Methods and products for diagnosing seafood allergy

Abstract

The present invention relates to a diagnostically useful carrier, which includes a tool for specifically capturing antibodies against a polypeptide in a sample from a subject, the polypeptide being selected from squid MLC1 or squid MLC2 or a variant thereof; a method comprising detecting the presence or absence of antibodies against squid MLC1 or squid MLC2 in a sample from a subject, and use of a polypeptide or a carrier according to the present invention or a polypeptide that specifically binds to IgE antibodies against squid MLC1 or squid MLC2 in a sample from a patient for preparing a diagnostic kit (preferably for allergy diagnosis).

Description

Technical Field

[0001] The present invention relates to a diagnostically useful carrier, which includes a tool for specifically capturing antibodies against a polypeptide in a sample from a subject, wherein the polypeptide is selected from squid MLC1 or squid MLC2 or a variant thereof; a method comprising the step of detecting whether antibodies against squid MLC1 or squid MLC2 are present in a sample from a subject; and use of a polypeptide or a carrier according to the present invention or a polypeptide that specifically binds to IgE antibodies against squid MLC1 or squid MLC2 in a sample from a patient for preparing a diagnostic kit (preferably for allergy diagnosis). Background Art

[0002] Allergic reactions to seafood range from mild to systemic reactions, including severe anaphylaxis. In addition to allergic reactions when consuming identifiable seafood products, allergens may be unidentifiable ingredients in various foods, where the nature of the allergen and the risk of allergic reaction are hidden.

[0003] Despite the high prevalence of seafood allergy, few treatment options are available and dietary abstinence is often the only possible option. Of course, this requires precise information about the nature of the antigen or antigens to which the patient is reacting.

[0004] A battery of tests is commercially available, particularly for the most prevalent seafood ingredients, especially crustaceans (including shrimp, prawns, crab, lobster and crayfish). For example, the muscle protein tropomyosin has been identified as a major cross-reactive allergen in various shrimp species.

[0005] These diagnostic tests for seafood allergies involve the detection of IgE antibodies from the patient that are specific for proteins from the allergen. However, a positive IgE test (i.e., IgE sensitization) does not always correlate with clinical manifestations. In other words, if IgE is detected against one allergen, this observation is not sufficient to provide a conclusive diagnosis.

[0006] Rather, it is the first sign that the tested allergen is more likely to be relevant than the others. In clinical practice, the physician's diagnosis of an allergen is usually based on positive IgE sensitization to the allergen in question and a plausible clinical history of an allergic reaction to that allergen. In addition, the results of complementary tests such as the skin prick test (SPT), performed by topical application of specific extracts on the patient's skin, may be considered for the final diagnosis.

[0007] However, a significant number of patients remain undiagnosed because they react to hitherto unknown allergens.

[0008] Once allergy to a specific allergen has been successfully identified, treatment can be initiated. While antihistamines can be used to improve treatment, long-term and definitive treatment is possible through specific immunotherapy based on controlled allergen administration. Although the exact mechanism is not fully understood, this specific activation of the immune system alleviates symptoms upon subsequent exposure to the same allergen environment. Successful treatment has been demonstrated for a variety of plant and animal allergens.

[0009] Yadzir et al. performed a proteomic analysis of extracts from white squid, including 2D SDS PAGE, followed by mass spectrometry and bioinformatics analysis (Yadzir, ZHM, Misnan, R. and Murad, S. (2012) Identification of tropomyosin as major allergen of white squid (Loligo edulis) by two-dimensional immunoblotting and mass spectrometry, Southeast Asian J Trop Med Public Health, 43 (1), 185). Although more than 50 different proteins were identified, the only consistent reactive protein spot was attributed to tropomyosin.

[0010] Tong, SW et al. disclosed myosin light chains from different species as allergens, but did not disclose those from squid (Tong, SW, Yuen, AWT, Wai, CYY, Leung, NYH, Chu, KH, Leung, PSC (2018) Diagnosis of fish and shellfish allergies, J. Asthma and Allergy, 11, 247-260).

[0011] US2015 / 0153358 discloses various shrimp allergens, including shrimp myosin light chain. Squid myosin light chain has not been disclosed.

[0012] Miayazawa et al. disclosed the purification of squid allergens (Miyazawa, MT, Fukamachi, F., Inakagi, Q., Reese, G., Daul, CB, Lehrer, SC Inouye, S and Sakaguchi, M. (1996) J. Allergy Clin. Immunol. 98, 948-953). Squid tropomyosin was identified, and allergic proteins with molecular weights between 70 kDa and 90 kDa were also found, but no reactive band of about 18 kDa (molecular weight of squid myosin light chain) was found in immunoblotting, and squid myosin light chain was not disclosed, let alone as an antigen.

[0013] Therefore, there remains a continuing need to provide reagents and methods for diagnosing, treating, and preventing seafood allergic reactions. Summary of the invention

[0014] The problem underlying the present invention is to provide new tests and related reagents for diagnosing or assisting in the diagnosis of seafood allergy, preferably mollusc allergy, more preferably squid allergy.

[0015] Another basic problem of the present invention is to provide new tests and related reagents for differential diagnosis or auxiliary differential diagnosis of seafood allergy, so as to distinguish squid allergy from other seafood allergies (especially shrimp allergy).

[0016] The problem underlying the invention is solved by the subject-matter of the attached independent and dependent claims.

[0017] In a first aspect, this problem is solved by a diagnostically useful carrier comprising means for specifically capturing antibodies against a polypeptide selected from squid MLC1 or squid MLC2 or a variant thereof in a sample from a patient.

[0018] In a preferred embodiment, the carrier is selected from beads, test strips, microtiter plates, microarrays, blots (preferably western blots, line blots and dot blots), glass surfaces, polymers (e.g. cellulose derivatives), slides, biochips and membranes, and most preferably microtiter plates or line blots.

[0019] In a preferred embodiment, the carrier further comprises means for capturing antibodies against squid TM.

[0020] In a second aspect, the problem is solved by a kit comprising a diagnostically useful carrier according to any one of claims 1 to 3, wherein the kit preferably additionally comprises one or more, preferably all, selected from a calibrator (preferably a set of calibrators), a wash buffer and means for detecting IgE or IgG (preferably IgG4 antibodies) (preferably a labeled secondary antibody).

[0021] In a third aspect, the problem is solved by a method comprising the step of detecting the presence of antibodies against squid MLC1 or squid MLC2 in a sample from a subject.

[0022] In a preferred embodiment, the method further comprises detecting the presence of antibodies against squid TM in the sample.

[0023] In a fourth aspect, the problem is solved by a pharmaceutical composition comprising squid MLC1 or squid MLC2 or a variant thereof, preferably further comprising an adjuvant.

[0024] In a fifth aspect, the problem is solved by a polypeptide comprising squid MLC1 or squid MLC2 or a variant thereof, wherein the polypeptide is isolated or immobilized.

[0025] In a preferred embodiment, the polypeptide is complexed with an antibody (preferably an IgE or IgG, preferably an IgG4 class antibody) that specifically binds to said polypeptide.

[0026] In a preferred embodiment, the polypeptide is expressed in eukaryotic cells.

[0027] In a sixth aspect, the problem is solved by the use of the polypeptide, the vector or the kit according to the invention for allergy diagnosis.

[0028] In a seventh aspect, the problem is solved by use of a polypeptide or a carrier according to the invention or a polypeptide specifically binding to IgE or IgG (preferably IgG4) antibodies against squid MLC1 or squid MLC2 in a patient sample for the preparation of a diagnostic kit (preferably for allergy diagnosis).

[0029] In a preferred embodiment, the allergy is a shellfish allergy, preferably a squid allergy.

[0030] In a preferred embodiment, the diagnosis is a differential diagnosis of shellfish allergy, wherein shrimp and squid allergies are preferably distinguished.

[0031] In another preferred embodiment, the diagnosis is a differential diagnosis of allergy, wherein preferably insect and shellfish allergies are distinguished, wherein the insects may more preferably be selected from house crickets and mealworms.

[0032] In an eighth aspect, the problem is solved by a method comprising the step of contacting a medical or diagnostic device comprising a polypeptide with a solution, said solution preferably comprising antibodies against the polypeptide, preferably IgE or IgG (preferably IgG4) antibodies against the polypeptide, in a known concentration, wherein the polypeptide is selected from squid MLC1 and squid MLC2 or variants thereof.

[0033] In a ninth aspect, the problem is solved by an antibody (preferably of the IgE or IgG, preferably IgG4 class) that specifically binds to squid MLC1 or squid MLC2, which antibody is preferably isolated. In a preferred embodiment, the antibody is a monoclonal antibody.

[0034] The present invention is based on the surprising discovery of the inventors that squid myosin light chain 1 (MLC1) and myosin light chain 2 (MLC2) polypeptides are allergens, and that antibodies against squid MLC1 and squid MLC2 can be detected in samples from patients suffering from squid allergy. In addition, the detection of such antibodies is combined with the detection of antibodies against one or more antigens selected from squid tropomyosin, shrimp MLC and shrimp tropomyosin (TM), increasing the usefulness of the diagnosis. Moreover, squid allergy can be distinguished from other seafood allergies, particularly shrimp allergies.

[0035] In a preferred embodiment, the term "squid MLC1" as used herein refers to SEQ ID NO: 8 or a variant thereof or Uniprot accession number P05945, preferably SEQ ID NO: 8 or a variant thereof. In a preferred embodiment, the term "squid MLC2" as used herein refers to SEQ ID NO: 10 or a variant thereof. In a preferred embodiment, the term "squid TM" as used herein refers to SEQ ID NO: 6 or a variant thereof. In a preferred embodiment, the term "shrimp MLC" as used herein refers to SEQ ID NO: 7 or a variant thereof. In a preferred embodiment, the term "shrimp TM" as used herein refers to SEQ ID NO: 4 or accession number Q3Y8M6, preferably SEQ ID NO: 4 or a variant thereof. Any database code mentioned throughout this application refers to the polypeptide sequence obtained online through the corresponding database (e.g. Uniprot) at the filing date of this application or its earliest priority date.

[0036] The present invention relates to a diagnostically useful support, preferably a solid support, for contacting a bodily fluid sample from a subject, preferably a mammalian subject, more preferably a human subject, with means for a specific capture antibody bound to said support.

[0037] According to the present invention, the carrier includes one or more tools for specific capture of antibodies, preferably one or more, more preferably two or more, more preferably three or more such tools, each of which is capable of specific capture of different antibodies. In a most preferred embodiment, the carrier includes a tool for specific detection of antibodies against one or more antigens selected from squid MLC1, squid MLC2 and squid TM. The tool is preferably fixed on the carrier. In a preferred embodiment, the tool for specific capture of antibodies is an antigen or variant thereof (e.g., selected from squid MLC1, squid MLC2 and squid TM or variants thereof) or a polypeptide consisting thereof to which the antibody to be captured or detected binds. The polypeptide can be a linear peptide or a folded polypeptide, the latter preferably using a variant of the folding substantially the same as the natural protein, which can be determined by circular dichroism. In a preferred embodiment, the peptide or polypeptide comprises at least 7, preferably 10, more preferably 15 amino acids of the epitope of the antibody to be captured or detected. The antigen and the insoluble carrier to which it is attached can be separated from the reaction mixture in a direct manner, wherein the antigen is contacted with the sample, for example, by filtration, centrifugation or decantation.

[0038] Any fixation according to the present invention can be in a reversible or irreversible manner. For example, if the polypeptide to be immobilized interacts with the carrier through ionic interactions (which can be masked by adding high concentrations of salt), or if the polypeptide is bound by a breakable covalent bond, the immobilization is reversible. On the contrary, if such a polypeptide is connected to the carrier by a covalent bond that cannot be cleaved in an aqueous solution, the immobilization is irreversible. The polypeptide can be indirectly fixed, for example, by immobilizing antibodies or other entities with affinity to the polypeptide, then adding the polypeptide and forming a polypeptide-antibody complex. The captured antibody can be detected using a labeled secondary antibody or a labeling tool that specifically captures the antibody to be detected. The immobilization method is described in Wong, SS, Chemistry of Protein Conjugatino and Cross-Linking, 1991, CRC Press and Rosenberg, IM, Protein Analysis and Purification, Second Edition, Springer, 2005.

[0039] In a preferred embodiment, the diagnostically useful carrier is a blot, preferably a linear blot (Raoult, D., and Dasch, GA (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 "linear blot" as used herein refers to a test strip coated with one or more specific capture antibody tools (preferably each is a polypeptide), more preferably a membrane-based test strip. If two or more tools are used, they are preferably spatially separated on the carrier. Preferably, the width of the strip is at least 30% of the width of the test strip, more preferably 40%, 50%, 60%, 70% or 80%. The linear blot may include one or more control strips for confirming 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 or a secondary antibody, respectively. Various blots are described in the prior art, for example in van Oss, JC, and Regenmortel, MHV, Immunochemistry, 1994, Marcell Dekker, in particular Chapter 35.

[0040] In a preferred embodiment, the carrier can be a lateral flow immunoassay device. The lateral flow device can include a membrane, the membrane includes a sample pad, the sample liquid is placed on the pad, the particle conjugate at the downstream position includes an antigen to which the antibody to be detected is bound and a detectable label (e.g., colloidal gold), a test line with a means for immobilizing the particle conjugate bound to the antibody to be detected, and a control line. Lateral flow immunoassay is described in Wong, R., Tse, H. Lateral Flow Immunoassay, Springer, 2009.

[0041] In a preferred embodiment, the carrier is a cellulose derivative on which a polypeptide comprising MLC1 and / or MLC2 or a variant thereof is immobilized. Such derivatives are described in the prior art, for example in Adeleke, OA Premium ethylcellulose polymer based architectures at work in drug delivery, Int. J. Pharm. X, doi10.1016 / j.ijpx.2019.100023.

