Anti-wheat allergen IgE monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-wheat allergen IgE monoclonal antibody as well as a preparation method and application thereof. The human F4 IgE monoclonal antibody with strong reactivity is obtained by screening with a phage screening technology by adopting an F4 natural antigen, the monoclonal antibody can specifically recognize a wheat F4 antigen and can generate a remarkable enzyme-linked immunosorbent assay reaction with the antigen, and the accelerated stability at 37 DEG C meets the use requirement of the kit; as a quality control product, the monoclonal antibody can play an important role in wheat allergen F4 item detection, and has a wide market application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to anti-wheat allergen IgE monoclonal antibodies, their preparation methods and applications. Background Art

[0002] Food allergy or Food hypersensitivity is a general term for a series of symptoms caused by the body's adverse immune response to food proteins, which can be mediated by IgE or non-IgE, and the IgE-mediated immediate allergic reaction accounts for the vast majority. It is reported that more than 170 kinds of foods can cause IgE-mediated food allergic reactions at present.

[0003] Wheat is one of the important foods that cause IgE-mediated allergic reactions in children. The IgE-mediated allergic reaction to ingested wheat proteins includes gastrointestinal, respiratory and skin symptoms. The reaction typically occurs within one hour after ingesting wheat. Sensitized patients are allergic to wheat in their infancy, but many of them are tolerant to wheat in adulthood. In the preparation of in vitro diagnostic kits, positive plasma is required as a quality control product. However, it is difficult to collect and obtain high-concentration wheat IgE samples; most of the magnetic particle wheat allergen-specific IgE antibody detection kits on the market use the indirect method for evaluation, that is, coating antigens and using anti-human IgE antibody as the secondary antibody to detect IgE antibodies. In view of this, it is necessary to research and develop a wheat F4 IgE human monoclonal antibody. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide anti-wheat allergen IgE monoclonal antibodies, their preparation methods and applications.

[0005] The present invention provides a monoclonal antibody against wheat F4 allergen,

[0006] The amino acid sequence of CDR1 in its heavy chain variable region is as shown in SEQ ID NO:1, SEQ ID NO:9 and / or SEQ ID NO:17;

[0007] The amino acid sequence of CDR2 in the heavy chain variable region is as shown in SEQ ID NO:2, SEQ ID NO:10 and / or SEQ ID NO:18;

[0008] The amino acid sequence of CDR3 in the heavy chain variable region is as shown in SEQ ID NO:3, SEQ ID NO:11 and / or SEQ ID NO:19;

[0009] The amino acid sequence of CDR1 of its light chain variable region is as shown in SEQ ID NO:4, SEQ ID NO:12, and / or SEQ ID NO:20;

[0010] The amino acid sequence of CDR2 of the light chain variable region is as shown in SEQ ID NO:5, SEQ ID NO:13, and / or SEQ ID NO:21;

[0011] The amino acid sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO:6, SEQ ID NO:14, and / or SEQ ID NO:22.

[0012] Furthermore, for the monoclonal antibody of the present invention,

[0013] The heavy chain variable region includes CDR1 with the amino acid sequence as shown in SEQ ID NO:1, CDR2 with the amino acid sequence as shown in SEQ ID NO:2, and CDR3 with the amino acid sequence as shown in SEQ ID NO:3 (antibody 1H7); and / or

[0014] The heavy chain variable region includes CDR1 with the amino acid sequence as shown in SEQ ID NO:9, CDR2 with the amino acid sequence as shown in SEQ ID NO:10, and CDR3 with the amino acid sequence as shown in SEQ ID NO:11 (antibody 6A8); and / or

[0015] The heavy chain variable region includes CDR1 with the amino acid sequence as shown in SEQ ID NO:17, CDR2 with the amino acid sequence as shown in SEQ ID NO:18, and CDR3 with the amino acid sequence as shown in SEQ ID NO:19 (antibody 3B6);

[0016] The light chain variable region includes CDR1 with the amino acid sequence as shown in SEQ ID NO:4, CDR2 with the amino acid sequence as shown in SEQ ID NO:5, and CDR3 with the amino acid sequence as shown in SEQ ID NO:6 (antibody 1H7); and / or

[0017] The light chain variable region includes CDR1 with the amino acid sequence as shown in SEQ ID NO:12, CDR2 with the amino acid sequence as shown in SEQ ID NO:13, and CDR3 with the amino acid sequence as shown in SEQ ID NO:14 (antibody 6A8); and / or

[0018] The light chain variable region includes CDR1 with the amino acid sequence as shown in SEQ ID NO:20, CDR2 with the amino acid sequence as shown in SEQ ID NO:21, and CDR3 with the amino acid sequence as shown in SEQ ID NO:22 (antibody 3B6).

