Patent Application
20180340933
The invention relates to a compilation comprising at least five different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented. The invention further relates to an in vitro method for determining a patient's immune status to soybean allergens, to a method for detecting at least one soybean allergen in a substance and to a method for determining the allergenicity of a soybean variety. Additionally, the invention relates to a kit comprising at least one composition containing a compound comprising at least five different sequence elements each corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented. Furthermore, the invention relates to the use of a peptide comprising a sequence element corresponding to an epitope for providing a molecule binding to a protein or peptide comprising the epitope. The invention further relates to a method for producing an immunoassay product and to a method for generating a variant of an allergenic soybean protein having a reduced allergenic potential.
Within the last decades, soybean seeds have become an important source of protein in many countries of the world. In developing countries, soybean is increasingly used to provide sufficient nutrition to the population, whereas in industrial countries it has become a major ingredient of processed food products. Moreover, soybean food products are specifically promoted for having health benefits and are believed to lower incidences of high plasma cholesterol, cancer, diabetes mellitus and obesity. Indeed, soybean products provide all essential amino acids and are rich in vitamins and minerals, making them an interesting source for protein rich nutrition. Despite these advantages, soybean is one of the most allergenic foods. Soybean allergies usually arise spontaneously during childhood with symptoms such as atopic dermatitis, enterocolitis and other IgE-mediated multisystem reactions (Masilamani et al., 2012).
Moreover, they often occur together with allergies to other food products, in particular peanut and milk protein (Masilamani et al., 2012). Thus, affected patients not only have to cope with the risk of allergic reactions for almost their entire life but need to be particularly careful about their diet in general.
Although the reaction threshold of soybean protein (400 mg) is rather high compared to peanut (0.1 mg), most sensitive persons need to avoid soybean proteins completely to prevent allergic reaction (Masilamani et al., 2012). Unfortunately, obviating soybean protein containing food products is increasingly difficult, since soybean protein became an ubiquitous ingredient of vegetable as well as meat containing foods. In particular processed or partially processed food products usually contain at least traces of at least some soybean proteins. To increase security for persons suffering from allergies, governments, in particular within the European Union and the United States of America, implement increasing requirements regarding the identification of food ingredients. To meet these requirements, food producing companies need comprehensive and reliable methods for analyzing the content of their food products.
Additionally, patients need to know about the primary cause and extent of their allergic reaction to be able to benefit from the increasing information provided about food product ingredients.
Therefore, an unmet need exists for improved products and methods to determine the sensitivity of patients to soybean protein as well as to identify the presence of soybean proteins and their allergenic potential in food and other products.
In a first aspect, the invention relates to a compilation comprising at least five different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented.
In a further aspect, the invention relates to an in vitro method for determining a patient's immune status to soybean allergens, comprising the steps providing at least five different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented, contacting each peptide with a sample comprising antibodies derived from the patient, and detecting an interaction of each peptide with the sample.
In a further aspect, the invention relates to a kit comprising at least one composition containing a compound comprising at least five different sequence elements each corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented.
In a further aspect, the invention relates to a method for detecting at least one soybean allergen in a substance, comprising the steps providing at least two different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, raising at least one antibody against each peptide, contacting the substance with the antibodies, and determining binding of the antibodies to the substance.
In a further aspect, the invention relates to a method for producing an immunoassay product, comprising the steps providing at least two different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, raising at least one antibody against each peptide, and compiling the antibodies to provide the immunoassay product.
In a further aspect, the invention relates to a method for determining the allergenicity of a soybean variety by detecting the presence of at least two epitopes in a sample of the variety, wherein the epitopes are selected from the group consisting of SEQ ID NO.: 1-354.
In a further aspect, the invention relates to the use of a peptide comprising a sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354 for providing a molecule binding to a protein or peptide comprising the epitope.
In a further aspect, the invention relates to a method for generating a variant of an allergenic soybean protein having a reduced allergenic potential, comprising the steps providing an amino acid sequence of at least one epitope selected from the group consisting of SEQ ID NO.: 1-354, altering the amino acid sequence of the at least one epitope as to eliminating the structure of the epitope, and generating a protein or peptide comprising the altered amino acid sequence.
In a first aspect, the invention relates to a compilation comprising at least five different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented.
Several proteins of soybean seeds have been found to trigger immunoglobulin E (IgE) mediated allergic responses in sensitive persons. Strong immunodominant allergic proteins, such as P34 thiol protease (Gly m Bd 30K), are known to induce allergic reactions, whereas other soybean proteins, although no potent inducers of a primary allergic response, can nevertheless trigger effector responses due to their binding by IgE. Interestingly, affected persons show varying sensitivity to different soybean proteins. For example, only 65% of soybean sensitive individuals carry IgE recognizing P34 thiol protease (Gly m Bd 30K), which is considered one of the strongest allergenic soybean proteins (Yang et al., 2011). Finally, as antibodies usually do not recognize an entire protein but interact with distinct structures of limited numbers of amino acids within a molecule, sensitivity between patients varies distinctly due to their genetic background and different results of antibody maturation. To identify many specific epitopes, i.e. amino acid sequences of soybean proteins, recognized by antibodies of sensitive patients, with high resolution down to the individual amino acids involved, the inventors applied a novel approach using a specific random phage library (EP 14 166 662.8). This phage library is generated using triplet codons with specific limitations, such that most amino acids are regularly represented throughout the entire library. In consequence, the library covers an unusually broad range of sequence variability and allows particular reliable statistical analysis. The library was contacted with serum samples derived from a biobank of 50 different soybean protein sensitive patients. After phage selection, the amino acid sequences that were bound by the patients' antibodies (anti-[allergen antibody] amino acid sequence) were identified by next generation sequencing and the results statistically analyzed. To confirm the allergy relevance of the identified amino acid sequences, their reactivity with patient IgE was determined by peptide arrays. Thereby, the inventors identified more than 300 potentially allergenic epitopes from different soybean proteins. The identified epitopes could be assigned to a total of 23 soybean proteins (Table 1) including P34 thiol protease (Gly m Bd 30K), β-conglycinin (Gly m Bd60 K) and Gly m Bd 28K, which are considered major soybean allergenic proteins (Yang et al., 2011).