[0042] In a preferred embodiment, the second antibody according to the present invention can be an antibody that is combined with all antibodies from the Ig (immunoglobulin) class. Preferably, the second antibody is combined with one or more different human antibody classes, preferably combined with IgE and / or IgG and / or IgM antibodies, preferably combined with IgG and / or IgE, preferably combined with IgE. The second antibody usually identifies the constant domain of the classification. Various second antibodies are all commercially available. In a preferred embodiment, the second antibody combined with IgM, IgG and IgE class antibodies can be a mixture of the second antibody combined with IgM class antibodies, the second antibody combined with IgG class antibodies and the second antibody combined with IgE class antibodies. In a preferred embodiment, the second antibody combined with IgE and IgG class antibodies can be a mixture of the second antibody combined with IgE class antibodies and the second antibody combined with IgG class antibodies. The second antibody is preferably labeled. Suitable secondary antibodies are commercially available, for example from EUROIMMUN Medizinische Labordiagnostika AG (products EI2606-9601-2 or "Conjugate" in 2260-9601) or from Rockland Immunochemicals Inc. (Dylight 609-145-006).

[0043] In a preferred embodiment, the diagnostically useful carrier is more preferably a linear blot, which includes a CCD control that allows identification of false positive results due to binding of IgE or similar antibodies to glycoprotein epitopes (e.g., α-1,3-fucose on N-glycans) (Altmann, F. (2016) Coping with cross-reactive carbohydrate determinants in allergy diagnosis, Allergo J Int 25 (4), 98-105). This can be an additional line in the linear blot containing the glycoprotein epitope, which, if negative, indicates that there are no false negative reactions in other bands. A large number of linear blots are commercially available, for example from EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany. In a preferred embodiment, the method according to the invention includes a step of detecting the presence of antibodies that bind to cross-reactive carbohydrate epitopes, thereby causing false positive results. Other test strips include dipsticks. Various test strips and blot-based carriers are described in the prior art, for example Khare, R., Guide to Clinical and Diagnostic Virology, Wiley 2019, in particular Chapter 14 and Fischbach, FT, Barnett, M., A Manual of Laboratory and Diagnostics Tests, 8th Edition, 2009, in particular Chapter 3.

[0044] In another preferred embodiment, the diagnostically useful carrier is a bead. Various beads for numerous applications are commercially available, and beads are mainly based on carbohydrates (e.g., agarose gel or agarose) or plastics. They contain active or activatable chemical groups (e.g., carboxyl or ester groups) that can be used to fix the means of specific capture antibodies. Preferably, the beads are beads with an average diameter of 0.1 μm to 10 μm, 0.5 μm to 8 μm, 0.75 μm to 7 μm, or 1 μm to 6 μm. The beads can be coated with the means of specific capture antibodies directly or through affinity ligands (e.g., biotin or glutathione and streptavidin and GST, respectively). For example, beads can be coated with biotin or glutathione, and the antigen can be fused with streptavidin or glutathione-S-transferase, or variants thereof, respectively. Preferably, the beads are provided as an aqueous suspension having a bead content of 10% to 90%, preferably 20% to 80%, preferably 30% to 70%, more preferably 40% to 60% (w / w).In a preferred embodiment, the beads are coated with a first detectable label, such as a fluorescent dye.

[0045] If more than one type of bead with different allergens coated thereon is used, each type may have a different type of detectable label to make the type of bead distinguishable, e.g. for identifying the type of bead and allergen in flow cytometry. If the presence of antibodies to the allergen is detected using a fluorescent label, the first detectable label (if also a fluorescent label) is chosen so that the two can be distinguished, e.g. using separate lasers (e.g. green and red lasers).

[0046] In a particularly preferred embodiment, the beads are paramagnetic beads that can be easily concentrated on the surface by means of a magnet. For this purpose, commercial paramagnetic beads generally contain paramagnetic minerals, such as iron oxide. A variety of suitable paramagnetic beads are commercially available. The beads can be labeled with a detectable label. The techniques of coating antigens or antibodies on beads and using them for diagnostic assays are known in the prior art and are described in textbooks, such as Kontermann, RE, Dübel, S., Antibody Engineering, 2nd edition, Springer 2010 and Wild, D. The Immunoassay Handbook, 3rd edition, 2005, Elsevier.

[0047] In another preferred embodiment, the carrier is a microtiter plate that can be used for ELISA, preferably including at least 8 holes. At least one hole is coated with a tool for directly or indirectly specifically capturing antibodies. At least 3 (preferably 4, more preferably 5) calibrators are provided, which include antibodies for antigens (preferably one or more antigens selected from squid MLC1, squid MLC2 and squid TM or its variants of the concentration determined), and can be used to establish a calibration curve for semi-quantitative analysis. When carrying out the method of the present invention, the calibrator can be processed and developed in parallel with the sample. A second antibody comprising a detectable label (such as enzyme activity, chemiluminescence or fluorescent label) can be provided, such as a label with horseradish peroxidase activity or alkaline phosphatase activity or an enzyme capable of chemiluminescence. The experimental protocol for performing ELISA analysis and reagent preparation (including coated microtiter plates) is well known in the prior art and is described in, for example, Crowther, JR, The ELISA Guidebook, 2001, Humana Press, particularly Chapter 3.

[0048] In another preferred embodiment, the carrier is a microarray. In a preferred embodiment, the term "microarray" as used herein refers to a chip with various (preferably at least 20, preferably 30, 40, 50, 80 or 100) spatially separated allergens. They can include additional mollusk allergens, such as those disclosed in Tong, SW, Yuen, AWT, Wai, CYY, Leung, NYH, Chu, KH, Leung, PSC (2018) Diagnosis of fish and shellfish allergies, J. Asthma and Allergy, 11, 247-260; US2015 / 0153358 and Miyazawa, MT, Fukamachi, F., Inakagi, Q., Reese, G., Daul, CB, Lehrer, SC Inouye, Sand Sakaguchi, M. (1996) J. Allergy Clin. Immunol. 98, 948-953. Microarrays are known in the art and are described in Schena, M., Protein Microarrays, 2005, Jones and Bartlett.

[0049] The sample from the experimenter for implementing the present invention comprises antibody, also referred to as immunoglobulin.Typically, sample is a kind of body fluid, and it comprises the representative set of experimenter's whole immunoglobulin.But, once sample is provided, further processing can be carried out, and processing can comprise whole immunoglobulin or any immunoglobulin class (preferably IgE or IgG, preferably IgG4) of classification, centrifugation, enrichment or separation experimenter, and this can affect the relative distribution of different classes of immunoglobulin.Sample can be selected from whole blood, serum, blood plasma, cerebrospinal fluid and saliva, and preferably serum.In the most preferred embodiment, sample comprises IgE class antibody.In a more preferred embodiment, sample comprises the representative set of experimenter's antibody from IgA, IgG and IgE class (preferably IgG and IgE, more preferably IgG1, IgG4 and IgE), wherein most preferably antibody and different antigen ratio are compared with the ratio obtained from experimenter sample and do not change substantially, particularly IgE class antibody.Sample can be from the animal or people that can produce antibody when being exposed to antigen, preferably mammal, more preferably primate, most preferably human experimenter. The subject can be an experimental animal, preferably selected from mice, rats, rabbits, hamsters, chickens, goats, primates or horses, and more preferably rodents. These methods and reagents can be used to test whether the experimental animal has been successfully immunized, for example for the purpose of developing candidate vaccines or drugs or for antiserum production.

[0050] The teaching of the present invention can be carried out not only using polypeptides having the exact sequence mentioned explicitly (eg by function, name, sequence number or accession number) or implicitly in this application, but also using variants of these polypeptides.

[0051] In a preferred embodiment, the term "variant" as used herein can refer to at least one fragment of the full-length sequence, more specifically one or more amino acids or nucleic acid sequences after truncating one or more amino acids at one or both ends relative to the full-length sequence. Such a fragment contains or encodes a peptide of at least 6, 7, 8, 9, 10, 15, 25, 50, 75, 100, 110, 120 or 130 continuous amino acids of the original sequence or its variant. The total length of the variant can be 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or 250 or more amino acids.

[0052] 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 100 consecutive amino acids, which are at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the 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 for the folding or structure of the polypeptide are deleted and / or substituted, and / or one or more such essential amino acids are substituted 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 prior 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, ClustalW software (Larkin, MA, Blackshields, G., Brown, NP, Chenna, R., McGettigan, PA, McWilliam, H., Valentin, F., Wallace, IM, Wilm, A., Lopez, R., Thompson, JD, Gibson, TJ, Higgins, DG (2007): Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948) is used with default settings.

[0053] In a preferred embodiment, the variant may also include chemical modifications, such as labeling (e.g., isotope labeling or detectable labeling) or covalent modifications (e.g., glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation, etc.). Those skilled in the art are familiar with methods for modifying polypeptides. In addition, variants may also be formed by fusion with other known polypeptides or variants thereof, such as with artificial linkers (preferably not derived from Plasmodium polypeptides), affinity tags, other antigens, etc.

[0054] According to the present invention, the preparation of medical or diagnostic devices (e.g., diagnostically useful vectors) can be by expressing a recombinant variant (optionally with an artificial linker that can include a protease cleavage site) containing an affinity tag in a cell (e.g., a eukaryotic cell or a prokaryotic cell), contacting the expressed variant with a ligand that specifically binds to the affinity tag and is fixed on a solid phase, washing the solid phase to remove non-specific binding material from the cell, and preferably eluting the expressed variant from the solid phase by adding an excess of non-immobilized ligand. The variant can then be fixed on the device. Optionally, the affinity tag can be removed by contacting the variant with a protease (preferably a protease that recognizes the protease cleavage site) before immobilization. Affinity tags can be selected from the group of tags including 18A, ACP, Aldehyd, Avi, BCCP, calmodulin, chitin binding protein, E tag, ELK16, FLAG, flash, polyglutamic acid, polyaspartic acid, GST, GFP, HA, Isope, maltose binding protein, myc, nus, NE, ProtA, ProtC, Thold4, S-Tag, SnoopTag, SpyTag, SofTag, streptavidin, Strep-tag II, T7 epitope tag, TAP, TC, thioredoxin, Ty, V5, VSV and Xpress tags. Available proteases include but are not limited to TEV, thrombin, Faktor-Xa or enteropeptidase.

[0055] In a preferred embodiment, the eukaryotic cell is an insect or mammalian cell, more preferably a mammalian cell, most preferably a human cell. Immortalized artificial cell lines (eg CHO or HEK293) can be used to produce any protein.

[0056] The variant of the polypeptide has biological activity. In a preferred embodiment, this biological activity is the ability to bind to the corresponding antibody. In a preferred embodiment, it comprises an epitope that can bind to the corresponding antibody of a sample preferably from a patient suffering from seafood allergy (more preferably squid allergy), wherein more preferably the epitope comprises a sequence of at least 5, 6, 7 or 8 amino acid residues from an antigen, and the antigen is preferably selected from squid MLC1, squid MLC2 and squid TM. More specifically, the variants of squid MLC1, squid MLC2 and squid TM can specifically bind or specifically capture antibodies that bind to antibodies from samples of patients suffering from seafood allergy (more preferably squid allergy).

[0057] In a preferred embodiment, the diagnostically useful carrier comprises one or more additional means for specifically capturing antibodies directed against a polypeptide, more preferably a polypeptide selected from the group consisting of: crustacean MLC, crustacean sarcoplasmic calcium binding protein, crustacean TM, crustacean troponin C, crustacean arginine kinase, triosephosphate isomerase, preferably selected from the group consisting of Der f 1, Der f 2, Der f 3, Der f 4, Der f 6, Der f 7, Der f 8, Der f 10, Der f 11, Der f 13, Der f 14, Der f 15, Der f 16, Der f 17, Der f 18, Der f 20, Der f 21, Der f 22, Der f 23, Der f 24, Der f 25, Der f 26, Der f 27, Der f 28, Der f 29, Der f 30 f30, Der f 31, Der f 32, Der f 33, Der f 34, Der f 35, Der f 36, Der f 37, Der f 38, Der f 39, Der m 1, Der p 1, Der p 2, Der p 3, Der p 4, Der p 6, Der p 7, Der p 8, Derp 10. Der p 11, Der p 13, Der p 14, Der p 15, Der p 18, Der p 20, Der p 21, Der p23, Der p 24, Der p 25, Der p 36, Der p 37, Der p 38, Der p 39, Eur m 1, Eur m 2, Eurm 3. Eur m 4. Eur m The house dust mite allergen of 14 is preferably selected from the group consisting of Bla g 1, Bla g 2, Bla g 3, Bla g 4, Bla g 5, Bla g 6, Bla g 7, Bla g 8, Bla g 9, Bla g 11, Per a 1, Per a 2, Per a 3, Per a 5, Per a 6, Per a 7, Per a 9, Per a 10, Per a 11, Per a 12, and Per a 13, wherein the crustacean protein is preferably shrimp protein, and most preferably selected from the group consisting of Pen m 1 (tropomyosin), Pen m 2 (arginine kinase), Pen m 3 (myosin light chain), Pen m 4 (sarcoplasmic calcium binding protein), Cra c 6 (troponin c) and Cra c 8 (triosephosphate isomerase).

[0058] Comprehensive information including shrimp allergen epitopes can be found in Pascal, M., Grishina, G., Yang, A.C., Sanchez-Garcia, S., Lin, J., Towle, D., Ibanez, MD, Sastre, J., Sampson, HA, and Ayuso, R. (2015) Molecular Diagnosis of Shrimp Allergy: Efficiency of Several Allergens to Predict Clinical Reactivity, J. Allergy Clin. Immunol. Pract., 521-529e10, which is incorporated herein by reference.