[0019] Furthermore, for the monoclonal antibody of the present invention,

[0020] the amino acid sequence of the heavy chain variable region is at least one or more of those shown in SEQ ID NO:7, SEQ ID NO:15, and / or SEQ ID NO:23;

[0021] the amino acid sequence of the light chain variable region is at least one or more of those shown in SEQ ID NO:8, SEQ ID NO:16, and / or SEQ ID NO:24.

[0022] In a specific embodiment of the present invention,

[0023] the amino acid sequence of the heavy chain variable region of monoclonal antibody 1H7 is as shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8;

[0024] the amino acid sequence of the heavy chain variable region of monoclonal antibody 6A8 is as shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:16;

[0025] the amino acid sequence of the heavy chain variable region of monoclonal antibody 3B6 is as shown in SEQ ID NO:23, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:24;

[0026] In the present invention, for the monoclonal antibody described, its constant region is of the IgE subtype;

[0027] The antibody of the present invention can be derived from humans, rats, mice, rabbits, cows, sheep, etc., and the present invention does not make any limitation in this regard; in a specific embodiment of the present invention, the antibody is derived from humans, and the constant region is of the human IgE subtype.

[0028] In the present invention, using the F4 natural antigen, 190 positive phage clones were obtained after screening and panning on a human antibody library. Secondly, through phage monoclonal Elisa detection, phages containing highly reactive monoclonal antibodies 1H7, 3B6, and 6A8 were obtained; through the construction and expression of complete monoclonal antibodies, it was found that: 1H7 and 6A8 IgE antibodies have obvious reactivity, and 3B6 IgE has weak reactivity; and the expression levels of 1H7 and 6A8 full-length IgE antibodies after purification are 11.2 mg / L and 15.4 mg / L respectively; after the titer detection of the purified antibodies, the titers of 1H7 and 6A8 IgE antibodies are 365210 IU / mL and 234667 IU / mL respectively, with strong reactivity, both meeting the reactivity requirements for quality control products.

[0029] The present invention provides nucleic acids, which include nucleic acids encoding the monoclonal antibodies described in the present invention.

[0030] In the present invention,

[0031] The nucleotide sequence of the nucleic acid encoding the heavy chain variable region of 1H7 is as shown in SEQ ID NO:25;

[0032] The nucleotide sequence of the nucleic acid encoding the light chain variable region of 1H7 is as shown in SEQ ID NO:26;

[0033] The nucleotide sequence of the nucleic acid encoding the heavy chain variable region of 3B6 is as shown in SEQ ID NO:27;

[0034] The nucleotide sequence of the nucleic acid encoding the light chain variable region of 3B6 is as shown in SEQ ID NO:28;

[0035] The nucleotide sequence of the nucleic acid encoding the heavy chain variable region of 6A8 is as shown in SEQ ID NO:29;

[0036] The nucleotide sequence of the nucleic acid encoding the light chain variable region of 6A8 is as shown in SEQ ID NO:30.

[0037] The nucleic acids described in the present invention can be DNA, RNA, cDNA or PNA. In the embodiments of the present invention, the nucleic acids are in the form of DNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be linear or circular in topology. The nucleic acid can be a part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be directly obtained from natural sources, or can be prepared with the assistance of recombinant, enzymatic or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0038] Furthermore, the nucleic acids described in the present invention can be optimized or unoptimized. The optimization includes, but is not limited to: codon usage bias, eliminating secondary structures (such as hairpin structures) that are unfavorable for expression, changing GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA instability regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0039] The present invention provides recombinant vectors, which contain the nucleic acids described in the present invention.

[0040] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences or elements essential for the expression of the operably linked coding gene in a specific host organism. In this specification, "plasmid" and "vector" can sometimes be used interchangeably, because plasmids are the most commonly used form of vectors at present. However, the present invention is intended to include such other forms of expression vectors that play an equivalent role and are known or will become known in the art, including but not limited to: plasmids, bacteriophage particles, viral vectors, and / or merely potential genomic inserts.

[0041] The present invention provides a host cell transfected or transformed with the recombinant vector of the present invention, or its genome integrated with the nucleic acid as described in the present invention.