The epitopes identified by the inventors provide the most comprehensive data on potentially allergenic proteins and peptides derived from soybean that are available so far. These data, i.e. the identified epitopes, provide the essential basis for assessing both, the immune status of an individual to soybean protein and the allergenic potential of food and other products suspected of containing soybean protein. The compilation of the invention provides a collection of peptides of different amino acid sequences with each peptide comprising a short element of about three to six amino acids (sequence element), which corresponds to one of the identified epitopes (table 1). The sequence element may also have a length of about three to ten amino acids. Since the sequence element corresponds to one of the identified epitopes, each peptide presents a potential epitope for a soybean-allergen antibody (IgE). The term “corresponding” refers to the exact reproduction of the epitope sequence as well as variants thereof differing from the identified epitope in one or two amino acids. The variation may be due to one or two missing amino acids and/or to the exchange of one or two amino acids in comparison to the identified epitope. It is well established in the art that the secondary structure and the physical-chemical properties of a peptide (e.g. its antibody binding properties due to size, charge and/or polarity) may be maintained despite of minor changes to the amino acid sequence. In particular, single amino acids with side chains of little polarity or other reactivity, may be omitted or replaced by other amino acids of similar reactivity (e.g. lysine-arginine or leucine-isoleucine). The possibility of minor amino acid changes within the amino acid sequence corresponding to the epitope was also confirmed by the data obtained from the phage display and the peptide arrays. The antibodies contained in the patients' sera recognized several variants of individual epitopes, e.g. overlapping epitopes such as EDEEQQ (SEQ ID NO.: 32), EEQQRES (SEQ ID NO.: 33), RESEES (SEQ ID NO.: 34), (see table 1, and SEQ ID NO.: 385) of beta conglycinin alpha chain.
Accordingly, comprehensive analysis of interactions of antibody sera derived from various patients allowed to determine variations within the epitope sequences. Table 2 depicts the epitopes within the endogenous soybean protein sequence (epitopes underlined) and indicates the variability of the epitope (minimum amino acids/all amino acids of the epitope).
VFASQVVVQTEGRVC
KEECEEGEIPRPRPR
LSIVDMNEGALLLPHF
HKQEKHQGKESEEEEE
NKRSQQLQNLRDYRI
QDIFVIPAGYPVMVN
ESYFVDAQPQQKEEG
SHNILETSFHSEFEE
RPSYTNGPQEIYIQQ
REGDLIAVPTGVAWW
ESQQRGRSQRPQDRH
PDCDEKDKHCQSQSR
HNIGQTSSPDIFNPQ
RNGLHSPSYSPYPRM
CQLNNLNALEPDHRV
RVFYLAGNPDIEHPE
EGGLSVISPKWQEQE
THHNAVSSYIKDVFR
AYVDDHLLCGIEGNH
VENVEGNGGPGTIKK
EIHVEKHRVPKMATH
EYTAKKKEEAQRELE
QPQEAEERPSEGIGE
In the compilation of the invention, at least five different epitopes are represented. The compilation of the invention is for example suitable for determining the immune status of a patient to soybean proteins by analysing the patient's immunological reaction to selected epitopes. As each peptide of the compilation presents a potentially allergenic soybean epitope, interaction of the patient's antibodies (IgE) with the peptides is representative for the patient's immune status to soybean allergens.
Additionally, a peptide comprising at least one sequence element corresponding to a soybean epitope is suitable for identifying or generating molecules specifically binding to the respective epitope and peptides or proteins comprising the same. The resulting molecules, e.g. antibodies, provide a significant advantage for detecting soybean proteins. Common anti-soybean antibodies or sera are raised against whole soybean proteins or even proteomes and therefore inevitably include antibodies directed against non-allergen proteins/parts of proteins. In contrast, utilizing peptides with sequence elements corresponding to specific allergenic soybean epitopes allows for the generation of antibodies distinctly directed against allergenic soybean proteins. Moreover, these antibodies specifically bind to the allergenic part (i.e epitope) of a soybean protein. Finally, such antibodies, or other binding molecules generated by using the peptides of the invention, allow the detection of allergenic remnants of soybean proteins that could be missed using antibodies binding to non-allergenic parts of a protein.
The term “compilation” refers to a collection of at least five different peptides, which includes that each peptide may be present in several or even hundreds of identical copies. Each peptide comprises at least one sequence element corresponding to an epitope, but may also comprise more, e.g. two or three sequence elements corresponding to identical and/or different epitopes. For example, each peptide may comprise multiple sequence elements corresponding to different epitopes. A peptide comprising sequence elements corresponding to the same epitope may comprise the same sequence element (thus representing the same epitope) but different additional amino acids. In this case, the same sequence element is presented in various different molecular environments, e.g. secondary structures. Therefore, it is to be understood that the compilation may comprise both, peptides comprising sequence elements corresponding to different epitopes as well as peptides comprising identical sequence elements, thus corresponding to the same epitope, but comprising different additional amino acids.
The compilation may be provided by immobilizing the peptides on a carrier, e.g. a chip, slide, well plate or on beads. Likewise, the compilation may be provided as a multitude of compositions, each composition containing identical copies of a peptide or a mixture of different peptides. Alternatively, the compilation may be provided as a single composition containing different peptides comprising sequence elements corresponding to the same or different epitopes, wherein at least five different epitopes are represented.
In a preferred embodiment, at least 10, preferably at least 20, more preferred at least 50 different epitopes are represented in the compilation.
In a preferred embodiment, each of the at least five different peptides comprises a sequence element corresponding to a different epitope.
In a preferred embodiment, the compilation comprises at least 10, preferably at least 20, more preferred at least 50 different peptides. Depending on the application, the advantageous number of different peptides may vary. For example, in a simple diagnostic application, a few peptides, e.g. five to ten peptides, may be sufficient to investigate sera for the general presence of antibodies against soybean proteins. In such cases, the sequence elements are preferably selected to correspond to epitopes of the most allergenic proteins such as Gly m Bd 28K, glycinin, hydrophobic seed protein (Gly m 1a), kunitz trypsin inhibitor, profilin (Gly m3), β-conglycinin (Gly m Bd 60K) and defensin (Gly 2m). For more detailed analyses, the compilation may comprise about 20 to 500 different peptides, preferably representing at least one epitope of each identified allergenic soybean protein. For example, such analysis could be used for providing a more personalized analysis of a patient's health condition. For such comprehensive analysis, a peptide array or a bead-based multiplex assay may be provided covering sequence elements corresponding to each of the identified epitopes.
