln Ser Gln Ser Gln Ser Pro Ser Ser Pro Glu Lys Glu Ser Pro Glu

580 585 590

Lys Glu Asp Gln Glu Glu Glu Asn Gln Gly Gly Lys Gly Pro Leu Leu

595 600 605

Ser Ile Leu Lys Ala Phe Asn

610 615

<210> 17

<211> 527

<212> PRT

<213> Artificial sequence

<220>

<223> SEQ ID NO17: Ara h 3.01.01 as used in Example 3

<400> 17

Met Ser His His His His His His His Leu Glu Val Leu Phe Gln

1 5 10 15

Gly Pro Ser Met Arg Gln Gln Pro Glu Glu Asn Ala Cys Gln Phe Gln

20 25 30

Arg Leu Asn Ala Gln Arg Pro Asp Asn Arg Ile Glu Ser Glu Gly Gly

35 40 45

Tyr Ile Glu Thr Trp Asn Pro Asn Asn Gln Glu Phe Glu Cys Ala Gly

50 55 60

Val Ala Leu Ser Arg Leu Val Leu Arg Arg Asn Ala Leu Arg Arg Pro

65 70 75 80

Phe Tyr Ser Asn Ala Pro Gln Glu Ile Phe Ile Gln Gln Gly Arg Gly

85 90 95

Tyr Phe Gly Leu Ile Phe Pro Gly Cys Pro Arg His Tyr Glu Glu Pro

100 105 110

His Thr Gln Gly Arg Arg Ser Gln Ser Gln Arg Pro Pro Arg Arg Leu

115 120 125

Gln Gly Glu Asp Gln Ser Gln Gln Gln Arg Asp Ser His Gln Lys Val

130 135 140

His Arg Phe Asp Glu Gly Asp Leu Ile Ala Val Pro Thr Gly Val Ala

145 150 155 160

Phe Trp Leu Tyr Asn Asp His Asp Thr Asp Val Val Ala Val Ser Leu

165 170 175

Thr Asp Thr Asn Asn Asn Asp Asn Gln Leu Asp Gln Phe Pro Arg Arg

180 185 190

Phe Asn Leu Ala Gly Asn Thr Glu Gln Glu Phe Leu Arg Tyr Gln Gln

195 200 205

Gln Ser Arg Gln Ser Arg Arg Arg Ser Leu Pro Tyr Ser Pro Tyr Ser

210 215 220

Pro Gln Ser Gln Pro Arg Gln Glu Glu Arg Glu Phe Ser Pro Arg Gly

225 230 235 240

Gln His Ser Arg Arg Glu Arg Ala Gly Gln Glu Glu Glu Asn Glu Gly

245 250 255

Gly Asn Ile Phe Ser Gly Phe Thr Pro Glu Phe Leu Glu Gln Ala Phe

260 265 270

Gln Val Asp Asp Arg Gln Ile Val Gln Asn Leu Arg Gly Glu Thr Glu

275 280 285

Ser Glu Glu Glu Gly Ala Ile Val Thr Val Arg Gly Gly Leu Arg Ile

290 295 300

Leu Ser Pro Asp Arg Lys Arg Arg Ala Asp Glu Glu Glu Glu Tyr Asp

305 310 315 320

Glu Asp Glu Tyr Glu Tyr Asp Glu Glu Asp Arg Arg Arg Gly Arg Gly

325 330 335

Ser Arg Gly Arg Gly Asn Gly Ile Glu Glu Thr Ile Cys Thr Ala Ser

340 345 350

Ala Lys Lys Asn Ile Gly Arg Asn Arg Ser Pro Asp Ile Tyr Asn Pro

355 360 365

Gln Ala Gly Ser Leu Lys Thr Ala Asn Asp Leu Asn Leu Leu Ile Leu

370 375 380

Arg Trp Leu Gly Pro Ser Ala Glu Tyr Gly Asn Leu Tyr Arg Asn Ala

385 390 395 400

Leu Phe Val Ala His Tyr Asn Thr Asn Ala His Ser Ile Ile Tyr Arg

405 410 415

Leu Arg Gly Arg Ala His Val Gln Val Val Asp Ser Asn Gly Asn Arg

420 425 430

Val Tyr Asp Glu Glu Leu Gln Glu Gly His Val Leu Val Val Pro Gln

435 440 445

Asn Phe Ala Val Ala Gly Lys Ser Gln Ser Glu Asn Phe Glu Tyr Val

450 455 460

Ala Phe Lys Thr Asp Ser Arg Pro Ser Ile Ala Asn Leu Ala Gly Glu

465 470 475 480

Asn Ser Val Ile Asp Asn Leu Pro Glu Glu Val Val Ala Asn Ser Tyr

485 490 495

Gly Leu Gln Arg Glu Gln Ala Arg Gln Leu Lys Asn Asn Asn Pro Phe

500 505 510

Lys Phe Phe Val Pro Pro Ser Gln Gln Ser Pro Arg Ala Val Ala

515 520 525

Claims (31)