[0042] Furthermore, the transformation methods include: chemical transformation and electrotransformation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0043] The host cells provided by the present invention can be derived from plants, animals, bacteria, fungi, bacteriophages or viruses, and the present invention does not limit this. The present invention uses a vector constructed by recombinant DNA technology to transform or transfect host cells, and such transformed host cells are capable of replicating the vector encoding the protein or expressing the desired protein, or playing the corresponding role.

[0044] The present invention provides a method for preparing the antibody, which is to culture the host cell as described in the present invention to obtain a mixture containing the antibody as described in the present invention.

[0045] The present invention provides a solid object labeled, fixed, linked or attached, adhered with the antibody as described in the present invention;

[0046] The antibody described in the present application can be labeled, fixed, linked or attached, adhered to a solid object, and the solid object can be a solid phase carrier such as a microtiter plate, magnetic beads, nanomaterials, etc.; the linking can be direct or indirect coupling, such as linking to the solid object by covalent coupling, and the present invention does not limit this.

[0047] The present invention provides the antibody as described in the present invention labeled with a biological label or chemical label; in the present invention, the biological label or chemical label includes but is not limited to enzymes, biotin, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, and / or magnetic beads, etc.;

[0048] The present invention provides a reagent containing a monoclonal antibody, which includes at least one of the monoclonal antibody as described in the present invention or the monoclonal antibody obtained by culturing the host cell as described in the present invention and excipients;

[0049] Furthermore, the excipients include those for preserving the monoclonal antibody, or maintaining the activity of the monoclonal antibody, or assisting the monoclonal antibody to exert its function; specifically, the excipients include buffer solutions, antioxidants, stabilizers, and / or preservatives, etc.

[0050] The present invention provides a kit for wheat allergen immunoassay, which is characterized by comprising reagents for wheat allergen immunoassay and at least one of the following a) - c):

[0051] a) The monoclonal antibody of the present invention;

[0052] b) The monoclonal antibody obtained by culturing the host cell of the present invention;

[0053] c) The reagent of the present invention.

[0054] Wheat flour F4, as a food allergen that may cause allergies, can determine whether the body has an allergic reaction to it by detecting the content of allergen-specific IgE antibodies; the monoclonal antibody of the present invention can be used as a quality control product in the process of allergen-specific IgE antibodies; in addition, as an antibody, it can specifically bind to the F4 antigen and be used for the detection of antigens.

[0055] The present invention provides the application of at least one of the following I) - VII) in wheat allergen immunoassay:

[0056] I) The monoclonal antibody of the present invention;

[0057] II) The nucleic acid of the present invention;

[0058] III) The recombinant vector of the present invention;

[0059] IV) The host cell of the present invention;

[0060] V) The monoclonal antibody obtained by culturing the host cell of the present invention;

[0061] VI) The reagent of the present invention;

[0062] VII) The kit of the present invention.

[0063] The present invention provides a method for wheat allergen immunoassay, which is characterized by comprising detecting by using at least one of the following A) - G):

[0064] A) The monoclonal antibody of the present invention;

[0065] B) The nucleic acid of the present invention;

[0066] C), the recombinant vector of the present invention;

[0067] D), the host cell of the present invention;

[0068] E), the monoclonal antibody obtained by culturing the host cell of the present invention;

[0069] F), the reagent of the present invention;

[0070] G), the kit of the present invention.

[0071] The present invention uses the F4 natural antigen and, by means of phage screening technology, screens and obtains a human-derived F4 IgE monoclonal antibody with strong reactivity. The monoclonal antibody can specifically recognize the wheat F4 antigen, can have a significant enzyme-linked immunosorbent reaction with the antigen, and the accelerated stability at 37°C meets the usage requirements of the kit. As a quality control product, it can play an important role in the detection of the wheat allergen F4 project and has broad market application prospects. Description of the Drawings

[0072] Figure 1 The SDS-PAGE diagram is shown, where 1 is the reduced band of 1H7 (treated with the reducing agent DTT); 2 is the purified flow-through sample of 1H7; 3 is the non-reduced band of 1H7; 4 is the non-reduced band of 6A8; 5 is the purified flow-through sample of 6A8; 6 is the reduced band of 6A8 (treated with the reducing agent DTT). Detailed Embodiments

[0073] The present invention provides an anti-wheat allergen IgE monoclonal antibody, its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0074] The term "comprising", "having" or "containing", including the use of its grammatical synonyms, should generally be understood as open and non-restrictive, for example, it does not exclude other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0075] At present, there are few studies on wheat allergenic components in China. However, wheat allergy is relatively common in Europe. In Germany, wheat dimeric α-amylase inhibitor (Tri a28) and wheat tetrameric α-amylase inhibitor (Tri a29) are the main allergenic components. Among patients with baker's asthma in Spain, the positive rate of α-amylase inhibitor subunit (WTAI-CM16, TriaCM16) is the highest.