In a preferred embodiment, the peptide consists of about 5 to 30 amino acids, preferably 8 to 20 amino acids, more preferred 8 to 15 amino acids. Besides the sequence element corresponding to the epitope, usually four, five or six amino acids, each peptide may comprise further amino acids on one or both sides of the sequence element. Such additional amino acids may be added for example for immobilizing the peptide on a substrate or for embedding the sequence element corresponding to the epitope into a specific molecular environment (secondary structure). The additional amino acids may be chosen depending on the use of the compilation. For example, to present the epitope in its natural molecular environment, the sequence element corresponding to the epitope may be flanked by amino acids as found in the naturally occurring protein sequence of the soybean protein. In case the epitope is flanked by different amino acid sequences in different soybean varieties, corresponding peptides may be used to evaluate the influence of the adjacent amino acids to the allergenic potential of the epitope. Additionally, functional molecules, e.g. for coupling or readout, may be added at the N- or C-terminus of the peptide.
In a preferred embodiment, each epitope is selected from one of the groups comprising group 1 (β-conglycinin) consisting of SEQ ID NO.: 1-93, group 2 (defensin) consisting of SEQ ID NO.: 94-103, group 3 (Gly m Bd 28K) consisting of SEQ ID NO.: 104, group 4 (glycinin) consisting of SEQ ID NO.: 105-250, group 5 (hydrophobic seed protein) consisting of SEQ ID NO.: 251-253, group 6 (kunitz trypsin inhibitor) consisting of SEQ ID NO.: 254-257, group 7 (P34 thiol protease) consisting of SEQ ID NO.:258-259, group 8 (profilin) consisting of SEQ ID NO.: 260-274, and group 9 (Pr-10) consisting of SEQ ID NO.: 275-283. Although the sensitivity to soybean proteins is known to vary distinctly between different patients, several proteins are considered major allergens, in particular P34 (Gly ml), β-conglycinin (Gly m5) and glycinin (Gly m6). β-conglycinin and glycinin are storage proteins and constitute 70 to 80% of total seed protein. Both are regarded good diagnostic markers for soybean protein sensitivity (Masilamani et al., 2012). β-conglycinin comprises three individual subunits (α, α′, β), which were all found to react with IgE. Interestingly, wild and cultivated soybean plants were found to vary distinctly in these subunits (Natarajan 2014). Glycinin comprises five subunits, which were also all found to be recognized by IgE from soybean protein sensitive persons. P34 is a soybean vacuole protein (hull protein) with high sequence similarity to thiol proteases of the papain family, which are known to potentially induce allergic reactions in human. In general, several soybean allergens show distinct similarity to allergens of other organisms. For example, P34 is related to DERp1 from dust mite, profilin (Gly m3) is related to the profilin family of proteins in birch pollen, Gly m5/6 is closely related to ARAh1/3 of peanut and Pr10 (Gly m4) is highly homolog to BETV1 and other major allergens of cross reactivity with birch pollen. Thus, compilations covering sequence elements corresponding to epitopes of the above mentioned groups are suitable to provide detailed information not only on soybean sensitivity but also regarding sensitivity to allergens from other organisms. In particular, when providing screening tests to identify soybean protein sensitive patients, the compilation of peptides may cover at least five, preferably all of these proteins. Accordingly, in a preferred embodiment, each of the at least five different epitopes is derived from a different soybean protein. Thus, each epitope is preferably selected from a different group of the groups comprising group 1 (β-conglycinin) consisting of SEQ ID NO.: 1-93, group 2 (defensin) consisting of SEQ ID NO.: 94-103, group 3 (Gly m Bd 28K) consisting of SEQ ID NO.: 104, group 4 (glycinin) consisting of SEQ ID NO.: 105-250, group 5 (hydrophobic seed protein) consisting of SEQ ID NO.: 251-253, group 6 (kunitz trypsin inhibitor) consisting of SEQ ID NO.: 254-257, group 7 (P34 thiol protease) consisting of SEQ ID NO.:258-259, group 8 (profilin) consisting of SEQ ID NO.: 260-274, and group 9 (Pr-10) consisting of SEQ ID NO.: 275-283.
In a preferred embodiment, at least nine different epitopes are represented in the compilation, wherein each epitope is selected from a different group of the groups comprising group 1 (β-conglycinin) consisting of SEQ ID NO.: 1-93, group 2 (defensin) consisting of SEQ ID NO.: 94-103, group 3 (Gly m Bd 28K) consisting of SEQ ID NO.: 104, group 4 (glycinin) consisting of SEQ ID NO.: 105-250, group 5 (hydrophobic seed protein) consisting of SEQ ID NO.: 251-253, group 6 (kunitz trypsin inhibitor) consisting of SEQ ID NO.: 254-257, group 7 (P34 thiol protease) consisting of SEQ ID NO.:258-259, group 8 (profilin) consisting of SEQ ID NO.: 260-274, and group 9 (Pr-10) consisting of SEQ ID NO.: 275-283.
In a particularly preferred embodiment, the compilation comprises at least nine different peptides and each peptide comprising at least one sequence element corresponding to an epitope selected from each of the groups comprising group 1 (β-conglycinin) consisting of SEQ ID NO.: 1-93, group 2 (defensin) consisting of SEQ ID NO.: 94-103, group 3 (Gly m Bd 28K) consisting of SEQ ID NO.: 104, group 4 (glycinin) consisting of SEQ ID NO.: 105-250, group 5 (hydrophobic seed protein) consisting of SEQ ID NO.: 251-253, group 6 (kunitz trypsin inhibitor) consisting of SEQ ID NO.: 254-257, group 7 (P34 thiol protease) consisting of SEQ ID NO.:258-259, group 8 (profilin) consisting of SEQ ID NO.: 260-274, and group 9 (Pr-10) consisting of SEQ ID NO.: 275-283.
In a preferred embodiment, each epitope is selected from group 10 consisting of SEQ ID NO.: 18, 22, 29, 34, 37, 60, 65, 67, 79, 85, 87, 91, 99, 95, 94, 92, 103, 117, 131, 110, 126, 152, 153, 168, 173, 172, 183, 188, 200, 227, 233, 237, 250, 251, 252, 253, 254, 255, 257, 258, 262, 271, 261, 276, 281, 274, 309, 311, 319, 307, 294, 305, 298, 284, 291, 290, 328, 322, 323, 331, 332, 333, 334, 335, 336, 337, 339, 340, 345, 346 and 351. Although the immune status to major allergenic soybean proteins varies distinctly between patients, statistic analyses enabled by the use of the specific phage display library, made it possible to identify certain epitopes that are recognized by the majority of patients (Table 3).