[0059] Zhang et al. have studied the epitopes of MLC allergens (Zhang, YX, Chen, HL, Maleki, SJ, Cao, MJ, Zhang, LJ, Su, WJ, and Liu, GM (2015) Purification, Characterization and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii (2015) J. Agr. Food Chem., 63, 6271-6282). If a variant of sequence X is desired, one skilled in the art can design a biologically active variant by starting with sequence X, introducing modifications (e.g., point mutations, truncations, etc.), and then confirming that the variant still has biological activity by testing whether the variant binds to antibodies from such a sample.

[0060] Those skilled in the art may consider that epitopes from squid MLC1 antigen may include GGTKKMGE (SEQ ID NO: 18), SSKDTGTA (SEQ ID NO: 19) and DREGQ (SEQ ID NO: 20) and variants thereof.

[0061] sequence

[0062] GGTKKMGEKAYKLEEILPIYEEMSSKDTGTAADEFMEAFKTFDREGQ (SEQ ID NO: 21) and variants thereof are also highly antigenic.

[0063] Those skilled in the art may consider that epitopes from squid MLC2 antigen include ALRNAFSMFD (SEQ ID NO: 30), more preferably NAFSMF (SEQ ID NO: 31), FIPEDYLKDL (SEQ ID NO: 42), more preferably IPEDYLKDL (SEQ ID NO: 43) and FSKEEIKNVWKD (SEQ ID NO: 51).

[0064] Epitopes of shrimp MLC allergens have been described in Pascal et al., 2015. One skilled in the art may consider epitopes from squid TM, including DESERGRKVLENRSQGDEER ​​(SEQ ID NO: 26), IDLLEKQLEEAKWIAEDADR (SEQ ID NO: 27), KFDEAARKLAITEVDLERAEARLE (SEQ ID NO: 28), KEVDRLEDELLAEKERYKTISDELDQTFAELAGY (SEQ ID NO: 29) and variants thereof.

[0065] Therefore, variants according to the present invention include polypeptides and peptides comprising these epitopes. In a preferred embodiment, the variant of MLC2 represented by SEQ ID NO: 10 includes a peptide or polypeptide comprising one or more selected from SEQ ID NO: 30, SEQ ID NO: 42 and SEQ ID NO: 51, preferably comprising all, and preferably comprising one or two selected from SEQ ID NO: 43 and SEQ ID NO: 31. In a more preferred embodiment, the variant comprises an amino acid sequence of at least 5, 6, 7, 8, 9, 10, 12 or 15 amino acid stretches from SEQ ID NO: 10, wherein the amino acid stretch comprises one or more selected from SEQ ID NO: 30, SEQ ID NO: 42 and SEQ ID NO: 51, preferably comprising all, and preferably comprising one or two selected from SEQ ID NO: 43 and SEQ ID NO: 31. When designing variants of MLC2, the results described in Example 2 will help guide those skilled in the art.

[0066] In a preferred embodiment, it is preferred to use a linear blot assay as described in the examples comprising the variant as a tool for specific capture of antibodies to test whether the variant has such activity. The variant may also comprise N-terminal and / or C-terminal peptide or polypeptide fusions, as long as they do not have specific binding activity for the antigen sequence portion and / or do not bind to other sequence portions and mask the antigen sequence so that its binding properties are significantly changed and / or are no longer able to bind to the antibody.

[0067] The polypeptide may be provided in any form and to any degree of purification from tissues, fluids or cells containing the polypeptide in endogenous form, more preferably cells overexpressing the polypeptide, crude or enriched lysates of these cells, up to purified and / or isolated polypeptides (which may be substantially pure). 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 cell (more preferably a prokaryotic cell or a eukaryotic cell, preferably a mammalian cell). Glycosylated forms of the polypeptide may be used.

[0068] According to the present invention, polypeptide can be a recombinant protein, wherein the term "recombination" as used herein refers to a polypeptide produced using a genetic engineering method at any stage of the production process, such as by fusing the nucleic acid encoding the polypeptide with a strong promoter to overexpress in a cell or tissue, or by transforming the sequence of the polypeptide itself. Those skilled in the art are familiar with methods for transforming nucleic acids and encoded polypeptides (such as described in Sambrook, J., Fritsch, EF and Maniatis, T. (1989), Molecular Cloning, CSH or described in Brown TA (1986), Gene Cloning-anintroduction, Chapman & Hall) and methods for producing and purifying natural or recombinant polypeptides (such as the manual "Strategies for Protein Purification" published by GE Healthcare Life Sciences, "Antibody Purification", and in Burgess, RR, Deutscher, MP (2009): Guide to Protein Purification). In another preferred embodiment, the polypeptide is an isolated polypeptide, wherein the term "isolated" means that the polypeptide has been enriched compared to its state when produced using biotechnology or synthetic methods, and is preferably pure, i.e. at least 60%, 70%, 80%, 90%, 95% or 99% of the polypeptide in the corresponding liquid consists of the polypeptide, as judged by SDS polyacrylamide gel electrophoresis followed by Coomassie blue staining and visual inspection. Preferably, any polypeptide on the carrier used as a means for capturing antibodies is pure.

[0069] The teachings of the present invention provide a kit, preferably for diagnosing seafood allergies. Such a kit is a container comprising specific reagents required for implementing the method of the present invention, in particular a diagnostically useful carrier according to the present invention, optionally in addition to one or more solutions or reagents required for implementing the method of the present invention, preferably selected from or all selected from a sample dilution buffer, a wash buffer, and a buffer containing tools or reagents for detecting any specific captured antibodies (e.g., a secondary antibody, which may contain a detectable label and optionally a tool for detecting the latter). In addition, it may include instructions for using the kit in detail. It may include a diagnostically useful carrier for contacting the polypeptide of the present invention with a body fluid sample from a subject (preferably a human subject), such as a linear blot. In addition, the kit may include a positive control, such as a recombinant or polyclonal antibody known to bind to one or more of the group including squid MLC1, squid MLC2, and squid TM and variants thereof, and a negative control, such as a protein (e.g., bovine serum albumin) that has no detectable affinity for squid MLC1 or MLC2 or TM. The positive control may be a diluted sample from a patient, which contains antibodies to squid MLC1, MLC2 and / or TM, preferably IgE or IgG (e.g., IgG4). Finally, such a kit may include one or more standard solutions, also referred to as calibrants, comprising one or more antibodies comprising antibodies binding to squid MLC1, antibodies binding to squid MLC2 and antibodies binding to squid TM and antibodies binding to polypeptides comprising antigens selected from squid MLC1, squid MLC2 and squid TM or variants thereof, which are included in the kit, for example, fixed on a diagnostically useful carrier, for preparing a calibration curve, wherein the absolute or relative concentration of the antibodies in each standard solution is preferably known. The concentrations in the standard solutions may be different so as to cover the concentration range of the calibration curve. For example, the relative concentrations of the antibodies in the two standard solutions may be 1:2, 1:5, 1:10, 1:20, 1:50 or 1:100 or more. In a more preferred embodiment, the antibody has a constant region or a variant thereof or the secondary antibody has a binding affinity similar to the antibody to be detected, and / or any secondary antibody used for detection binds to the antibody and the antibody to be detected. The antibody can be a monoclonal antibody, preferably a hybrid comprising human IgE or IgG (preferably IgG4), more preferably an IgE constant region or a variant thereof that is bound to a second antibody, the second antibody being bound to human IgE or IgG (preferably IgG4, more preferably an IgE class antibody). The kit can comprise a polypeptide according to the present invention or a purified polypeptide comprising an antigen selected from squid MLC1, squid MLC2 and squid TM or a variant thereof, the polypeptide optionally comprising a detectable label. The kit can also comprise a washing solution. The kit can comprise a suitable watertight container for contacting a diagnostically useful carrier with a sample in the presence of other liquids (e.g., reaction buffer). For example, a linear blot can be provided in an incubation tray or in combination with an incubation tray, or a microtiter plate can be provided.Suitable containers are described in the prior art, for example EP3025780 or EP3025779. The kit may comprise reagents for carrying out a method according to the invention, for example a method according to aspect 8. Calibrators and their use are described in The Immunoassay Handbook by David Wild, 3rd edition, Elsevier 2005, in particular Chapter 9.

[0070] According to the present invention, a tool or reagent or polypeptide for detecting the captured antibody can be provided or used to detect one or more capture antibodies selected from squid MLC1, squid MLC2 and squid TM. The tool or reagent or polypeptide can be a secondary antibody or a fragment or variant thereof that binds to the constant region of the target type antibody. For example, if the captured human IgE or IgG (e.g., IgG4) class antibodies are to be detected, antibodies that specifically bind to the constant region of human IgE or IgG (e.g., IgG4) class antibodies can be used. Antibodies (preferably monoclonal antibodies that bind specifically to the constant regions of these types and can be used to capture all antibodies of this type in solution, regardless of the binding specificity determined by their variable regions) are commercially available, for example from sources described in Berlina, AN, Taranova, NA, Zherdev, AV, Sankov, MN, Andreev, IV, Martynov, AI and Dzantiev, BB (2013) Quantum-Dot-Based Immunochromatographic Assay for Total IgE in Human Serum. PLoS ONE 8(10):e77485.doi:10.1371 / journal.pone.0077485. The means or reagents may be labeled directly or indirectly.

[0071] In a preferred embodiment, any binding or detection or capture of a means or reagent or antibody, preferably the binding of an antibody to be detected to an antigen (e.g. MLC1 or MLC2, preferably MLC2), means specific binding, which preferably means that the binding reaction (e.g. between the means and the captured antibody) is characterized by a dissociation constant of 1 x 10 -5 M, more preferably 1x 10 -7 M, more preferably 1x 10 -8 M, more preferably 1x 10 -9 M, more preferably 1x 10 -10 M, more preferably 1x 10 -11 M, more preferably 1x10 -12The binding response of M was stronger as determined by surface plasmon resonance performed at 25°C in PBS buffer, pH 7, using a Biacore instrument.

[0072] The method or product according to the present invention can be used to diagnose allergies, preferably seafood allergies, more preferably squid allergies. In a preferred embodiment, the term "diagnosis" as used herein refers to any type of method intended to obtain information that helps to assess whether a patient suffers from or is likely or more likely to suffer from allergies in the past, at the time of diagnosis, or in the future than an average subject or a comparison subject (the latter preferably having similar symptoms), to find out how allergies develop or may develop in the future, or to assess the responsiveness of a patient to treatment or to measure the efficacy of a compound in treating a subject suffering from allergies. In other words, the term "diagnosis" includes not only diagnosis, but also the process of predicting and / or monitoring a disease or condition. If antibodies to at least one antigen selected from squid MLC1, squid MLC2 and squid TM, as shown by detection of antibodies to a polypeptide according to the present invention, are detected in a sample from a subject, the subject is likely or more likely to suffer from an allergy.

[0073] Therefore, the term "diagnosis" preferably does not mean that the diagnostic method or product according to the present invention will be definitive and sufficient to complete the diagnosis based on a single test (let alone parameters), but may refer to the contribution to the so-called "differential diagnosis" (i.e. a systematic diagnostic process that considers the likelihood of a series of possible conditions based on a series of diagnostic parameters). In a preferred embodiment, the term "diagnosis" means that the method or product or use can be used to help diagnose allergies or identify subjects at risk of suffering from allergies. The term "diagnosis" may also refer to a method or medicament for selecting the most promising treatment regimen for a patient. The allergy may be a seafood allergy, wherein the term "seafood" as used herein preferably refers to crustaceans and molluscs, preferably molluscs, preferably squid. According to the present invention, allergies to crustaceans (preferably shrimps) can be distinguished from allergies to molluscs (preferably squid).

[0074] The present invention relates to a method comprising the following steps: detecting the presence of an antibody selected from the group consisting of an antibody against squid MLC1, an antibody against squid MLC2 and an antibody against squid TM in a liquid (e.g., a sample from a subject). Such a method may comprise the following steps: a) providing a liquid, b) contacting the liquid with a polypeptide selected from squid MLC1, squid MLC2 and squid TM under conditions compatible with the formation of a complex comprising a diagnostically useful carrier and an antibody (more specifically a polypeptide and an antibody), c) optionally separating any of the complexes, e.g., by removing the liquid, d) optionally washing the complexes, and e) optionally detecting the complexes after contact with one or more corresponding antigens selected from squid MLC1, squid MLC2 and squid TM, which may be labeled in the case of a competitive assay format. The method is preferably an in vitro method.

[0075] Detection of antibodies or complexes for use in the prognosis, diagnosis, methods or kits according to the invention includes the use of methods selected from immunodiffusion techniques, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, labeled immunoassays (e.g., selected from radiolabeled immunoassays, enzyme immunoassays (e.g., colorimetric assays), chemiluminescent immunoassays, and immunofluorescent techniques). These methods are familiar to those skilled in the art and are also described in the prior art, for example in Zane, HD (2001): Immunology-Theoretical & Practical Concepts in Laboratory Medicine, WB Saunders Company, in particular in Chapter 14. In a preferred embodiment, the term "presence" of an antibody as used herein means that the antibody can be detected using such a method (preferably selected from an enzyme immunoassay, more preferably a colorimetric assay or a chemiluminescent immunoassay, most preferably a method based on a linear blot and a colorimetric assay using alkaline phosphatase activity detection as described in the Examples).