[0076] Immunoglobulin E (IgE) antibodies are only present in mammals. IgE is synthesized by plasma cells. The IgE monomer consists of two heavy chains (ε chains) and two light chains. The ε chain contains 4 Ig-like constant regions (Cε1-Cε4). The molecular weight of the IgE antibody is 160 kD. Its important feature is that it is a cytophilic antibody. Its CH2 and CH3 domains can bind to the high-affinity FcεR I on mast cells and basophils. When it binds to the antigen that enters the body again, it can cause type I hypersensitivity reactions. In addition, IgE antibodies may be related to the body's anti-parasitic immunity.

[0077] Humanized anti-IgE monoclonal antibodies are currently used in the treatment of allergic asthma. After treatment, the symptoms are significantly improved, and the feasibility of treating food allergies has been continuously explored. Omalizumab monoclonal antibody has been approved in the United States, Australia and other countries for the treatment of poorly controlled persistent allergic asthma and food allergic diseases. Its binding site to IgE is the high-affinity binding site FcεRI of the IgE receptor, which exists on the membranes of mast cells and basophils. When the allergen enters the body and binds to IgE and mast cells, it activates the mast cells and causes degranulation, resulting in type I allergic reactions. When the Omalizumab monoclonal antibody binds to the FcεRI site of IgE, IgE can no longer bind to effector cells, thereby blocking type I allergic reactions and achieving the effect of treating food allergies. Based on this, in this study, this antibody was tried to be used as a purified affinity medium to explore the purification conditions to achieve the purpose of purifying IgE antibodies.

[0078] Allergen detection is crucial for accurate diagnosis, treatment planning and avoidance strategies. The IgE monoclonal antibody developed in this study against F4 allergen has strong reactivity and meets the requirements for the reactivity of quality control products.

[0079] CDR1 of the heavy chain variable region of 1H7: GLTFSSL (SEQ ID NO:1);

[0080] CDR2 of the heavy chain variable region of 1H7: GTGGD (SEQ ID NO:2);

[0081] CDR3 of the heavy chain variable region of 1H7: ELADSTSWGWHLDL (SEQ ID NO:3);

[0082] CDR1 of the variable region of the 1H7 light chain: RASQDISSALA (SEQ ID NO:4);

[0083] CDR2 of the variable region of the 1H7 light chain: DGSTLES (SEQ ID NO:5);

[0084] CDR3 of the variable region of the 1H7 light chain: QHFNRNPPGVT (SEQ ID NO:6);

[0085] Amino acid sequence of the variable region of the 1H7 heavy chain: EVQLVESGGGLAQPGGSLRLSCAVSGLTFSSLDMHWVRQATGKGLEWVSAIGTGGDTYYAGSVKGRFTISRENAKNSLYLQMNDLRAGDTAVYYCARELADSTSWGWHLDLWGRGTLVTVSS (SEQ ID NO:7);

[0086] Amino acid sequence of the variable region of the 1H7 light chain: NIQMTQSPSSLSASVGDRVAITCRASQDISSALAWYQQKPGKPPELLIFDGSTLESGVPARFSGSGSGTDFTLIISSLRPEDFATYYCQHFNRNPPGVTFGPGTKLDIK (SEQ ID NO:8);

[0087] CDR1 of the variable region of the 6A8 heavy chain: GGSISSSSY (SEQ ID NO:9);

[0088] CDR2 of the variable region of the 6A8 heavy chain: YYTGT (SEQ ID NO:10);

[0089] CDR3 of the variable region of the 6A8 heavy chain: GTGSYWKFGYYFDS (SEQ ID NO:11);

[0090] CDR1 of the variable region of the 6A8 light chain: RASQSVSRSYLA (SEQ ID NO:12);

[0091] CDR2 of the variable region of the 6A8 light chain: GASSRAT (SEQ ID NO:13);

[0092] CDR3 of the variable region of the 6A8 light chain: QQYGSSPLT (SEQ ID NO:14);