These epitopes are particularly preferred for providing a general immune status test, because it can be expected that every sensitive person will respond to at least one of these epitopes. For example the epitopes RNQACHA (SEQ ID NO.: 17), EIPRP (SEQ ID NO.: 20), NSERD (SEQ ID NO.: 15) have been recognized by 15/23 patients resp. 3/23 resp 7/23. All three epitopes have been recognized just by one patient, two epitopes (SEQ ID NO.: 15, SEQ ID NO.: 17) by 5/23 patients.
In a further aspect, the invention relates to an analysis tool comprising at least five different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented. The term “analysis tool” as used herein refers to means and products for chemical, biochemical or medical analysis of samples, e.g. biological samples. Such tools include devices (e.g. microarray, multi-well plates), as well as reagents (e.g. compositions comprising free or bead-bound peptides) suitable for single or multiplex analyses. When presented on the surface of a chip or beads, the peptides can interact with substances, e.g. other molecules, brought into contact with the surface. In a peptide array, for example, multitudes of identical peptides are deposited in spots, each spot representing a different peptide. In case of a bead based analysis tool, identical peptides may be conjugated to the surface of a single bead. The peptides can be attached to a surface using hydrophilic linker moieties to avoid sterical hindrance, which might influence the interaction between the peptide and the sample. The analysis tools of the invention are for example suitable for diagnostic applications such as determining a patient's immune status to soybean proteins. Serum derived from a person suspected of being sensitive to soybean protein is brought into contact with the analysis tool to allow the IgE or other antibody classes of the patient to interact with and bind to the peptides. The binding of the antibodies can be specifically detected and analyzed. Depending on the aim of the diagnosis, the analysis tool may cover epitopes of different soybean allergens. For example, for a general initial screening, the analysis tool may contain peptides comprising sequence elements corresponding to epitopes of some or all major allergens of soybean. Additionally, the analysis tool may contain peptides comprising sequence elements corresponding to epitopes of allergens of other organisms to provide a more comprehensive survey. For a more detailed analysis, all major soybean allergens should be represented by the analysis tool. When used in analysis tools such as microarrays or bead based assays, the peptides preferably consist of 5 to 30, more preferred of 8 to 20, most preferred of 8 to 15 amino acids.
In a further aspect, the invention relates to an in vitro method for determining a patient's immune status to soybean allergens, comprising the steps providing at least five different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented, contacting each peptide with a sample comprising antibodies derived from the patient, and detecting an interaction of each peptide with the sample. The term “immune status” refers to the presence of antibodies in the blood of a person, in particular to the presence of antibodies directed to soybean proteins. The method allows revealing whether a patient produces antibodies recognizing soybean proteins and, thus, is expected to show allergic reactions when consuming soybean containing products and at least one of these antibodies is an IgE antibody. Moreover, the method is suitable to specifically identify the epitopes of soybean proteins, to which the patient reacts. This may provide further information about the strength of the allergy and may indicate whether the patient is likely to react to allergens from other organisms as well, since related allergenic epitopes are found in proteins of various organisms. Dependent on the distinct aim of using the method, various numbers of peptides comprising sequence elements corresponding to at least five different epitopes may be employed. For example, to merely test whether a person in question is likely to show any allergic reaction to soybean proteins, a few peptides (e.g. five to ten) representing epitopes of at least two, preferably five major allergenic proteins (epitopes of groups 1 to 9) may be sufficient. For a more detailed analysis on the antibody status of the patient, peptides comprising sequence elements corresponding to epitopes selected from all major soybean allergens, preferably from all potentially allergenic soybean proteins, may be covered. The results of such a comprehensive analysis can help to decide about a patient's personalized treatment, e.g. form the basis for a subsequent desensitization protocol.
For carrying out the method, the peptides can be provided as a microarray or bead-based multiplex assay. For a simpler approach, the method may be used in form of a lateral flow test. In any case, the peptides are incubated with the patient's sample such that the molecules contained in the sample, in particular the patient's antibodies, can interact and bind to the peptides. Binding of the antibodies is subsequently detected, e.g. using secondary antibodies directed against human immunoglobulins. In case the secondary antibodies are fluorescently labelled, the analysis can be performed using fluorescence scanners and suitable data analysis software, which allows for a fast and standardized readout providing reliable results in reasonable time.
In a preferred embodiment, the sample is a body fluid sample, preferably a blood sample, more preferred a serum sample. For analyzing a patient's immune status to soybean allergens, antibodies derived from the patient are contacted with peptides comprising sequence elements corresponding to the identified epitopes. The antibodies may be derived from the patient through a blood or serum sample, which are usually used for testing on IgE. Serum samples are particularly preferred since they are devoid of several blood compounds that could interfere with peptide binding. Moreover, blood shows high autofluorescence and is therefore disadvantageous in case a fluorescent readout is used. In addition, samples derived from lymphocyte transformation tests may be used (Pichler and Tilch, 2004).
Alternatively to determining the patient's immune status using a body fluid sample, allergenic sensitivity is commonly tested using skin allergy tests such as skin prick test, skin scratch test, intrademic test or patch test. In all these tests the potential allergen is applied onto or beneath the patient's skin. In case the patient is sensitive to the allergen, i.e. produces antibodies recognizing the allergen, the patient will develop rash and/or urticaria in the proximity of the applied allergen. Since the peptides of the invention comprise sequence elements corresponding to epitopes that can be specifically recognized by IgE of soybean protein sensitive patients, they are suitable for respective allergy tests. Accordingly, in a further aspect, the invention relates to a method for determining a patient's sensitivity to soybean allergens, comprising the steps providing a compound comprising at least five different sequence elements each corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented, contacting the patient with the compound, preferably by applying the compound to and/or beneath the patient's skin, and detecting a reaction, preferably rash and/or urticaria. Upon application, e.g. beneath the patient's skin, the epitope is recognized by IgE antibodies localized on mast cells, inducing dimerisation of the IgE receptors and subsequent degranulation of the mast cell. Thus, for application in allergy tests, at least two peptides comprising at least five different sequence elements corresponding to epitopes, preferably derived from the same allergenic protein, are combined into a single compound. This can be achieved, for example, by providing a polypeptide comprising the sequence elements corresponding to epitopes, e.g. a homo- or heterodimer or polymer, respectively. Alternatively, the compound may be provided by associating the peptides with a cargo molecule. Suitable cargo molecules include chemical compounds as well as surface molecules such as nanoparticles (e.g. iron oxide nanoparticles or albumin nanoparticles), liposomes, microparticles or microbubbles.