[0076] In many cases, detection preferably refers to detecting the presence or absence of an antibody, and optionally refers to determining whether the concentration of the antibody exceeds a certain threshold sufficient for diagnosis, preferably set by using an ELISA measurement in a liquid. If one or more antibodies can be detected, preferably selected from antibodies to squid MLC1, antibodies to squid MLC2, and antibodies to squid TM, this will be information that helps the clinician diagnose and indicates that the patient has an increased likelihood of suffering from an allergy. In a preferred embodiment, the relative concentration of the antibodies in the serum can be determined compared to the level that can be found in an average healthy subject. Similarly, if the relative concentration of one or more antibodies to squid antigens (preferably squid MLC1, MLC2 and / or TM) is higher than the relative concentration of one or more antibodies to shrimp allergens (preferably shrimp MLC1, MLC2 and / or TM), this may indicate that the patient is more likely to suffer from squid allergy than shrimp allergy. In a preferred embodiment, the term "detecting ... presence" as used herein means that the use of a suitable complex detection method that indicates the presence of the target antibody or the presence of more target antibodies than in a healthy subject is sufficient to check whether a signal sufficient to exceed any background level can be detected.

[0077] In a preferred embodiment, the presence or absence of two or more antibodies selected from an antibody against squid MLC1, an antibody against squid MLC2, and an antibody against squid TM are detected simultaneously (ie at the same time).

[0078] In a preferred embodiment, the presence or absence of two or more antibodies selected from antibodies against squid MLC1, antibodies against squid MLC2 and antibodies against squid TM are detected in spatially separated reactions, more preferably in different reaction mixtures in separate containers.

[0079] In a preferred embodiment, two or more antibodies selected from the group consisting of antibodies against squid MLC1, antibodies against squid MLC2 and antibodies against squid TM are detected. In a more preferred embodiment, this means not only detecting whether at least one of these antibodies is present, but also detecting which of these antibodies is present. In other words, being able to distinguish which of these antibodies is present in the sample.

[0080] In a preferred embodiment, the present invention provides the use of a reagent or a tool or a polypeptide for detecting one or more antibodies, the antibody being selected from an antibody against squid MLC1, an antibody against squid MLC2 and an antibody against squid TM, or a reagent binding to such one or more antibodies or a nucleic acid encoding squid MLC1 or squid MLC2 or squid TM or a variant thereof, or a nucleic acid that specifically hybridizes with the nucleic acid under stringent conditions, or a vector or cell containing the nucleic acid for the preparation of a kit for allergy diagnosis. In a preferred embodiment, a cell containing the vector is provided and cultured under conditions that allow nucleic acid expression, and then the expression product is isolated, which may be squid MLC1 or squid MLC2 or squid TM or a variant thereof, and the expression product is subsequently used as a tool for specifically capturing antibodies in a method or product according to the present invention.

[0081] In another preferred embodiment, the method can be used to confirm the reliability of an antibody detection assay and can involve detecting antibodies to squid allergens selected from antibodies to squid MLC1, antibodies to squid MLC2 and antibodies to squid TM in a solution that is not a sample from a patient but is known to contain (preferably in a known concentration) antibodies to squid allergens. Alternatively, the solution can be a negative control that does not contain antibodies to check background. This method can be run simultaneously with, after or before the diagnostic method.

[0082] In a preferred embodiment, the present invention provides a device for analyzing a sample from a patient to detect one or more antibodies indicative of an increased likelihood of allergy, wherein the antibodies are preferably selected from the group consisting of antibodies against squid MLC1, antibodies against squid MLC2, and antibodies against squid TM, more preferably all antibodies are selected from the group, the device comprising:

[0083] a. a support comprising means for capturing at least one antibody from a sample when the sample is contacted with the support,

[0084] b. when the detectable means is contacted with the carrier, a detectable means capable of binding to the antibody captured by the carrier, in particular, the detectable means is a labeled secondary antibody capable of binding to the antibody captured on the carrier,

[0085] c. optionally, means for removing any sample from the support and the detectable means, preferably by washing;

[0086] d. a detection device for detecting the presence of a detectable tool and converting the result into an electrical signal, and

[0087] e. Optionally, for receiving an electronic signal from a detection device and determining whether the level of the signal indicates an increased likelihood of squid allergy, in particular a higher likelihood of squid allergy than shrimp allergy, by comparing with the signal level detected in the background or an input reference value obtained from a sample of a healthy subject.

[0088] The present invention provides a pharmaceutical composition (preferably a vaccine) comprising one or more antigens selected from squid MLC1, squid MLC2 and squid TM or variants thereof, optionally in combination with one or more other seafood antigens, the composition preferably being suitable for administration to a subject, preferably a mammalian subject, more preferably to 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, buccally, vaginally or by 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 a capsule, tablet, aqueous suspension and solution, preferably in a sterile form. It may 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 present invention. Hypoallergenic variants of antigens may be used. Those skilled in the art are familiar with methods for producing hypoallergenic variants of known allergens. The pharmaceutical composition can be, for example, a PBS-based sterile liquid solution that can be used for injection. The production of such pharmaceutical compositions and variants is described in the prior art, for example in Curin et al. (2018) Next-Generation of Allergen-Specific Immunotherapies: Molecular Approaches, Curr. Allergy and Asthma Rep. 18, 39 (2018). https: / / doi.org / 10.1007 / s11882-018-0790-x Or in Valenta and Coffman, Vaccines against Allergies, Springer 2011.

[0089] According to the present invention, an antibody that specifically binds to a polypeptide comprising squid MLC1 or squid MLC2 or a variant thereof can be provided, and the antibody is preferably isolated. Such an antibody can be used not only as a positive control or calibrator, but also in various assay formats, such as competitive assays or sandwich assays for antibody testing, in which a complex comprising an antibody to be detected, a polypeptide comprising squid MLC1 or squid MLC2 or a variant thereof, and an antibody that specifically binds to a polypeptide comprising squid MLC1 or squid MLC2 is detected. More preferably, the antibody provided is an antibody against a sequence selected from the following group: ALRNAFSMFD (SEQ ID NO: 30), more preferably NAFSMF (SEQ ID NO: 31), FIPEDYLKDL (SEQ ID NO: 42), more preferably IPEDYLKDL (SEQ ID NO: 43) and FSKEEIKNVWKD (SEQ ID NO: 51). More preferably, the antibody is an antibody against an epitope from MLC2 comprising a sequence selected from the group consisting of ALRNAFSMFD (SEQ ID NO: 30), more preferably NAFSMF (SEQ ID NO: 31), FIPEDYLKDL (SEQ ID NO: 42), more preferably IPEDYLKDL (SEQ ID NO: 43) and FSKEEIKNVWKD (SEQ ID NO: 51). Alternatively, the antibody may be an antibody that binds to a binding site on MLC2 without binding to an epitope comprising a sequence selected from the group consisting of ALRNAFSMFD (SEQ ID NO: 30), more preferably NAFSMF (SEQ ID NO: 31), FIPEDYLKDL (SEQ ID NO: 42), more preferably IPEDYLKDL (SEQ ID NO: 43) and FSKEEIKNVWKD (SEQ ID NO: 51), for example for immobilization of a polypeptide comprising MLC2 or a variant thereof for competitive assays. The antibody may be a monoclonal antibody. The antibody may be a polyclonal antibody. The antibody may be a monoclonal antibody. The antibody may be an antibody of an animal selected from the group consisting of mice, rats, rabbits, hamsters, chickens, goats, primates or horses. These assays and other assays are described in David Wild's The Immunoassay Handbook, 3rd edition, Elsevier 2005, particularly Chapters 1 and 2.

[0090] According to the present invention, a polypeptide selected from squid MLC1 or squid MLC2 or a variant thereof can be used to prepare a polyclonal antibody to squid LC1 or squid MLC2 or a variant thereof. According to the present invention, the polypeptide can be injected into an animal capable of producing antibodies, preferably a mammal or a bird, and the antiserum is then recovered by a conventional experimental protocol. The reagents and methods according to the present invention can be used to determine the effectiveness of antibody (preferably IgE or IgG, preferably IgG4 class antibodies) production or the effectiveness of immunization or the efficacy of a drug or vaccine or a candidate drug or vaccine administered to a patient. For example, if the level of antibodies produced after administration of a candidate drug is reduced, it indicates that the drug is effective. These and other assays for the production of monoclonal and polyclonal antibodies are described in David Wild's The Immunoassay Handbook, 3rd edition, Elsevier 2005, particularly Chapter 8.

[0091] In a preferred embodiment, the products according to the invention (eg, vectors, devices, polypeptides, antibodies, calibrators, etc.) may be used in combination or in a composition comprising a stabilizer (eg, bovine serum albumin) or a preservative (eg, azide). BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 Depicted is the layout of the EUROLINE test with various shrimp and squid proteins based on example strips.

[0093] Figure 2 An example of a peptide chip incubated as described in this example is shown. The chip includes eight panels, with the green regular panel representing the control peptide. IgE reactions are shown in green and IgG4 reactions are shown in red.

[0094] Figure 3 Shown is the detection of antibody reactivity in sera of four allergic and two tolerant patients against peptides based on SEQ ID NO: 10 as discussed in Example 2. The Y-axis shows the C-score and the X-axis shows the separated peptides. The black line shows the cutoff value, the green bars represent IGE reactivity, and the red bars represent IgG4 reactivity.

[0095] Figure 4 Heatmaps of IgE and IgG4 serum responses to overlapping peptides based on SEQ ID NO: 10 are shown.

[0096] sequence:

[0097] The present invention includes a series of novel nucleic acid and polypeptide sequences, more specifically:

[0098] SEQ ID NO: 1: Nucleic acid sequence encoding Tod p MLC1 with an N-terminal His tag as used in the examples

[0099] ATGAGCCATCATCATCATCATCATCATTTGGAAGTGCTGTT TCAGGGTCCATCCATGTCTCAACTGACCAAAGACGAAATTGAGGAAGTCCGTGAAGTGTTTGACCTCTTCGACTTTTGGGATGGTC GTGATGGTGATGTTGACGCTGCGAAAGTTGGCGATCTGTTACG CTGTTTAGGGATGAATCCAACCGAAGCTCAGGTACACCAACATGGAGGCACGAAGAAAATGGGCGAGAAAGCGTATAAACTGGAA GAGATTCTGCCGATTTATGAGGAAATGAGCTCCAAAGATACTGGCACAGCAGCGGACGAATTCATGGAAGCCTTCAAAACGTTTGA TCGTGAAGGTCAGGGTTTGATCAGTTCAGCCGAAATTCGGAATGTGCTGAAAATGCTTGGCGAACGCATTACCGAGGATCAGTGCA ACGATATCTTCACCTTTTGCGACATTCGCGAAGATATCGATGG GAACATCAAATACGAGGATCTGATGAAGAAGGTGATGGCAGGACCTTTTCCGGACAAATCGGATTAA

[0100] SEQ ID NO: 2: Nucleic acid sequence encoding Tod p MLC2 with an N-terminal His tag as used in the examples

[0101] ATGAGCCATCATCATCATCATCATCATCATTTGGAAGTGCTGTT TCAGGGTCCATCCATGGCGGAAGAAGCTCCTCGTCGCGTGAAA CTGTCACAACGCCAGATGCAGGAGCTGAAAGAAGCGTTTACCATGATTGATCAGGACCGTGATGGGTTCATTGGCATGGAAGATCT GAAAGACATGTTCAGCTCTTTAGGACGGGTACCACCCGATGACGAACTGAATGCCATGCTGAAAGAATGCCCGGGTCAACTCAATT TCACGGCTTTTCTGACCTTGTTTGGGGAGAAAGTGAGTGGTAC TGACCCGGAAGATGCACTTCGCAATGCCTTTTCGATGTTCGAC GAAGATGGTCAGGGCTTTATCCCGGAAGATTATCTGAAAGACTTGCTGGAAAATATGGGCGATAACTTTTCCAAAGAGGAGATTAA GAACGTCTGGAAAGATGCACCGTTAAAGAACAAACAGTTCAACTACAACAAGATGGTTGACATCAAAGGCAAAGCGGAGGATGAAGATTAA

[0102] SEQ ID NO:3: Polypeptide sequence encoding Pen a 1.0101 (Penaeus aztecus tropomyosin) with an N-terminal His tag as used in the examples

[0103] MSHHHHHHHHLEVLFQGPSMDAIKKKMQAMKLEKDNAMDRAD TLEQQNKEANNRAEKSEEEVHNLQKRMQQLENDLDQVQESLLKANIQLVEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATT KLAEASQAADESERMRKVLENRSLSDEERMDALENQLKEARFLAEEADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLTNKLKAAEARAEFAE RSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGY

[0104] SEQ ID NO:4: polypeptide sequence encoding Pen a 1.0101 (red shrimp tropomyosin); accession number Q3Y8M6

[0105] MDAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKSEEE VHNLQKRMQQLENDLDQVQESLLKANIQLVEKDKALSNAEGEVA ALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERMRKVLENRSLSDEERMDALENQLKEARFLAEEADRKYDEVARKLAMVEAD LERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLTNKLKAAEARAEFAERSVQKLQKEVDRLEDELVNEK EKYKSITDELDQTFSELSGY

[0106] SEQ ID NO:5: Polypeptide sequence encoding Sep 1 1.0101 (Sepioteuthis lessoniana tropomyosin) with an N-terminal His tag as used in the Examples MSHHHHHHHHLEVLFQGPSMDAIKKKMLAMKMEKEVATDKAE QTEQSLRDLEDAKNKIEEDLSTLQKKYANLENDFDNANEQLTAANTNLEASEKRVAECESEIQGLNRRIQLLEEDLERSEERFSSAQSKLEDASKAADESERGRKVLENRSQGDEERIDLLEKQLEEAKWIAEDAD RKFDEAARKLAITEVDLERAEARLEAAEAKIVELEEELKVVGNNMKSLEISEQEASQREDSYEETIRDLTHRLKEAENRAAEAERTVSKLQ KEVDRLEDELLAEKERYKTISDELDQTFAELAGY