[0093] Amino acid sequence of the heavy chain variable region of 6A8: QVQLQESGPGLVKPSETLSLTCSVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYTGTTYYNPPLKSRLTISVDTSKKQFSLRLTSVTAADTAVYYCVRGTGSYWKFGYYFDSWGQGTLVTVSS (SEQ ID NO:15);

[0094] Amino acid sequence of the light chain variable region of 6A8: EIVMTQSPDTLSLSPGERATLPCRASQSVSRSYLAWYQQKPGQAPRLLFYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVDIK (SEQ ID NO:16);

[0095] CDR1 of the heavy chain variable region of 3B6: GYTFTNY (SEQ ID NO:17);

[0096] CDR2 of the heavy chain variable region of 3B6: YPGDSD (SEQ ID NO:18);

[0097] CDR3 of the heavy chain variable region of 3B6: HTGGSYST (SEQ ID NO:19);

[0098] CDR1 of the light chain variable region of 3B6: RSSQSLVYGDGNTYLS (SEQ ID NO:20);

[0099] CDR2 of the light chain variable region of 3B6: KVSNWDS (SEQ ID NO:21);

[0100] CDR3 of the light chain variable region of 3B6: MQSTLWPPT (SEQ ID NO:22);

[0101] Amino acid sequence of the heavy chain variable region of 3B6: EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIGWVRQMPGKGLEWMGFIYPGDSDTKYNPSFEGQVTISADTSINTAYLQWSSLRASDTAMYYCARHTGGSYSTWGRETLVTVSS (SEQID NO:23);

[0102] Amino acid sequence of the variable region of the 3B6 light chain: DIVMTQSPLSLPVTLGQPASISCRSSQSLVYGDGNTYLSWFQQRPGQSPRRLIYKVSNWDSGVPDRFSGSGSGTYFTLKISRVEAEDVGIYYCMQSTLWPPTFGLGTRLEIK (SEQ ID NO:24);

[0103] Nucleotide sequence of the variable region of the 1H7 heavy chain: gaagtgcagctggtggagtctgggggaggcttggcacagcctggggggtccctgagactctcctgtgcagtctctggactaaccttcagtagcctcgacatgcactgggtccgccaagctacaggaaaaggtctggagtgggtctcagctattggtactggcggtgacacatactatgcaggctccgtgaagggccgattcaccatctccagagaaaacgccaagaactccttgtatcttcaaatgaacgacctgagagccggggacacggctgtgtattactgtgcaagagagttggccgatagcaccagctggggctggcacctcgatctgtggggccgtggcaccctggtcaccgtctcctca (SEQ ID NO:25);

[0104] Nucleotide sequence of the variable region of the 1H7 light chain: aacatccagatgacccagtctccatcctccctgtctgcatctgtgggagacagagtcgccatcacttgccgggcaagtcaggacattagcagtgctttggcctggtatcagcagaaaccagggaaacctcctgagctcctgatctttgatggctccactttggaaagtggggtcccagcaaggttcagcggcagtggatctgggacagacttcactctcatcatcagcagcctgcggcctgaagactttgcaacttattactgtcaacactttaatcgtaaccctccaggagtcactttcggccctgggaccaaactggatatcaaa (SEQ ID NO:26);

[0105] 3B6 heavy chain variable region nucleotide sequence: gaggtgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctcctgtaagggttctggatacacctttaccaactactggatcggctgggtgcgccagatgcccgggaaaggcctggagtggatggggttcatctatcctggtgactctgataccaaatacaacccgtccttcgaaggccaggtcaccatctcagccgacacgtccatcaacaccgcctacctgcagtggagcagcctgagggcctcggacaccgccatgtattactgtgcgagacatacgggtgggagttattcgacctggggcagggaaaccctggtcaccgtctcctca (SEQ ID NO:27);

[0106] 3B6 light chain variable region nucleotide sequence: gatattgtgatgactcagtctccactctccctgcccgtcacccttggccagccggcctccatctcctgcaggtctagtcaaagcctcgtatatggtgacggaaacacctacctgagttggtttcagcagaggccaggccaatctccaaggcgcctaatttataaggtttccaactgggactctggggtcccagacagattcagcggcagtggatcaggcacttatttcacactgaaaatcagcagggtggaggctgaggacgttggcatttattactgcatgcagagtacactctggcctccaaccttcggcctagggacacgactggagattaaa (SEQ ID NO:28);