In a further aspect, the invention relates to a kit comprising at least one composition, containing a compound comprising at least five different sequence elements each corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented. The composition of the kit may represent various epitopes of a single soybean protein by containing compounds that comprise various sequence elements corresponding to different epitopes of the soybean protein of interest. The various sequence elements may be comprised in a single compound or allocated to different compounds. The composition of the kit may also cover epitopes of several or even all allergenic soybean proteins. The kit is suitable to provide potential soybean protein allergens for various applications, in particular skin allergy tests. Accordingly, the composition may be a composition suitable for diagnostic tests such as skin prick test or patch test. The concentration, in which the peptides are provided and applied to the skin, should be according to biological standardization, e.g histamine equivalent potency (HEP/ml) or allergy units (AU/ml) as defined by the Food and Drug Administration of the United States.
In a further aspect, the invention relates to a method for detecting at least one soybean allergen in a substance, comprising the steps providing at least two different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, raising at least one antibody against each peptide, contacting the substance with the antibodies, and determining binding of the antibodies to the substance. Detection of the soybean allergen may include a qualitative detection and/or a quantitative analysis of the amount of soybean protein present in the substance. In a first step, epitopes of interest are selected, preferably representing one or more of the major allergenic soybean proteins. Depending on the aim of the analysis, different epitopes of various soybean products may be covered. To gain comprehensive information on the presence of potentially allergenic proteins in a sample, e.g. of a food product, at least one epitope of each major allergenic protein, preferably of each potentially allergenic protein should be detected. To exclude the presence of any soybean remnants e.g. for baby nutrition, the method may be performed using antibodies for the most frequently recognized or each of the identified epitopes. Accordingly, depending on the number of epitopes and allergens to be covered by the method, at least 5 different peptides, preferably at least 10 different peptides, more preferred at least 20 different peptides, most preferred at least 50 different peptides are provided and antibodies raised against them. In a preferred embodiment, at least five different epitopes, preferably at least 10 different epitopes, more preferred at least 20 different epitopes, most preferred at least 50 different epitopes are represented by the peptides. In a preferred embodiment, each peptide comprises a sequence element corresponding to a different epitope. Finally, epitopes may be used that are recognized by most patients (group 10, table 3) giving a good first impression on the immune status of the patient.
After selecting the epitopes, peptides are produced comprising at least one of the selected epitopes. Each peptide may consist of the epitope sequence(s) or may comprise additional amino acids on one or both sides of the epitope(s). The additional amino acids may for example correspond to the amino acids flanking the epitope in the natural protein or in the original peptide identified by the phage display. Alternatively, they may be individually selected, e.g. to optimize antibody formation. For example, to avoid hindrance of interactions between the peptide and an antibody, flanking amino acids may be selected having small residues, such as glycine, alanine or serine. The size of the peptide amounts preferably to 5 to 30, more preferred to 8 to 20, most preferred to 8 to 15 amino acids. For raising antibodies, preferably monoclonal antibodies, methods well known in the art may be employed. To detect soybean proteins or protein remnants in a substance, the substance is contacted with the antibodies to allow interaction and binding, which is then determined or even quantified, e.g. by use of fluorescent secondary antibodies and fluorescence scanners. In summary, the method allows determining the allergenic potential of the substance by detecting allergenic proteins or remnants of soybean proteins therein.
In a preferred embodiment, the substance is a human or animal food product, a dietary supplement or a cosmetic product. Soybean protein and preparations thereof are not only processed in human and animal food products but also in dietary supplements and cosmetics, e.g. in the form of soybean extracts or soybean oil. Since allergy reactions do not only occur upon oral ingestion but also upon contact to skin, a test for potentially allergenic proteins and protein remnants is likewise needed for cosmetic products and ingredients.
In a preferred embodiment, the soybean allergen is selected from the group consisting of profilin, P34 thiol protease, kunitz trypsin inhibitor, hydrophobic seed protein, glycinin, Gly m BD 28K, defensin, β-glycinin and PR10. These major allergenic soybean proteins are known to induce strong allergic reactions and are thus considered most crucial. Therefore, preferably each of these proteins is represented by at least one epitope. For testing products with particularly strict safety requirements, e.g. baby nutrition, all epitopes of the major allergenic soybean proteins or even of all potentially allergenic soybean proteins (Table 1) may be detected.
In a further aspect, the invention relates to a method for producing an immunoassay product, comprising the steps providing at least two different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, raising at least one antibody against each peptide, and compiling the antibodies to provide the immunoassay product. Processed food and other industrial products contain soybean protein in various quantities and different conditions. For example, processing of soybean seeds or extracted protein leads to denaturation and modification of the soybean proteins, which can eliminate epitopes and thereby reduce the allergenic potential of the product. Therefore, methods are already used and continuously developed that aim on reducing the allergenic soybean proteins, e.g. by food processing techniques (Wilson et al., 2005) or genetic modification of soybean plants (WO 2010/087888). Moreover, wild (glycine soja) and cultivated (glycine max) soybean plants as well as different varieties of glycine max were found to have different variants of potentially allergenic proteins. The proteins differ at least slightly in their amino acid sequences and therefore vary in their allergenic potential. This prompted the development of specific glycine max varieties, which express reduced amounts of potentially allergenic proteins, e.g. P34 (Joseph et al., 2006). Despite these efforts, a reliable and complete destruction or removal of all allergenic proteins from soybean products has not yet been achieved. Therefore, it is necessary to assess the remaining allergenic potential of food and other products comprising soybean proteins. So far, soybean protein tests are mainly based on antibodies raised against the complete soybean proteome. Sometimes polyclonal antibodies are even directly derived from animals or humans sensitive to soybean proteins. However, such antibodies or sera do not necessarily provide reliable results. Antibodies usually only recognize the particular protein or amino acid residues they have been raised against. Therefore, antibodies raised against native proteins may no longer recognize soybean proteins having an altered confirmation due to industrial processing techniques. Thus, the allergenic epitope, although present, may not be bound by the respective antibodies. Moreover, antibodies or sera raised against the entire proteome of a specific variety may fail to detect allergenic proteins of a different variety. In contrast, antibodies raised against individual and defined epitopes of about four to ten amino acids have a high potential to recognize allergenic proteins or amino acid residues independently of conformational changes in the molecule (e.g. due to food processing techniques) or adjacent amino acid sequences (that may vary according to soybean variety). Thus, the identified epitopes and peptides comprising sequence elements corresponding to these epitopes are particularly suitable for providing an immunoassay product for detecting potentially allergenic soybean proteins. The term “antibody” as used herein refers to natural as well as artificial immunoglobulins and derivatives thereof, including polyclonal antibodies, monoclonal antibodies, antibody fragments, antigen-binding fragments (Fab) and single chain variable fragments (scFv).