[0107] SEQ ID NO:6: Polypeptide sequence encoding Sep 1 1.0101 (Pseudomonas leishmaniformis tropomyosin) MDAIKKKMLAMKMEKEVATDKAEQTEQSLRDLEDAKNKIEEDLSTLQKKYANLENDFDNANEQLTAANTNLEASEKRVAECESEIQGLNRRIQLLEEDLERSEERFSSAQSKLEDASKAADESERGRKVLENRSQGDEERIDLLEKQLEEAKWIAEDADRKFDEAARKLAITEVDLERAEARLEAAEAKIVELEEELKVVGNNMKSLEISEQEASQREDSYEETIRDLTRLKEAENRAAEAERTVSKLQKEVDRLEDELLAEKERYKTISDELDQTFAELAGY

[0108] SEQ ID NO:7: Polypeptide sequence encoding Lit v 3.0101 (Litopenaeus vannamei myosin light chain 2) [His-tagged version is SEQ ID NO:17]MSRKSGSRSSSKRSKKSGGGSNVFDMFTQRQVAEFKEGFQLMDRDKDGVIGKTDLRGTFDEIGRIATDQELDEMLADAPAPINFTMLLN MFAERQTGESDDDDVVAKAFLAFADEEGNIDCDTFRHALMTWGDKFSSQEADDALDQMDIDDGGKIDVQGVIQMLTAGGGDDAAAEE A

[0109] SEQ ID NO:8: Polypeptide sequence encoding Pacific squid (Todarodes pacificus) Tod p MLC1 (myosin light chain 1); derived from accession number P05945

[0110] SQLTKDEIEEVREVFDLFDFWDGRDGDVDAAKVGDLLRCLGMNP TEAQVHQHGGTKKMGEKAYKLEEILPIYEEMSSKDTGTAADEFMEAFKTFDREGQGLISSAEIRNVLKMLGERITEDQCNDIFTFCDIREDIDGNIKYEDLMKKVMAGPFPDKSD

[0111] SEQ ID NO: 9: A polypeptide sequence encoding Tod p MLC1 (Pacific squid myosin light chain 1) with an N-terminal His tag as used in the Examples

[0112] MSHHHHHHHHLEVLFQGPSMSQLTKDEIEEVREVFDLFDFWDGR DGDVDAAKVGDLLRCLGMNPTEAQVHQHGGTKKMGEKAYKLEEILPIYEEMSSKDTGTAADEFMEAFKTFDREGQGLISSAEIRNVLK MLGERITEDQCNDIFTFCDIREDIDGNIKYEDLMKKVMAGPFPDK SD

[0113] SEQ ID NO: 10: Polypeptide sequence encoding Tod p MLC2 (Pacific squid myosin light chain 2)

[0114] AEEAPRRVKLSQRQMQELKEAFTMIDQDRDGFIGMEDLKDMFSS LGRVPDDELNAMLKECPGQLNFTAFLLTLFGEKVSGTDPEDALRNAFSMFDEDGQGFIPEDYLKDLLENMGDNFSKEEIKNVWKDAPLKNKQFNYNKMVDIKGKAEDED

[0115] SEQ ID NO: 11: A polypeptide sequence encoding Tod p MLC2 (Pacific squid myosin light chain 2) with an N-terminal His tag as used in the Examples

[0116] MSHHHHHHHHLEVLFQGPSMAEEAPRRVKLSQRQMQELKEAFT MIDQDRDGFIGMEDLKDMFSSLGRVPPDDELNAMLKECPGQLNFTAFLTLFGEKVSGTDPEDALRNAFSMFDEDGQGFIPEDYLKDLLENMGDNFSKEEIKNVWKDAPLKNKQFNYNKMVDIKGKAEDED SEQ ID NO: 12: Nucleic acid sequence of vector pET24d-Lit v3.0101-(PSc)-His comprising a sequence encoding Lit v 3.0101 (myosin light chain 2)

[0117] GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTT TGTTTAACTTTAAGAAGGAGATATACCATGTCTCGCAAAAGTGGCTCACGTTCCAGCTCCAAACGCAGCAAGAAATCGGGTGGTG GCTCGAATGTCTTTGACATGTTCACGCAACGCCAAGTTGCGGAGTTCAAAGAAGGGTTTCAGCTGATGGATCGCGACAAAGATGG GGTGATTGGCAAAACCGATCTGCGTGGTACCTTTGACGAGATT GGCCGCATTGCGACCGATCAGGAACTGGATGAGATGCTTGCAGATGCCCCAGCACCGATCAATTTCACGATGCTCCTGAACATGT TTGCGGAACGTCAGACTGGCGAATCTGACGACGATGACGTGGTTGCCAAAGCGTTCTTAGCCTTTGCCGATGAAGAGGGAAACAT CGATTGCGATACCTTTCGGCATGCTCTGATGACTTGGGGCGAC AAGTTCAGCAGTCAAGAAGCGGATGATGCCTTGGATCAGATGGACATTGATGACGGCGGGAAAATCGACGTACAGGGTGTGATTCA GATGCTGACAGCTGGTGGAGGTGATGATGCAGCAGCGGAAGAAGCTCTCGAGGTCCTGTTCCAAGGACCCCACCACCATCACCAT CACTGATCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCT GAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGC GAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCC CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAA AACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTA TCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACG CGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTA AATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAA GGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACC TATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTT ATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCG TGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAA GGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATG GTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTC AGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATA CCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTG TATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAA AATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGC GCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCC TTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTG GCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGC CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAG CGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTT CCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGT TATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGA GTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGT GCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGC CCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTT GTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAA CGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGT TGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCC TCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGA ACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCG GTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTA GCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATT CTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTC AACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGG GACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGC GACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGG GTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCG CTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCA GGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATT GCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTG GTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTC GGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCA TGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGG CCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGAT GCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCC GGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGT CATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTT CGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCA GGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAG CTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAA GAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCA TGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGG ATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGG AATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATG AGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATC CCGCGAAATTAATACGACTCACTATA

[0118] SEQ ID NO: 13: Nucleic acid sequence of vector pET24d-N-(PSc)-Pen_a1.0101 containing a sequence encoding Pen a 1.0101

[0119] GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTT TGTTTAACTTTAAGAAGGAGATATACCATGAGCCATCATCATC ATCATCATCATCATTTGGAAGTGCTGTTTCAGGGTCCATCCATGGATGCCATCAAGAAGAAGATGCAGGCCATGAAACTGGAGAA AGACAACGCCATGGATCGTGCGGATACACTGGAACAGCAGAA CAAAGAAGCGAATAATCGTGCGGAAAAGTCTGAAGAAGAAGTGCATAATCTGCAGAAACGCATGCAACAGTTGGAAAACGATCTG GATCAAGTGCAGGAAAGTCTCCTGAAAGCGAACATTCAGCTGGTAGAGAAAGACAAAGCGCTTTCGAATGCAGAAGGCGAAGTTG CCGCCTTGAATCGTCGCATTCAACTGTTGGAAGAAGATCTGGAACGCTCAGAAGAGCGGTTAAATACCGCCACCACCAAATTAGCT GAAGCAAGCCAAGCAGCCGACGAATCTGAACGTATGCGCAAAGTGCTGGAGAATCGCTCACTCTCGGATGAAGAACGCATGGAC GCGTTAGAGAACCAGCTGAAAGAGGCTCGTTTTCTCGCGGAG GAAGCTGACCGGAAATACGATGAAGTGGCGCGCAAACTGGCCATGGTTGAGGCCGACCTTGAGCGCGCTGAAGAACGCGCAGAG ACTGGTGAGAGCAAAATCGTAGAACTGGAAGAGGAGTTACGTGTCGTTGGCAATAACCTGAAATCCCTGGAAGTTTCCGAGGAGA AAGCTAACCAGCGCGAAGAAGCGTATAAAGAACAGATCAAGA CTCTGACGAACAAACTGAAAGCGGCAGAGGCACGTGCGGAATTTGCAGAACGTAGCGTGCAAAAGCTGCAGAAAGAAGTCGATC GCTTGGAAGATGAACTGGTCAACGAGAAAGAAAAGTACAAATCGATTACCGATGAACTCGACCAAACGTTCAGCGAACTTAGTGGGTATTAATCGAGTAAGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCT GCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTA AACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGC GCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCT TTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCG GGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGC CATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTC CACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTT GCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAA AAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTT ATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTT TCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAAT GGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACC GTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGA TCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGA ATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAA AATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTT TAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATA CAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAA TCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGT TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCT TTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTAT GAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGT ATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGG GTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCC TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCG CCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGAT GCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATA GTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCA TGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGT GACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCG TGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTC ACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAA TGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAA CAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTC CACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACA TAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCA GACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAG CCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGG GGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGC AAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTG CCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGA GCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCC CGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGC ATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAG CAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGG TATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTG ATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGT CGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAG ATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAAT GCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAG AAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATG GCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTC GACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACG GCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCA ACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTT TCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGG TGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTA GGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTC CCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGT GATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCC GGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATA

[0120] SEQ ID NO:14: Nucleic acid sequence of vector pET24d-N-(PSc)-Sepl1.0101 containing the sequence encoding Sep l1.0101

[0121] GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTT TGTTTAACTTTAAGAAGGAGATATACCATGAGCCATCATCATC ATCATCATCATCATTTGGAAGTGCTGTTTCAGGGTCCATCCATGGATGCCATCAAGAAGAAAATGCTGGCTATGAAAATGGAGAAA GAGGTTGCAACCGACAAAGCGGAGCAAACCGAACAGTCACTGCGTGATCTGGAAGATGCGAAGAACAAAATCGAAGAAGATCTGA GCACATTGCAGAAGAAATATGCCAACCTCGAAAACGATTTCGACAATGCGAATGAACAGCTGACTGCAGCGAACACGAACTTAGAA GCGTCCGAGAAACGTGTCGCAGAATGCGAATCGGAGATTCAA GGCCTTAATCGGCGCATTCAGCTGCTTGAAGAGGACTTAGAGCGTAGTGAAGAACGCTTTTCTAGCGCTCAGAGCAAACTGGAAGA TGCCTCGAAAGCCGCTGATGAATCCGAACGTGGTCGCAAAGTGCTGGAGAATCGCAGTCAAGGGGACGAAGAACGCATTGATCTG CTCGAGAAACAGTTGGAAGAAGCGAAATGGATTGCCGAAGAT GCGGATCGCAAATTCGACGAAGCTGCGCGTAAACTGGCCATTACCGAAGTCGATCTCGAACGTGCGGAAGCACGTTTGGAAGCAG CTGAGGCGAAAATCGTGGAACTGGAGGAGGAACTGAAAGTAGTTGGCAACAACATGAAATCGCTGGAAATCTCAGAGCAGGAAGC TTCTCAACGTGAGGACAGCTATGAGGAAACCATTCGCGATCTTACTCATCGCCTGAAAGAGGCCGAAAATCGGGCAGCAGAAGCC GAACGCACAGTGTCCAAACTGCAGAAAGAGGTTGATCGCTTAG AGGACGAACTGCTGGCCGAAAAGGAACGCTACAAGACCATCAGTGACGAATTGGATCAGACGTTTGCGGAATTAGCGGGTTATTAATGATCGAGTAAGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTG CCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCC GGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTG CCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGA CCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCA CGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGC CGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAA ATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTT ATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATT CATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGG CAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATG GCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCG TTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGAT CGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAA TCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAA ATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTT GCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAG CCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAA CACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTT CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTT TTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACT CTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTG TTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGT CGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATG AGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTA TCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCT TTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGAT GCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCT GACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGT GACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCG TGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTC ACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTAC TGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACT CAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTC CACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGA AACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCG GTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGG GGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGT TTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGC TCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCAT AAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGA GCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGC TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAG CAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGT TTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAG CCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTC AGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGT CGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGA ACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGG AGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATG GCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGA CGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCC AGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCA ACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTT TCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGG TGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTA GGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTC CCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGT GATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCC GGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATA

[0122] SEQ ID NO:15: Nucleic acid sequence of vector pET24d-N-(PSc)-Todp MLC1 containing the sequence encoding Tod p MLC1 (light chain 1)

[0123] GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTT TGTTTAACTTTAAGAAGGAGATATACCATGAGCCATCATCATC ATCATCATCATCATTTGGAAGTGCTGTTTCAGGGTCCATCCATGTCTCAACTGACCAAAGACGAAATTGAGGAAGTCCGTGAAGTG TTTGACCTCTTCGACTTTTGGGATGGTCGTGATGGTGATGTTGACGCTGCGAAAGTTGGCGATCTGTTACGCTGTTTAGGGATGAA TCCAACCGAAGCTCAGGTACACCAACATGGAGGCACGAAGAAAATGGGCGAGAAAGCGTATAAACTGGAAGAGATTCTGCCGATT TATGAGGAAATGAGCTCCAAAGATACTGGCACAGCAGCGGAC GAATTCATGGAAGCCTTCAAAACGTTTGATCGTGAAGGTCAGGGTTTGATCAGTTCAGCCGAAATTCGGAATGTGCTGAAAATGCT TGGCGAACGCATTACCGAGGATCAGTGCAACGATATCTTCACCTTTTGCGACATTCGCGAAGATATCGATGGGAACATCAAATACG AGGATCTGATGAAGAAGGTGATGGCAGGACCTTTTCCGGACAA ATCGGATTAATGATCGAGTAAGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTT GGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAA CTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGC TACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTC CCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGT TGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGG AACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT TTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTA CAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCA TGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAA AGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCG TCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGG TGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAA CAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAAT ACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTC ACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCC CGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCT TCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTG GAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATAT GGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTC CACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAG CTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCA CCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTG CTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGC GCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGT GAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGC ACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTG ATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCG TATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAG CGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTT CACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGA CTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGA CAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCT CATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTC ATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCT GTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGAT ACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGT GAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATG TAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGAT CCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTC AGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGC ACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCG AGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCC AGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAA GACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGG TCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGT GCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCG AAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCAC CAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCA GCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATG GCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCG CCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCG TCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTC CACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCAC GCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACA ATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGC GGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACAT CGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCC ATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGA GCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGC CGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTT CCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGA GGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATA

[0124] SEQ ID NO: 16: Nucleic acid sequence of pET24d-derived vector pET24d-N-(PSc)-Tod p MLC2 containing a sequence encoding Tod p MLC2 (light chain 2)

[0125] GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTT TGTTTAACTTTAAGAAGGAGATATACCATGAGCCATCATCATCATCATCATCATCATTTGGAAGTGCTGTTTCAGGGTCCATCCAT GGCGGAAGAAGCTCCTCGTCGCGTGAAACTGTCACAACGCCAGATGCAGGAGCTGAAAGAAGCGTTTACCATGATTGATCAGGAC CGTGATGGGTTCATTGGCATGGAAGATCTGAAAGACATGTTCA GCTCTTTAGGACGGGTACCACCCGATGACGAACTGAATGCCAT GCTGAAAGAATGCCCGGGTCAACTCAATTTCACGGCTTTTCTGACCTTGTTTGGGGAGAAAGTGAGTGGTACTGACCCGGAAGAT GCACTTCGCAATGCCTTTTCGATGTTCGACGAAGATGGTCAGGGCTTTATCCCGGAAGATTATCTGAAAGACTTGCTGGAAAATAT GGGCGATAACTTTTCCAAAGAGGAGATTAAGAACGTCTGGAAA GATGCACCGTTAAAGAACAAACAGTTCAACTACAACAAGATGGTTGACATCAAAGGCAAAGCGGAGGATGAAGATTAATGATCGAG TAAGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTG AGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTT GAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTG TGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGT TCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGAT AGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATC TCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCG AATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTT TCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAA ACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAG GAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTG GTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAAT CACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTT ATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCG TGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAA GGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATT CTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTC AGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGA TTGTCGCCACCTGATTGCCCGACATATTATCGGACCCATTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGCCTA GAGCAAGACGTTTCCCGTTGAATATAGGCTCATAACACA AATCCCTTAACGTGAGTTTTCGTTCACTGAGCGTCAGACCCC GTAGAAAGATCAAAGGATCTTTCTGGATCTCTTTTTCTGCGCGTAATCTGCTGCTTGCTTGCAAACAAAAAAACCACCGCTACCAGC GGTGGTTTGTTGCCGATCAAGAGCTACCAACTCTTTTCCGAAGGTAACTGGCTTCCAGAGCGCGAGATACCAAATACTGTCC TTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTGCTCTCAATCCTGTTACCAGTG GCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTCACGGATAAGGCGCAGCGGTCGGGCTGGAACGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCT ACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCCCACGCTTCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAG CGGCAGGTCGGAAACAGGAGCGCACGAGGGAGCTTCCAGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGT TATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTG AGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTT TCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCA GTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTT GTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTC CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGA AGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCG GGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCC TCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGA ACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAA TGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCA GGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATT CTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATG CCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGG GACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAA AGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGC GACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAA TGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCG CTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCA GGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGT ATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCA ACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCG TTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGAT GCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCC GGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGT CATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTT CGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCA GGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAG CTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAA ACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTT TCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGG ATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAG CCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCC ACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCG GCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATA

[0126] SEQ ID NO: 17: A polypeptide sequence encoding Lit v 3.0101 (Litopenaeus vannamei myosin light chain 2) with a C-terminal His tag as used in the Examples

[0127] MSRKSGSRSSSKRSKKSGGGSNVFDMFTQRQVAEFKEGFQLMDR DKDGVIGKTDLRGTFDEIGRIATDQELDEMLADAPAPINFTMLLNMFAERQTGESDDDDVVAKAFLAFADEEGNIDCDTFRHALMTWG DKFSSQEADDALDQMDIDDGGKIDVQGVIQMLTAGGGDDAAAEE ALEVLFQGPHHHHHH

[0128] SEQ ID NO: 18: Epitope sequence of Tod MLC1

[0129] GGTKKMGE

[0130] SEQ ID NO: 19: Epitope sequence of Tod MLC1

[0131] SSKDTGTA

[0132] SEQ ID NO:20: Epitope sequence of Tod MLC1

[0133] DREGQ

[0134] SEQ ID NO:21: Epitope sequence of Tod MLC1

[0135] GGTKKMGEKAYKLEEILPIYEEMSSKDTGTAADEFMEAFKTFDR EGQ

[0136] SEQ ID NO:22: Epitope sequence of Tod MLC2

[0137] QGPSMA

[0138] SEQ ID NO:23: Epitope sequence of Tod MLC2

[0139] KVSGTDPE

[0140] SEQ ID NO:24: Epitope sequence of Tod MLC2

[0141] DREGQ

[0142] SEQ ID NO:25: Epitope sequence of Tod MLC2

[0143] QELKEAFTMIDQDRDGFIGMEDLKDMFSSLGRVPPDDELNAMLK ECPGQLNFT

[0144] SEQ ID NO:26: Epitope sequence of Tod TM

[0145] DESERGRKVLENRSQGDEER

[0146] SEQ ID NO:27: Epitope sequence of Tod TM

[0147] IDLLEKQLEEAKWIAEDADR

[0148] SEQ ID NO:28: Epitope sequence of Tod TM

[0149] KFDEAARKLAITEVDLERAEARLE

[0150] SEQ ID NO:29: Epitope sequence of Tod TM

[0151] KEVDRLEDELLAEKERYKTISDELDQTFAELAGY

[0152] SEQ ID NO:30: MLC2 epitope sequence 1

[0153] ALRNAFSMFD

[0154] SEQ ID NO:31: Shorter epitope sequence 1 of MLC2

[0155] NAFSMF

[0156] SEQ ID NO:32: Microarray peptide 1 for identifying MLC2 epitope sequence 1

[0157] GTDPEDALRNAFSMF

[0158] SEQ ID NO:33: Microarray peptide 2 for recognizing MLC2 epitope sequence 1

[0159] TDPEDALRNAFSMFD

[0160] SEQ ID NO:34: Microarray peptide 3 for recognizing MLC2 epitope sequence 1

[0161] DPEDALRNAFSMFDE

[0162] SEQ ID NO:35: Microarray peptide 4 for recognizing MLC2 epitope sequence 1

[0163] PEDALRNAFSMFDED

[0164] SEQ ID NO:36: Microarray peptide 5 for recognizing MLC2 epitope sequence 1

[0165] EDALRNAFSMFDEDG

[0166] SEQ ID NO:37: Microarray peptide 6 for recognizing MLC2 epitope sequence 1

[0167] DALRNAFSMFDEDGQ

[0168] SEQ ID NO:38: Microarray peptide 7 for recognizing MLC2 epitope sequence 1

[0169] ALRNAFSMFDEDGQG

[0170] SEQ ID NO:39: Microarray peptide 8 for recognizing MLC2 epitope sequence 1

[0171] LRNAFSMFDEDGQGF

[0172] SEQ ID NO:40: Microarray peptide 9 for recognizing MLC2 epitope sequence 1

[0173] RNAFSMFDEDGQGFI

[0174] SEQ ID NO:41: Microarray peptide 20 for recognizing MLC2 epitope sequence 1

[0175] NAFSMFDEDGQGFIP

[0176] SEQ ID NO:42: MLC2 epitope sequence 2

[0177] FIPEDYLKDL

[0178] SEQ ID NO:43: Shorter epitope sequence 2 of MLC2

[0179] IPEDYLKDL

[0180] SEQ ID NO:44: Microarray peptide 1 for identifying MLC2 epitope sequence 2

[0181] EDGQGFIPEDYLKDL

[0182] SEQ ID NO:45: Microarray peptide 2 for identifying MLC2 epitope sequence 2

[0183] DGQGFIPEDYLKDLL

[0184] SEQ ID NO:46: Microarray peptide 3 for recognizing MLC2 epitope sequence 2

[0185] GQGFIPEDYLKDLLE

[0186] SEQ ID NO:47: Microarray peptide 4 for recognizing MLC2 epitope sequence 2

[0187] QGFIPEDYLKDLLEN

[0188] SEQ ID NO:48: Microarray peptide 5 for recognizing MLC2 epitope sequence 2

[0189] GFIPEDYLKDLLENM

[0190] SEQ ID NO:49: Microarray peptide 6 for recognizing MLC2 epitope sequence 2

[0191] FIPEDYLKDLLENMG

[0192] SEQ ID NO:50: Microarray peptide 7 for recognizing MLC2 epitope sequence 2

[0193] IPEDYLKDLLENMGD

[0194] SEQ ID NO:51: Epitope sequence 3 of MLC2

[0195] FSKEEIKNVWKD

[0196] SEQ ID NO:52: Microarray peptide 1 for recognizing MLC2 epitope sequence 3

[0197] GDNFSKEEIKNVWKD

[0198] SEQ ID NO:53: Microarray peptide 1 for recognizing MLC2 epitope sequence 3

[0199] DNFSKEEIKNVWKDA

[0200] SEQ ID NO:54: Microarray peptide 2 for recognizing MLC2 epitope sequence 3

[0201] NFSKEEIKNVWKDAP

[0202] SEQ ID NO:55: Microarray peptide 3 for identifying MLC2 epitope sequence 3

[0203] FSKEEIKNVWKDAPL

[0204] SEQ ID NO:56: Microarray peptide NO 8:

[0205] GSGSGSGAEEAPRRV

[0206] SEQ ID NO:57: Microarray peptide NO 9:

[0207] SGSGSGAEEAPRRVK

[0208] SEQ ID NO:58: Microarray Peptide NO 10:

[0209] GSGSGAEEAPRRVKL

[0210] SEQ ID NO:59: Microarray peptide NO 11:

[0211] SGSGAEEAPRRVKLS

[0212] SEQ ID NO:60: Microarray Peptide NO 12:

[0213] GSGAEEAPRRVKLSQ

[0214] SEQ ID NO:61: Microarray peptide NO 13:

[0215] SGAEEAPRRVKLSQR

[0216] SEQ ID NO:62: Microarray peptide NO 14:

[0217] GAEEAPRRVKLSQRQ

[0218] SEQ ID NO:63: Microarray peptide NO 15:

[0219] AEEAPRRVKLSQRQM

[0220] SEQ ID NO:64: Microarray peptide NO 16:

[0221] EEAPRRVKLSQRQMQ

[0222] SEQ ID NO:65: Microarray peptide NO 17:

[0223] EAPRRVKLSQRQMQE

[0224] SEQ ID NO:66: Microarray peptide NO 18:

[0225] APRRVKLSQRQMQEL

[0226] SEQ ID NO:67: Microarray peptide NO 19:

[0227] PRRVKLSQRQMQELK

[0228] SEQ ID NO:68: Microarray peptide NO 20:

[0229] RRVKLSQRQMQELKE

[0230] SEQ ID NO:69: Microarray peptide NO 21:

[0231] RVKLSQRQMQELKEA

[0232] SEQ ID NO:70: Microarray Peptide NO 22:

[0233] VKLSQRQMQELKEAF

[0234] SEQ ID NO:71: Microarray peptide NO 23:

[0235] KLSQRQMQELKEAFT

[0236] SEQ ID NO:72: Microarray peptide NO 24:

[0237] LSQRQMQELKEAFTM

[0238] SEQ ID NO:73: Microarray peptide NO 25:

[0239] SQRQMQELKEAFTMI

[0240] SEQ ID NO:74: Microarray peptide NO 26:

[0241] QRQMQELKEAFTMID

[0242] SEQ ID NO:75: Microarray peptide NO 27:

[0243] RQMQELKEAFTMIDQ

[0244] SEQ ID NO:76: Microarray peptide NO 28:

[0245] QMQELKEAFTMIDQD

[0246] SEQ ID NO:77: Microarray peptide NO 29:

[0247] MQELKEAFTMIDQDR

[0248] SEQ ID NO:78: Microarray peptide NO 30:

[0249] QELKEAFTMIDQDRD

[0250] SEQ ID NO:79: Microarray peptide NO 31:

[0251] ELKEAFTMIDQDRDG

[0252] SEQ ID NO:80: Microarray peptide NO 32:

[0253] LKEAFTMIDQDRDGF

[0254] SEQ ID NO:81: Microarray peptide NO 33:

[0255] KEAFTMIDQDRDGFI

[0256] SEQ ID NO:82: Microarray peptide NO 34:

[0257] EAFTMIDQDRDGFIG

[0258] SEQ ID NO:83: Microarray peptide NO 35:

[0259] AFTMIDQDRDGFIGM

[0260] SEQ ID NO:84: Microarray peptide NO 36:

[0261] FTMIDQDRDGFIGME

[0262] SEQ ID NO:85: Microarray peptide NO 37:

[0263] TMIDQDRDGFIGMED

[0264] SEQ ID NO:86: Microarray peptide NO 38:

[0265] MIDQDRDGFIGMEDL

[0266] SEQ ID NO:87: Microarray peptide NO 39:

[0267] IDQDRDGFIGMEDLK

[0268] SEQ ID NO:88: Microarray peptide NO 40:

[0269] DQDRDGFIGMEDLKD

[0270] SEQ ID NO:89: Microarray Peptide NO 41:

[0271] QDRDGFIGMEDLKDM

[0272] SEQ ID NO:90: ​​Microarray Peptide NO 42:

[0273] DRDGFIGMEDLKDMF

[0274] SEQ ID NO:91: Microarray peptide NO 43:

[0275] RDGFIGMEDLKDMFS

[0276] SEQ ID NO:92: Microarray peptide NO 44:

[0277] DGFIGMEDLKDMFSS

[0278] SEQ ID NO:93: Microarray peptide NO 45:

[0279] GFIGMEDLKDMFSSL

[0280] SEQ ID NO:94: Microarray Peptide NO 46:

[0281] FIGMEDLKDMFSSLG

[0282] SEQ ID NO:95: Microarray Peptide NO 47:

[0283] IGMEDLKDMFSSLGR

[0284] SEQ ID NO:96: Microarray peptide NO 48:

[0285] GMEDLKDMFSSLGRV

[0286] SEQ ID NO:97: Microarray peptide NO 49:

[0287] MEDLKDMFSSLGRVP

[0288] SEQ ID NO:98: Microarray Peptide NO 50:

[0289] EDLKDMFSSLGRVPP

[0290] SEQ ID NO:99: Microarray peptide NO 51:

[0291] DLKDMFSSLGRVPPD

[0292] SEQ ID NO: 100: Microarray peptide NO 52:

[0293] LKDMFSSLGRVPPDD

[0294] SEQ ID NO:101: Microarray peptide NO 53:

[0295] KDMFSSLGRVPPDDE

[0296] SEQ ID NO:102: Microarray peptide NO 54:

[0297] DMFSSLGRVPPDDEL

[0298] SEQ ID NO:103: Microarray peptide NO 55:

[0299] MFSSLGRVPPDDELN

[0300] SEQ ID NO:104: Microarray peptide NO 56:

[0301] FSSLGRVPPDDELNA

[0302] SEQ ID NO:105: Microarray peptide NO 57:

[0303] SSLGRVPPDDELNAM

[0304] SEQ ID NO:106: Microarray peptide NO 58:

[0305] SLGRVPPDDELNAML

[0306] SEQ ID NO:107: Microarray peptide NO 59:

[0307] LGRVPPDDELNAMLK

[0308] SEQ ID NO:108: Microarray peptide NO 60:

[0309] GRVPPDDELNAMLKE

[0310] SEQ ID NO:109: Microarray peptide NO 61:

[0311] RVPPDDELNAMLKEC

[0312] SEQ ID NO:110: Microarray peptide NO 62:

[0313] VPPDDELNAMLKECP

[0314] SEQ ID NO:111: Microarray peptide NO 63:

[0315] PPDDELNAMLKECPG

[0316] SEQ ID NO:112: Microarray peptide NO 64:

[0317] PDDELNAMLKECPGQ

[0318] SEQ ID NO:113: Microarray peptide NO 65:

[0319] DDELNAMLKECPGQL

[0320] SEQ ID NO:114: Microarray peptide NO 66:

[0321] DELNAMLKECPGQLN

[0322] SEQ ID NO:115: Microarray peptide NO 67:

[0323] ELNAMLKECPGQLNF

[0324] SEQ ID NO:116: Microarray peptide NO 68:

[0325] LNAMLKECPGQLNFT

[0326] SEQ ID NO:117: Microarray peptide NO 69:

[0327] NAMLKECPGQLNFTA

[0328] SEQ ID NO:118: Microarray peptide NO 70:

[0329] AMLKECPGQLNFTAF

[0330] SEQ ID NO:119: Microarray peptide NO 71:

[0331] MLKECPGQLNFTAFL

[0332] SEQ ID NO:120: Microarray peptide NO 72:

[0333] LKECPGQLNFTAFLT

[0334] SEQ ID NO:121: Microarray peptide NO 73:

[0335] KECPGQLNFTAFLTL

[0336] SEQ ID NO:122: Microarray peptide NO 74:

[0337] ECPGQLNFTAFLTLF

[0338] SEQ ID NO:123: Microarray peptide NO 75:

[0339] CPGQLNFTAFLTLFG

[0340] SEQ ID NO:124: Microarray peptide NO 76:

[0341] PGQLNFTAFLTLFGE

[0342] SEQ ID NO:125: Microarray peptide NO 77:

[0343] GQLNFTAFLTLFGEK

[0344] SEQ ID NO:126: Microarray peptide NO 78:

[0345] QLNFTAFLTLFGEKV

[0346] SEQ ID NO:127: Microarray peptide NO 79:

[0347] LNFTAFLTLFGEKVS

[0348] SEQ ID NO:128: Microarray peptide NO 80:

[0349] NFTAFLTLFGEKVSG

[0350] SEQ ID NO:129: Microarray peptide NO 81:

[0351] FTAFLTLFGEKVSGT

[0352] SEQ ID NO:130: Microarray peptide NO 82:

[0353] TAFLTLFGEKVSGTD

[0354] SEQ ID NO:131: Microarray peptide NO 83:

[0355] AFLTLFGEKVSGTDP

[0356] SEQ ID NO:132: Microarray peptide NO 84:

[0357] FLTLFGEKVSGTDPE

[0358] SEQ ID NO:133: Microarray peptide NO 85:

[0359] LTLFGEKVSGTDPED

[0360] SEQ ID NO:134: Microarray peptide NO 86:

[0361] TLFGEKVSGTDPEDA

[0362] SEQ ID NO:135: Microarray peptide NO 87:

[0363] LFGEKVSGTDPEDAL

[0364] SEQ ID NO:136: Microarray peptide NO 88:

[0365] FGEKVSGTDPEDALR

[0366] SEQ ID NO:137: Microarray peptide NO 89:

[0367] GEKVSGTDPEDALRN

[0368] SEQ ID NO:138: Microarray peptide NO 90:

[0369] EKVSGTDPEDALRNA

[0370] SEQ ID NO:139: Microarray peptide NO 91:

[0371] KVSGTDPEDALRNAF

[0372] SEQ ID NO:140: Microarray peptide NO 92:

[0373] VSGTDPEDALRNAFS

[0374] SEQ ID NO:141: Microarray peptide NO 93:

[0375] SGTDPEDALRNAFSM

[0376] SEQ ID NO:142: Microarray peptide NO 94:

[0377] GTDPEDALRNAFSMF

[0378] SEQ ID NO:143: Microarray peptide NO 95:

[0379] TDPEDALRNAFSMFD

[0380] SEQ ID NO:144: Microarray peptide NO 96:

[0381] DPEDALRNAFSMFDE

[0382] SEQ ID NO:145: Microarray peptide NO 97:

[0383] PEDALRNAFSMFDED

[0384] SEQ ID NO:146: Microarray peptide NO 98:

[0385] EDALRNAFSMFDEDG

[0386] SEQ ID NO:147: Microarray peptide NO 99:

[0387] DALRNAFSMFDEDGQ

[0388] SEQ ID NO:148: Microarray Peptide NO 100:

[0389] ALRNAFSMFDEDGQG

[0390] SEQ ID NO:149: Microarray peptide NO 101:

[0391] LRNAFSMFDEDGQGF

[0392] SEQ ID NO:150: Microarray Peptide NO 102:

[0393] RNAFSMFDEDGQGFI

[0394] SEQ ID NO:151: Microarray peptide NO 103:

[0395] NAFSMFDEDGQGFIP

[0396] SEQ ID NO:152: Microarray peptide NO 104:

[0397] AFSMFDEDGQGFIPE

[0398] SEQ ID NO:153: Microarray peptide NO 105:

[0399] FSMFDEDGQGFIPED

[0400] SEQ ID NO:154: Microarray peptide NO 106:

[0401] SMFDEDGQGFIPEDY

[0402] SEQ ID NO:155: Microarray peptide NO 107:

[0403] MFDEDGQGFIPEDYL

[0404] SEQ ID NO:156: Microarray peptide NO 108:

[0405] FDEDGQGFIPEDYLK

[0406] SEQ ID NO:157: Microarray peptide NO 109:

[0407] DEDGQGFIPEDYLKD

[0408] SEQ ID NO:158: Microarray peptide NO 110:

[0409] EDGQGFIPEDYLKDL

[0410] SEQ ID NO:159: Microarray peptide NO 111:

[0411] DGQGFIPEDYLKDLL

[0412] SEQ ID NO:160: Microarray peptide NO 112:

[0413] GQGFIPEDYLKDLLE

[0414] SEQ ID NO:161: Microarray peptide NO 113:

[0415] QGFIPEDYLKDLLEN

[0416] SEQ ID NO:162: Microarray peptide NO 114:

[0417] GFIPEDYLKDLLENM

[0418] SEQ ID NO:163: Microarray peptide NO 115:

[0419] FIPEDYLKDLLENMG

[0420] SEQ ID NO:164: Microarray peptide NO 116:

[0421] IPEDYLKDLLENMGD

[0422] SEQ ID NO:165: Microarray peptide NO 117:

[0423] PEDYLKDLLENMGDN

[0424] SEQ ID NO:166: Microarray peptide NO 118:

[0425] EDYLKDLLENMGDNF

[0426] SEQ ID NO:167: Microarray peptide NO 119:

[0427] DYLKDLLENMGDNFS

[0428] SEQ ID NO:168: Microarray peptide NO 120:

[0429] YLKDLLENMGDNFSK

[0430] SEQ ID NO:169: Microarray peptide NO 121:

[0431] LKDLLENMGDNFSKE

[0432] SEQ ID NO:170: Microarray peptide NO 122:

[0433] KDLLENMGDNFSKEE

[0434] SEQ ID NO:171: Microarray peptide NO 123:

[0435] DLLENMGDNFSKEEI

[0436] SEQ ID NO:172: Microarray peptide NO 124:

[0437] LLENMGDNFSKEEIK

[0438] SEQ ID NO:173: Microarray peptide NO 125:

[0439] LENMGDNFSKEEIKN

[0440] SEQ ID NO:174: Microarray peptide NO 126:

[0441] ENMGDNFSKEEIKNV

[0442] SEQ ID NO:175: Microarray peptide NO 127:

[0443] NMGDNFSKEEIKNVW

[0444] SEQ ID NO:176: Microarray peptide NO 128:

[0445] MGDNFSKEEIKNVWK

[0446] SEQ ID NO:177: Microarray peptide NO 129:

[0447] GDNFSKEEIKNVWKD

[0448] SEQ ID NO:178: Microarray peptide NO 130:

[0449] DNFSKEEIKNVWKDA

[0450] SEQ ID NO:179: Microarray peptide NO 131:

[0451] NFSKEEIKNVWKDAP

[0452] SEQ ID NO:180: Microarray peptide NO 132:

[0453] FSKEEIKNVWKDAPL

[0454] SEQ ID NO:181: Microarray peptide NO 133:

[0455] SKEEIKNVWKDAPLK

[0456] SEQ ID NO:182: Microarray peptide NO 134:

[0457] KEEIKNVWKDAPLKN

[0458] SEQ ID NO:183: Microarray peptide NO 135:

[0459] EEIKNVWKDAPLKNK

[0460] SEQ ID NO:184: Microarray Peptide NO 136:

[0461] EIKNVWKDAPLKNKQ

[0462] SEQ ID NO:185: Microarray peptide NO 137:

[0463] IKNVWKDAPLKNKQF

[0464] SEQ ID NO:186: Microarray peptide NO 138:

[0465] KNVWKDAPLKNKQFN

[0466] SEQ ID NO:187: Microarray peptide NO 139:

[0467] NVWKDAPLKNKQFNY

[0468] SEQ ID NO:188: Microarray Peptide NO 140:

[0469] VWKDAPLKNKQFNYN

[0470] SEQ ID NO:189: Microarray peptide NO 141:

[0471] WKDAPLKNKQFNYNK

[0472] SEQ ID NO:190: Microarray Peptide NO 142:

[0473] KDAPLKNKQFNYNKM

[0474] SEQ ID NO:191: Microarray peptide NO 143:

[0475] DAPLKNKQFNYNKMV

[0476] SEQ ID NO:192: Microarray peptide NO 144:

[0477] APLKNKQFNYNKMVD

[0478] SEQ ID NO:193: Microarray peptide NO 145:

[0479] PLKNKQFNYNKMVDI

[0480] SEQ ID NO:194: Microarray Peptide NO 146:

[0481] LKNKQFNYNKMVDIK

[0482] SEQ ID NO:195: Microarray peptide NO 147:

[0483] KNKQFNYNKMVDIKG

[0484] SEQ ID NO:196: Microarray peptide NO 148:

[0485] NKQFNYNKMVDIKGK

[0486] SEQ ID NO:197: Microarray peptide NO 149:

[0487] KQFNYNKMVDIKGKA

[0488] SEQ ID NO:198: Microarray Peptide NO 150:

[0489] QFNYNKMVDIKGKAE

[0490] SEQ ID NO:199: Microarray peptide NO 151:

[0491] FNYNKMVDIKGKAED

[0492] SEQ ID NO:200: Microarray peptide NO 152:

[0493] NYNKMVDIKGKAEDE

[0494] SEQ ID NO:201: Microarray peptide NO 153:

[0495] YNKMVDIKGKAEDED

[0496] SEQ ID NO:202: Microarray peptide NO 154:

[0497] NKMVDIKGKAEDEDG

[0498] SEQ ID NO:203: Microarray peptide NO 155:

[0499] KMVDIKGKAEDEDGS

[0500] SEQ ID NO:204: Microarray Peptide NO 156:

[0501] MVDIKGKAEDEDGSG

[0502] SEQ ID NO:205: Microarray peptide NO 157:

[0503] VDIKGKAEDEDGSGS

[0504] SEQ ID NO:206: Microarray peptide NO 158:

[0505] DIKGKAEDEDGSGSG

[0506] SEQ ID NO:207: Microarray peptide NO 159:

[0507] IKGKAEDEDGSGSGS

[0508] SEQ ID NO:208: Microarray peptide NO 160:

[0509] KGKAEDEDGSGSGSG

[0510] 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.