1. A diagnostically useful vector comprising a tool for specifically capturing antibodies against polypeptides selected from the sequences shown in SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, and SEQ ID NO10 in a sample from a subject. The carrier is selected from beads, test strips, microtiter plates, microarrays, cellulose-derived solid polymers, imprints, glass surfaces, biochips, and membranes. 2. The diagnostically useful vector of claim 1, wherein the vector comprises a tool for specifically capturing an antibody against the polypeptide represented by SEQ ID NO8. 3. The diagnostically useful carrier according to claim 1, wherein the carrier is a glass slide, protein blot, line blot, or dot blot. 4. The diagnostically useful carrier according to claim 1, wherein the carrier is a microtiter plate. 5. The diagnostically useful carrier according to claim 3, wherein the carrier is a line imprint. 6. The diagnostically useful vector of claim 1, wherein the vector further comprises a tool for specifically capturing antibodies against one or more other antigens selected from Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, Ara h 8 and Ara h 5. 7. The diagnostically useful vector of claim 6, wherein the vector further comprises a tool for specifically capturing antibodies against one or more other antigens selected from Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9. 8. The diagnostically useful vector of claim 6, wherein the vector further comprises tools for specifically capturing antibodies against Ara h 2 and/or antibodies against Ara h 6. 9. The diagnostically useful vector of claim 6, wherein the vector further comprises a tool for specifically capturing antibodies against Ara h 2. 10. A kit comprising a diagnostically useful vector according to any one of claims 1 to 9, and optionally a tool for the specific detection of captured antibodies. 11. The kit according to claim 10, The diagnostically useful vector comprises a tool for specifically capturing antibodies against one or more other antigens selected from Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, Ara h 8, and Ara h 5. The tools for specifically capturing antibodies against peptides selected from the sequences shown in SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, and SEQ ID NO10, and the tools for specifically capturing antibodies against one or more other antigens, are on separate vectors, or The tools for specifically capturing antibodies against peptides selected from the sequences shown in SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9 and SEQ ID NO10 and the tools for specifically capturing antibodies against one or more other antigens are on a single vector. 12. The kit of claim 11, wherein the tools for specifically capturing antibodies against polypeptides selected from the sequences shown in SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9 and SEQ ID NO10 and the tools for specifically capturing antibodies against one or more other antigens are covalently linked to a vector. 13. Use of the carrier of any one of claims 1-9 in the preparation of a kit for diagnosing peanut allergy. 14. The use according to claim 13, wherein the diagnostic includes the step of detecting the presence of an antibody against a polypeptide selected from the sequences of SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9 and SEQ ID NO10 in a sample from a subject. 15. The use according to claim 14, wherein the diagnostic includes the step of detecting the presence of an antibody against the polypeptide represented by SEQ ID NO8 in a sample from a subject. 16. The use according to claim 14, wherein the diagnostic further comprises the step of: detecting the presence of antibodies against one or more other antigens in a sample from the subject, said other antigens being selected from Ara h 2, Ara h 6, Arah 1, Ara h 3, Ara h 9, Ara h 8, and Ara h 5. 17. The use according to claim 16, wherein the diagnostic further comprises the step of: detecting the presence of antibodies against one or more other antigens selected from Ara h 2, Ara h 6, Arah 1, Ara h 3 and Ara h 9 in a sample from the subject. 18. The use according to claim 17, wherein the diagnostic further comprises the step of detecting the presence of antibodies against Ara h 2 and/or Ara h 6 in a sample from the subject. 19. The use according to any one of claims 14 and 16 to 18, wherein the presence or absence of antibodies against polypeptides represented by sequences selected from SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9 and SEQ ID NO10, and the presence of antibodies against one or more other antigens are detected simultaneously. 20. The use according to any one of claims 15 to 18, wherein the presence or absence of an antibody against the polypeptide shown in SEQ ID NO 8 and the presence of an antibody against one or more other antigens are detected in a spatially separated binding reaction. 21. The use according to any one of claims 14 and 16 to 18, wherein the presence of antibodies against polypeptides selected from the sequences shown in SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9 and SEQ ID NO10, and the presence of antibodies against one or more other antigens are detected in a one-pot reaction. 22. The vector according to any one of claims 1 to 9 and the kit according to any one of claims 10 to 12, wherein the antibody against the polypeptide represented by the sequence selected from SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9 and SEQ ID NO10 is a single-specific antibody against the polypeptide represented by the sequence selected from SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9 and SEQ ID NO10. 23. A pharmaceutical composition comprising a polypeptide for detecting an antibody against Ara h 7 isotype 7.0201, wherein the polypeptide comprises a polypeptide represented by a sequence selected from SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, and wherein the polypeptide is not Ara h 7 isotype 7.0201. 24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition further comprises one or more other antigens selected from Ara h 2, Ara h 6, Ara h 1, Ara h 3, Ara h 9, Ara h 8 and Ara h 5. 25. The pharmaceutical composition of claim 24, wherein the pharmaceutical composition further comprises one or more other antigens selected from Ara h 2, Arah 6, Ara h 1, Ara h 3 and Ara h 9. 26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition further comprises Ara h 2 and/or Arah 6. 27. A polypeptide for detecting an antibody against Ara h 7 isotype 7.0201, wherein the polypeptide comprises a polypeptide represented by a sequence selected from SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, wherein the polypeptide is not Ara h 7 isotype 7.0201, and wherein the polypeptide is a recombinant, immobilized, purified or fusion protein. 28. Use of the polypeptide according to claim 27 in the preparation of a kit or pharmaceutical composition for diagnosing peanut allergy. 29. Use of Ara h 7 isotype 7.0201 in the preparation of kits or pharmaceutical compositions for the diagnosis of peanut allergy. 30. The use according to claim 29, wherein the Ara h 7 isotype 7.0201 is as shown in SEQ ID NO2 or SEQ ID NO12. 31. The pharmaceutical composition of claim 23, the polypeptide of claim 27, or the use of claim 28, wherein the polypeptide comprising the polypeptide shown in the sequence selected from SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10 is in a composition comprising the polypeptide and Ara h 2 and/or Ara h 6.