For providing an immunoassay product, the peptides and antibodies may be selected and provided as described above. Depending on the number of epitopes and allergens to be detected by the immunoassay product, at least 5 different peptides, preferably at least 10 different peptides, more preferred at least 20 different peptides, most preferred at least 50 different peptides are provided and antibodies raised against them. In a preferred embodiment, at least five different epitopes, preferably at least 10 different epitopes, more preferred at least 20 different epitopes, most preferred at least 50 different epitopes are represented by the peptides. In a preferred embodiment, each peptide comprises a sequence element corresponding to a different epitope. The antibodies are compiled to provide the immunoassay product, which is suitable for testing a sample for the presence of soybean proteins, e.g. by contacting the sample with the antibodies and detecting possible interactions.
In a preferred embodiment, compiling the antibodies comprises immobilizing them onto a solid surface, e.g a chip, a multiwell plate or beads. Thereby, they can be easily contacted with the sample to be tested and the non-bound remnants of the sample removed by washing if necessary. This allows a fast and specific detection suitable to be implied into industrial processes.
In a preferred embodiment, the immunoassay product is a microarray, a bead-based assay product, an ELISA plate or a lateral flow test. Microarrays and bead-based assay products are particularly preferred for detecting the presence of a multitude of different epitopes, because they allow the simultaneous use and specific readout of many antibodies directed against different epitopes. ELISA (enzyme-linked immunosorbent assay) plates, usually multi-well or microtiter plates, also allow for a simultaneous testing of a substantial number of different antibodies. Lateral flow tests, in contrast, provide a particular fast readout, however, usually cover only one or few different antibodies. Depending on the number of epitopes to be detected and the time available, different immunoassay products may be advantageous. All of the assays are fully established standard methods allowing a sensitive and fast readout of antibody-antigen interaction. Moreover, readout systems have been developed, in particular for microarrays and ELISA that allow a fast and fully automated analysis.
In a further aspect, the invention relates to a method for determining the allergenicity of a soybean variety by detecting the presence of at least two epitopes in a sample of the variety, wherein the epitopes are selected from the group consisting of SEQ ID NO.: 1-354. In addition to wild (glycine soja) and cultivated (glycine max) soybean, several varieties of glycine max are known. Additionally, plant varieties expressing reduced levels of allergenic proteins (e.g. P34, Joseph et al., 2006) or alternative amino acid sequences of the allergenic proteins (e.g. WO 2010/087888) were specifically developed to reduce the allergenic potential. By detecting the presence and, preferably, also the quantity of the allergenic epitopes or proteins, the allergenicity of a particular soybean variety can be determined. This is of particular importance, since the use of soybean varieties with reduced allergenicity could contribute to a total reduction of newly developed allergies.
In a preferred embodiment, the presence of at least five different epitopes, preferably at least 10 different epitopes, more preferred at least 20 different epitopes, most preferred at least 50 different epitopes is detected.
In a preferred embodiment, the presence of the epitopes is detected by an immunoassay or mass spectrometry. Suitable immunoassays comprise, for example, ELISA and flow rate assays that may be provided by raising antibodies specific to the identified epitopes. In addition, mass spectrometry can be used to directly analyze the proteome of soybean varieties for the presence or the quantity of epitopes and the corresponding soybean proteins, respectively (e.g. Houston et al., 2011).
In a further aspect, the invention relates to the use of a peptide comprising a sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354 for providing a molecule binding to a protein or peptide comprising the epitope, in particular an antibody. The term “molecule binding to a protein or peptide comprising the epitope” refers to any molecule that is able to specifically form a complex with the epitope and/or a peptide or protein containing a sequence element corresponding to the epitope due to intermolecular forces. Peptides consisting of, or comprising one or more sequence elements corresponding to the epitopes of a specific soybean protein may be used to identify molecules interacting and binding to the epitope and the original protein, e.g. by screening molecule libraries. Likewise, the peptides may be used to raise antibodies specifically recognizing and binding the epitope. Thus, in contrast to commonly used anti-soybean antibodies or sera, the molecules provided by using the peptides of the invention, specifically detect the epitope(s) of the protein responsible for allergic reactions to soybean. Accordingly, the binding molecules are particularly suitable for detecting allergenic proteins of soybean, for example in food or cosmetic products. In order to test the binding specificity of a molecule binding to a protein or peptide comprising the epitope, in particular an antibody, a compilation comprising at least five different peptides, each comprising the same sequence element corresponding to the epitope but different additional amino acids, may be used.
In a further aspect, the invention relates to a method for generating a variant of an allergenic soybean protein having a reduced allergenic potential, comprising the steps providing an amino acid sequence of at least one epitope selected from the group consisting of SEQ ID NO.: 1-354, altering the amino acid sequence of the at least one epitope as to eliminating the structure of the epitope, and generating a protein or peptide comprising the altered amino acid sequence. The term “variant of an allergenic soybean protein” refers to a variant of a soybean protein, which is known to induce allergenic reactions, that comprises one or more epitopes that have a different amino acid sequence compared to the natural protein. The term “a protein having reduced allergenic potential” also comprises proteins having no allergenic potential at all. The identified soybean epitopes (Tables 1 and 2) are suitable to generate variants of natural soybean proteins with an amino acid sequence that is altered in a way that binding of IgE antibodies of a soybean sensitive patient do not bind or bind to a reduced extent.
This can be achieved by replacing or removing amino acids within one or more of the identified epitopes (table 1), which are essential for IgE binding. For example, to support the selection of suitable residues, epitopes from the group consisting of SEQ ID NO.: 1-354 can be mapped onto existing structures of the respective soy protein, or structures of homologous variants of the protein from other, favourably closely related, species available in the protein structure databases. One or more selected amino acids within the epitope's sequence may be replaced by amino acids of different physiochemical properties, such as size, charge or polarity. As a result, the modified epitope, and more importantly, proteins comprising the modified epitope are not recognized by epitope specific antibodies. In case an allergenic soybean protein was found to comprise several epitopes, one or more epitopes may be eliminated. For example, to determine the patient's antibody specificity, all epitopes found to induce cross-reactivity with antibodies against proteins of other species (e.g. birch pollen) may be eliminated.