[0511] Example 1: Evaluation of the diagnostic reliability of Tod p MLC1 and Tod p MLC2

[0512] Cloning, recombinant expression and purification of Tod p MLC1 and Tod p MLC2

[0513] DNA sequences encoding the proteins shown above fused to linker and His-tag sequences were generated by gene synthesis (Eurofins Genomics GmbH), amplified by polymerase chain reaction and cloned using pBAD / ThioFusion TM The resulting SEQ ID NO: 12 (containing an insert encoding shrimp MLC, Lit v 3.0101), SEQ ID NO: 13 (containing an insert encoding shrimp TM, Pen a 1.0101), SEQ ID NO: 14 (containing an insert encoding squid TM, Sep l1.0101), SEQ ID NO: 15 (containing an insert encoding squid MLC1) and SEQ ID NO: 16 (containing an insert encoding squid MLC2) were used. The fusion proteins were expressed in E. coli as described by the manufacturer and separated by affinity chromatography (IMAC) on a nickel matrix. Preparation of multiparameter linear blots (EUROLINE)

[0514] EUROLINE linear blots were generated by coating the isolated individual fusion proteins SEQ ID NO: 3 (His-tagged shrimp TM Pen a 1.0101), SEQ ID NO: 5 (Sep I 1.0101, His-tagged squid TM), SEQ ID NO: 17 (Lit v 3.0101, His-tagged shrimp MLC2), SEQ ID NO: 9 (His-tagged squid MLC1) and SEQ ID NO: 11 (His-tagged squid MLC2) onto nitrocellulose membranes (commercially available from Whatman) using a precision dispenser (commercially available from Zeta Corporation). After blocking and washing as recommended in the manufacturer's instructions, the membranes containing the individual proteins were aligned and cut into 3 mm strips to generate a multiparameter test. The final layout is as follows Figure 1 shown.

[0515] Evaluation of samples using the EUROLINE linearity assay

[0516] EUROLINE strips were incubated as recommended in the instructions of the manufacturer (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany) (Product No.: DP 3110-1601E). In detail, the strips were incubated with 1 ml of 1 / 11 (v / v) diluted patient samples (25 squid-allergic and 42 squid-tolerant patients) in wash buffer at room temperature for 16 h. After discarding the patient samples, the strips were washed 3 times for 5 min each with 1 ml of wash buffer per strip and subsequently incubated with 1 ml of anti-human IgE antibody conjugated with AP for 1 h. Afterwards, they were washed again 5 times with wash buffer and then incubated with BCIP / NBT substrate for 10 min.

[0517] Use a scanner and The results were evaluated using the ELISA program (EUROIMMUN AG, Lübeck, Germany). Semiquantitative analysis was possible by using an IgE calibration curve based on the WHO standard (75 / 502).

[0518] Statistical analysis

[0519] Assay performance statistics were calculated based on the patient's food challenge results and were defined as follows: TP: percentage of true-positive test results; TN: percentage of true-negative test results; FP: percentage of false-positive test results; FN: percentage of false-negative test results.

[0520] result

[0521] EUROLINE multiparametric strips containing various shrimp and squid proteins were incubated with samples from 25 squid-allergic and 42 squid-tolerant children to identify whether it is possible to predict squid allergy based on sensitization to a single allergen component.

[0522] The assay performance statistics are shown in Table 1. Both squid TM and shrimp TM showed high sensitivity and moderate specificity, yielding PPVs of 57% and 59%, respectively. As TM is widely accepted as an arthropod pan-allergen with high cross-reactivity, this result is not surprising and validates the assay performed.

[0523] In contrast, squid MLC2 showed higher sensitivity than shrimp MLC2 (44% vs. 16%) with similar specificity (86% vs. 95%), yielding PPV (65% vs. 67%) and NPV (72% vs. 66%) values. Overall, squid MLC2 showed the highest efficacy of all proteins used in the test.

[0524] Although the sensitivity of MLC1 was limited (8%), its specificity (100%) was outstanding.

[0525] In conclusion, the presented results clearly demonstrate that the claimed squid MLC2 provides a diagnostic benefit for predicting squid allergy compared to all currently available squid allergy testing systems.

[0526]

[0527] Table 1: Assay performance statistics for individual proteins. TM: tropomyosin; MLC: myosin light chain; PPV: positive predictive value; NPV: negative predictive value.

[0528] Example 2: Comparative epitope mapping of Tod pMLC2_V2

[0529] According to the description of Ehlers et al. (Ehlers, AM, Klinge, M., Suer, W., Weimann, Y., Knulst, AC, Besa, F., Le, TM, Otten, HG (2019) Ara h 7 isoforms share many linear epitopes: Are 3D epitopes crucial to elucidate divergent abilities?, Clin Exp Allergy 49 (11), 1512-1519), a peptide microarray chip containing overlapping peptides of the fifteen amino acid sequences represented by SEQ ID NO: 10 was prepared and incubated, the deviation was selected as one, and the measurement was performed in triplicate. The peptides used included SEQ ID NO: 56 to SEQ ID NO: 208. Each peptide contained 15 amino acids and the deviation was 1. The measurement was performed in triplicate.

[0530] The patient cohort included eleven sera from patients allergic to squid and eight samples from sensitized but tolerant patients and two sera from healthy donors.

[0531] Results were evaluated as described by Ehlers et al., with the following exceptions: the cutoff value was increased to 3 to increase the significance of the results. The epitope was considered validated if four subsequent peptides were identified, corresponding to an epitope length of at least four amino acids for the described anti-his tag monoclonal antibody 3D5 (Lindner, P., Bauer, K., Krebber, A., Nieba, L., Kremmer, E., Krebber, C., Honegger, A., Klinger, B., Mocikat, R., Plückthun, A. (1997) Specific detection of his-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions, Biotechniques 22(1), 140-9).

[0532] Figure 2 An example of a peptide chip incubated as described in this example is shown. The chip includes eight panels, with the green regular panel representing the control peptide. IgE reactions are shown in green and IgG4 reactions are shown in red.

[0533] Figure 3 Shown is the detection of antibody reactivity in sera of four allergic and two tolerant patients against peptides based on SEQ ID NO: 10 as discussed in Example 2. The Y-axis shows the C-score and the X-axis shows the separated peptides. The black line shows the cutoff value, the green bars represent IGE reactivity, and the red bars represent IgG4 reactivity.

[0534] Figure 4 Heatmaps of IgE and IgG4 serum reactivity to overlapping peptides based on SEQ ID NO: 10 are shown.

[0535] The first column describes the nomenclature of the patient's serum. The left column represents the peptides used. Reactions with a set score of at least three were considered positive. An epitope was considered valid if at least three subsequent peptides were recognized. Green signals show IgG reactivity with the corresponding peptide. Red signals show IgG4 reactivity. The stronger the color, the higher the signal.

[0536] result

[0537] A clear difference in reactivity between sera from allergic patients and sera from tolerant patients can be shown.

[0538] Although two of nine sera from tolerant patients reacted with the peptide represented by the n-terminal region of SEQ ID NO: 10, specific reactivity of allergic patients was demonstrated to peptides containing the following sequences: ALRNAFSMFD (SEQ ID NO: 30), NAFSMF (SEQ ID NO: 31), FIPEDYLKDL (SEQ ID NO: 42), IPEDYLKDL (SEQ ID NO: 43) and FSKEEIKNVWKD (SEQ ID NO: 51).

[0539] In general, it can be concluded that the epitopes comprising the sequences SEQ ID NO: 31, SEQ ID NO: 43 and SEQ ID NO: 51 and peptides or proteins comprising them are specifically recognized by antibodies from allergic patients. These epitopes are not recognized by antibodies from tolerant patients and can be used to design antigens that bind to them or antibodies that bind to them or do not bind to them as reagents for immunoassays.

Claims

1. A kit comprising a diagnostically useful carrier and means for detecting any specifically captured antibody, The carrier comprises means for specifically capturing antibodies against a polypeptide in a sample from a subject, the polypeptide being selected from squid MLC1 according to SEQ ID NO: 8 or squid MLC2 according to SEQ ID NO: 10, wherein the means for specifically capturing the antibody is a polypeptide comprising SEQ ID NO: 8 or a polypeptide comprising SEQ ID NO: 10 or a variant thereof, Wherein the means for detecting any specific captured antibody is a secondary antibody that binds one or more different human antibody classes, wherein the variant comprises an epitope capable of binding an antibody against squid MLC2 in a sample from a patient suffering from squid allergy, wherein the epitope is selected from the group comprising SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:

51.

2. A kit comprising a purified polypeptide comprising an antigen selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10 and variants thereof, wherein the polypeptide optionally comprises a detectable label and a secondary antibody that binds to one or more different human antibody classes, wherein the variant comprises an epitope capable of binding an antibody against squid MLC2 in a sample from a patient suffering from squid allergy, wherein the epitope is selected from the group comprising SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:

51.

3. The kit according to claim 1, wherein the carrier is selected from the group consisting of beads, test strips, microtiter plates, glass surfaces, biochips and membranes.

4. The kit according to claim 1, wherein the carrier is selected from the group consisting of a microarray and a slide.

5. The kit according to any one of claims 1, 3 or 4, wherein the carrier further comprises means for capturing antibodies against squid TM.

6. Use of a diagnostically useful vector or a polypeptide comprising SEQ ID NO: 8 or SEQ ID NO: 10 or a variant thereof in the manufacture of a diagnostic kit, wherein the diagnostically useful vector contains a polypeptide comprising SEQ ID NO: 8 or a polypeptide comprising SEQ ID NO: 10 or a variant thereof, wherein the kit further comprises means for detecting IgE antibodies, The tool for detecting IgE antibodies is a secondary antibody that binds to human IgE antibodies. wherein the variant comprises an epitope capable of binding an antibody against squid MLC2 in a sample from a patient suffering from squid allergy, wherein the epitope is selected from the group comprising SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:

51.

7. The use according to claim 6, wherein the means for detecting IgE antibodies is a labeled secondary antibody.

8. Use according to claim 6 or 7, wherein the kit comprises a calibrator.

9. The use according to claim 6 or 7, wherein the polypeptide is isolated or immobilized.

10. The use according to claim 6 or 7, wherein the kit or polypeptide is used for diagnosis of squid allergy.

11. Use according to claim 10, wherein the diagnosis is a differential diagnosis of shellfish allergy, wherein shrimp allergy and squid allergy are distinguished.

12. An in vitro method comprising the following steps: Contacting a diagnostically useful carrier as defined in any one of claims 1 and 3 to 4 with a solution comprising a known concentration of antibodies against a polypeptide, said solution not being a sample from a patient, and detecting antibodies against a squid allergen selected from antibodies against squid MLC1 and antibodies against squid MLC2, wherein the polypeptide is selected from squid MLC1 and squid MLC2 or variants thereof.

13. An in vitro method comprising the following steps: Contacting a diagnostically useful carrier as defined in claim 5 with a solution comprising known concentrations of antibodies against polypeptides, said solution not being a sample from a patient, and detecting antibodies against squid allergens selected from antibodies against squid MLC1, antibodies against squid MLC2 and antibodies against squid TM, wherein the polypeptides are selected from squid MLC1 and squid MLC2 or variants thereof and squid TM.

14. The method of claim 12 or 13, wherein the antibody is an IgE antibody.

15. The method of claim 12 or 13, wherein the method is performed simultaneously with, after or before a diagnostic method.

16. The method of claim 12 or 13, wherein the method is used to confirm the reliability of an antibody detection assay.

17. A device for analyzing a sample from a patient to detect one or more antibodies indicative of an increased likelihood of allergy, wherein the antibodies are selected from antibodies against squid MLC1 according to SEQ ID NO: 8 and antibodies against squid MLC2 according to SEQ ID NO: 10, the device comprising: A carrier comprising means for capturing at least one antibody from a sample when the sample is contacted with the carrier, wherein the means is a polypeptide comprising SEQ ID NO: 8 or a polypeptide comprising SEQ ID NO: 10 or a variant thereof, wherein the variant comprises an epitope of an antibody against squid MLC2 capable of binding to a sample from a patient suffering from squid allergy, wherein the epitope is selected from the group comprising SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 51, a detectable means, which, when contacted with the support, is capable of binding to the antibody captured by the support, wherein the detectable means is a secondary antibody capable of binding to one or more different human antibody classes, means for removing any sample from the carrier and the detectable means, A detection device is used to detect the presence of a detectable tool and convert the result into an electrical signal.

18. The device of claim 17, wherein the detectable means is a labeled secondary antibody capable of binding to the antibody captured on the support.

19. The apparatus according to claim 17 or 18, wherein the apparatus further comprises means for receiving the electronic signal from the detection device and determining whether the level of the signal indicates an increased likelihood of squid allergy by comparison with the signal level detected in the background or an input reference value obtained from a sample of a healthy subject.

20. The apparatus of claim 17 or 18, wherein the apparatus further comprises means for receiving an electronic signal from the detection device and determining whether the level of the signal indicates a higher likelihood of squid allergy than shrimp allergy by comparison with the level of the signal detected in the background or an input reference value obtained from a healthy subject sample.

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