Proteins with one or more epitopes eliminated, are suitable for analysing antibody-epitope interaction, as they allow determining the participation of individual amino acids in this interaction. Additionally, they provide important negative controls for analysis tools based on epitope specific antibodies. By including respective variants of soybean proteins, antibody binding and specificity can be evaluated in detail.
In addition, the method can be employed for generating derivatives of natural soybean proteins comprising one or more modified epitopes. Such derivates are, for example, suitable for immunotherapeutic applications. Modified allergens, i.e. derivatives of allergens suitable to provoke an IgG immunoresponse, but lacking the allergenic epitopes responsible for IgE reactivity, can be used as recombinant vaccines to reduce or even prevent allergenic reactions (Valenta and Niederberger, 2007).
In a further aspect, the invention relates to a peptide or protein comprising a modified variant of at least one epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein the original structure of the epitope was eliminated.
Affinity Selection and Panning
24 μg of serum protein from patients with soy allergy were diluted in 2.5 ml PBS and coated in protein binding Immuno™ Tubes with Maxisorp™ for 1 h at 4° C. at 18 rpm (negative control: PBS). The coated tubes were washed with 2.5 ml blocking buffer (5% NFDM/1×PBS) and incubated with 4 ml blocking buffer at 4° C. for at least 15 min (18 rpm). The tubes were washed three times with 4 ml wash buffer I (1×PBS). 1 ml blocking buffer containing 30 μg of non-allergic serum protein and the phage library ENTE1 was added to each tube. The tubes were incubated for 2 h at 4° C. (18 rpm). After incubation, the tubes were washed 5 times with 1 ml 0.1% Tween/1×PBS and subsequently with 0.5% Tween/1×PBS. Then the bound phages were eluted using 1 ml of elution buffer (0.1 M Glycin.HCl pH 2.2). The tubes were incubated 5 min and vortexed several times. The eluate was immediately neutralized with 200 μl neutralization buffer (1 M Tris.HCl pH 8.2). In the first selection round 4×1011 cfu of the ENTE1-library were used per tube. In the second selection round 1000×the recovered phages were inserted.
Infection of TG1 (λ) with Eluted Phage
TG1 (λ) cells were grown in 250 ml baffled culture flasks in 10 ml dYT at 37° C. until they reached an OD600 about 0.5-1.0. The cells were incubated with eluted phage for 20 min at 37° C. with 100 rpm agitation. After incubation, the solution was immediately poured on dYT-agar plates (25×25 cm) containing 200 μg/ml ampicillin and incubated overnight at 30° C.
Packaging of Phage from Infected Cells
After overnight incubation the colonies were resuspended in 25 ml dYT containing 1×1010 cfu/ml M13K07. The cells were diluted in 25 ml dYT containing 500 μg/ml ampicillin and 1×1010 cfu/ml M13K07 using a 250 ml baffled culture flasks to obtain a cell density of OD600 0.5. The cells were incubated for 1 h at 37° C. with 180 rpm agitation. Afterwards the culture was growing about 13 h at 30° C. with 180 rpm agitation.
Purification of Phage
The infected bacteria cells were centrifuged at 20,000×g for 20 min at 4° C. ¼ volume of cold PEG/NaCl (PEG/NaCl (20% (w/v) PEG 6000, 2.5 M NaCl) solution was added to the supernatant and incubated for at least 1 h on ice. Phages were collected by centrifugation (30 min, 4° C., 14,000×g) and the pellet was resuspended in 1 ml 1×PBS. The solution was centrifuged at 14,000×g for 10 min and the supernatant recovered and incubated with IA volume of PEG/NaCl (PEG/NaCl (20% (w/v) PEG 6000, 2.5 M NaCl) solution for at least 20 min on ice. Phages were again collected by 30 min centrifugation at 14,000×g, 4° C. and the pellet of phage particles resuspended in 1 ml PBS. The purified phage suspension was stored at 4° C. for several days.
Identification of Selected Epitopes
For data analysis the LibDB software was used. Due to the design of the trinucleotide based library, sequences with potential errors were removed. All valid sequences were written into a database and a database of 3-5mer motifs was generated. The observed frequency as well as statistically and theoretically expected values were provided for each motif in the database.
Validation of Epitopes Using a Peptide Array
The peptide micro arrays were designed to represent nine predominant soybean allergens, named beta-conglycinin, glycinin, defensin, lectin, PR-10, Gly m 1, P34, Kunitz trypsin-inhibitor and profilin. Each allergen amino acid sequence was divided into 15mer fragments with 4 amino acids overlap and a chip was designed bearing duplicates of every peptide. The slides were purchased from the company PEPperPRINT, which synthesized the peptides in a cycle of synthesis, where amino acid micro particles are printed directly on a glass slide based on solid phase Fmoc chemistry.
To analyze the IgE reactivity of soybean allergic patients to the soybean antigen peptides displayed on the array, the glass slides were prepared as follows. Each slide was washed in staining buffer (PBS, 0.05% (v/v) Tween 20, 0.1% (w/v) BSA, pH 7.4) for 10 min at room temperature and afterwards blocked for 30 min in blocking buffer (PBS, 0.05% (v/v) Tween 20, 1% (w/v) BSA, pH 7.4). Incubation of the slides in patient sera diluted 1:50 in staining buffer was performed over night at 4° C. After 3 times washing in standard buffer (PBS, 0.05% (v/v) Tween 20, pH 7.4) the slides were shaken at 200 rpm for 30 min in a goat-anti human IgE antibody solution (Invitrogen; 1:5000 diluted in staining buffer). Detection was performed by incubating the array after 3 additional washing steps in a solution containing the secondary donkey-anti goat antibody labelled with Cy5 (Abcam, 1:5000 diluted in staining buffer) for another 30 min at room temperature. The fluorescence signals on the array were detected in a micro array reader at 635 nm.
Data evaluation was performed using the PepSlide Analyser software from PEPperPRINT, where signal intensity was directly connected to the peptide spot. Additionally the software in a first step performed the subtraction of the local background signal for every spot, resulting in a normalization of the different background levels for the tested sera. Further for all sera an overall threshold level from 30 fluorescence signal intensity units was set, where signal intensities above this threshold were assumed as positively recognized by the patient sera. Peptides detected by a minimum of 25% of the 16 analyzed patient sera were defined as soybean related allergenic epitopes. Peptide sequences tested positive for IgE binding (as a measure of allergy relevance) in a peptide array were entered in the software and matched/aligned with the motives in the database. A sequence was considered to represent a potential epitope, if the enrichment value of the 4mer motives was different in comparison with the naïve library. The enrichment value of the motive in the serum was higher than the enrichment value of the motive in the naïve library. All sequences with the identified motive were saved in a fastq-file, which was aligned to soybean proteins using the software MegAlign Pro 12. If the 4mer motive could be expanded to a 5mer or 6mer motive, the potential epitope was validated for inclusion into table 1.
Identification of Novel Epitopes
Sequences of soybean proteins were matched with the motives in the database and the statistic listed. Then, a graph of enrichment values of the naïve library and the serum was generated in Excel. A positive difference of the values of the naïve library and the serum indicated a relevant epitope. In the second panning more than one sequence should be enriched. All sequences with the identified motive were saved in a fastq-file, which was aligned to soybean proteins using the software MegAlign Pro 12. If the 4mer motive could expand to a 5mer or 6mer motive, the potential epitope was validated.
For example, the motive “QHQQ” was identified in the first panning contained in
>2013_S1_2. Panning_seq_10 Icount=250: GIEPCSKYSQHQQHVQN (SEQ ID NO.: 706) and
>2013_S1_2. Panning_seq_11 Icount=122: GEIMCWREVVTQHQQHA (SEQ ID NO.: 707).
In the second panning, the motive was significantly enriched with a total of 825 counts in comparison to 407 counts in the first panning (Serum 1_1. Panning round: count 407, Serum 1_2. Panning round: count 825).
Validation of an Epitope Using Magnetic Beads
200 μl Dynabeads® M-270 Carboxylic Acid (Invitrogen) were washed 3 times with 1 ml PBS. The beads were activated by adding of 200 μl 0.4 M 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) and 200 μl 0.1 M N-hydroxysuccinimide (NHS). After 5 minute incubation at room temperature with agitation, the beads were washed once with 1 ml 10 mM sodium acetat, pH 4.5. 100 μg streptavidin in 100 μl 10 mM sodium acetat, pH 4.5 were added. After 30 min incubation at room temperature with agitation, beads were blocked with 500 μl 0.1 M ethanolamine pH 8.5 for 10 minutes at room temperature with agitation. The beads were washed 3 times with 1 ml PBS and resuspended in 200 μl PBS.
Potential epitopes were synthesized as peptides and coupled with 10 μl streptavidin coupled beads by adding of 3 μg (=3 μl) peptide for 1 h at room temperature with agitation. Beads were blocked with 500 μl blocking buffer (15 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween) for 30 minutes at room temperature with agitation. After three washing steps with 1 ml PBS, the beads were incubated with 10 μl serum from a patient with soy allergy for 1 h at room temperature with agitation and afterwards washed 3 times with 1 ml 0.1% Tween/PBS and resuspended in 10 μl SDS-loading Dye.
Immediately a SDS-Page with a 12% gel at 180 V for 50 minutes was performed. The western blot was performed with 0.2 μm nitrocellulose membrane and 260 mA and 200 V for 2 h at 4° C. After blotting the membrane was blocked with 0.1% Tween/PBS for 1 hour at room temperature. The antibodies (Anti-human polyvalent immunoglobulins) were diluted 1:10000 in 10 ml 0.1% Tween/PBS. After incubation for 1 h at room temperature, three washing steps with 0.1% Tween/PBS and one with PBS were performed. Antibodies were detected using DAB-solution (10 ml 0.1m Imidazole, pH 7, 50 μl 1% Diaminobenzidine, 5 μl 30% H2O2).
In Vitro Analysis of the Immune Status of Soybean-Sensitive Patients Using a Peptide Microarray
Epitope candidates were supplied externally (Neundorf, University of Cologne, Germany). The peptide is synthesised with the following structure: Ac-N-epitope sequence-C-Ebes-Lys(γ-N3)-amide (Ebes=amino-3,6-dioxaoctyl-succinamic acid). The peptide sequences were based on either the naïve soybean protein sequence (Pr) or the mimotope from phage display experiment (Ph). The peptide length varied between eight and nineteen amino acids. Glass slides were coated with Dibenzocyclooctyl (DBCO). The peptides were immobilized using different concentrations (81 ng/μl, 27 ng/μl or 9 ng/μl) on glymo-covered slides using click chemistry.
Each slide was blocked for 30 min with PBS containing 0.1% (v/v) Tween-20 and 1% (w/v) BSA, pH 7.4. For the incubation with sera cover slides (HybriWell™. 60 mm×21 mm×0.15) were used. Sera from 50 patients with differently intense prick reaction to soy milk were tested. The slides were incubated with 100 μl sera from the soybean-sensitive patients diluted 1:50 in blocking buffer overnight at 4° C. After two washes in washing buffer (PBS containing 0.1% (v/v) Tween-20, pH 7.4) the slides were shaken at 200 rpm for 60 min in a mouse-anti human IgE antibody (Abcam, Cambridge, UK, ab99834) diluted to 100 ng/ml or goat-anti human IgG antibody Cy5 (Abcam, Cambridge, UK, ab97172) diluted to 100 ng/ml in blocking buffer. After two further washes, bound IgG and IgE were detected by incubating the array for 60 min at room temperature with the secondary Cy5-labeled goat anti-mouse antibody (life technologies, Thermo Fisher Scientific, Waltham, Mass., USA, A10524) diluted to 400 ng/ml in blocking buffer. Fluorescence signals on the array were detected in a microarray reader (Geneprix 4200A) at 635 nm and the data were evaluated using GenePix® Pro 6 software to determine the signal intensity of each peptide spot. The software calculated the signal to noise ratio (SNR) for every spot. An overall threshold of 5 signal to noise ratio units was set, and signals above this threshold were assumed to indicate positive recognition by the patient sera.
VEEEEECEEGQI (SEQ ID NO.: 742)
The results show that the peptides are recognized by antibodies in the sera of soybean-protein sensitive patients. The results further show that the sensitivity to different soybean proteins and epitopes varies.
In contrast to commonly used methods for determining the immune status of a patient, the method disclosed facilitates a differentiation between IgE and IgG antibodies.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15184199.6 | Sep 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/071196 | 9/8/2016 | WO | 00 |