[0107] Nucleotide sequence of the heavy chain variable region of 6A8: caggtacagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcagtgtctctggtggctccatcagtagcagtagttactactggggctggattcgtcagcccccagggaagggcttggagtggattggcagtatctattacactgggacaacttactacaacccgcccctcaagagtcgactcaccatatccgtggacacgtccaagaagcagttctccctgaggctgacctctgtgaccgccgcagacacggctgtgtattattgtgtgagggggactgggagctattggaagtttgggtactactttgactcctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO:29);

[0108] Nucleotide sequence of the light chain variable region of 6A8: gaaattgtgatgacacagtctccagacaccctgtctttgtctccaggggagagagccaccctgccctgcagggccagtcagagtgttagtaggagctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcttctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcaacagtatggtagttcaccactcactttcggcggagggaccaaagtggatatcaaa (SEQ ID NO:30);

[0109] The materials used in the present invention are all ordinary commercially available products and can be purchased on the market. The present invention will be further described below in conjunction with the examples:

[0110] Example 1 Panning of the target antibody from a phage human antibody library

[0111] Using the F4 natural antigen, screening and panning were carried out on the fully human antibody library constructed in the early stage of the laboratory. The evaluation mode was the indirect method, that is, 1 mL of the F4 natural antigen was coated in an immunization tube. After blocking and washing, 1 mL of the phage supernatant of the natural antibody library was added. After incubating with shaking at room temperature for 2 h, it was washed 10 times with PBST. Acid dissociation was carried out using Gly-HCl (pH 2.2), incubated at room temperature for 10 min, and the phage dissociated was added to 2 M Tris base solution to neutralize the eluted phage. Subsequently, the TG1 bacterial solution in the logarithmic growth phase was added. After incubating statically at 37 °C for 30 min, 10 μL was taken and plated (containing Amp resistance), and the plate was cultured overnight at 37 °C to detect the output. The remaining bacterial solution was added with the helper phage M13K07. After incubating statically at 37 °C for 30 min, the solution was centrifuged and changed (2YT medium containing 1 μM IPTG and 100 μg / mL AMP), and cultured overnight on a shaker at 30 °C. The above screening process was repeated 3 rounds, that is, panned three rounds, to enrich the phage that binds to the F4 natural antigen.

[0112] Example 2 Induced Expression of Positive Phage Clones

[0113] 190 single colonies after 3 rounds of enrichment were selected and cultured overnight in 2YT / GA medium (containing 2 wt% glucose and 50 μg / mL Amp). The next day, when the activated bacteria reached an OD value of about 2, a 2YT + helper phage (MOI = 5) mixture was added and incubated statically at 37 °C for 30 min. After culturing on a shaker at 37 °C for 1.5 h, 3 times the volume of 2YT-AK medium (containing 100 μg / mL Amp and 50 μg / mL Kan+) was added to each well and cultured overnight for 16 - 18 h. The next day, the supernatant was taken by centrifugation for detection.

[0114] Example 3 Phage Monoclonal Elisa Detection

[0115] The F4 wheat natural antigen was coated on the enzyme-linked immunosorbent assay (ELISA) plate and incubated overnight at 4 °C; after blocking with Casion, monoclonal phage was added and incubated at 37 °C for 30 min. After washing 6 times with PBST washing solution, HRP-M13 was added and incubated at 37 °C for 30 min. After washing 5 times with PBST washing solution, color development was carried out for 15 min, and then 2M H 2 SO 4 The reaction was terminated, and the absorbance value at 450 nm was detected by an enzyme-linked immunosorbent assay reader. As shown in Table 1, 3 clones with strong reactivity (1H7, 3B6, 6A8) were selected from Table 1 and sent for sequencing.

[0116] Table 1. Phage Monoclonal Elisa Detection

[0117]

[0118] Example 4 Nucleic Acid Sequence Analysis of Variable Region Genes and Construction of Full-Length IgE Antibodies

[0119] Antibody 1H7: Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7 (the nucleotide sequence is as shown in SEQ ID NO:25); its light chain variable region has the amino acid sequence shown in SEQ ID NO:8 (the nucleotide sequence is as shown in SEQ ID NO:26).

[0120] Antibody 6A8: Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:15 (the nucleotide sequence is as shown in SEQ ID NO:29); its light chain variable region has the amino acid sequence shown in SEQ ID NO:16 (the nucleotide sequence is as shown in SEQ ID NO:30).

[0121] Antibody 3B6: Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:23 (the nucleotide sequence is as shown in SEQ ID NO:27); its light chain variable region has the amino acid sequence shown in SEQ ID NO:24 (the nucleotide sequence is as shown in SEQ ID NO:28).

[0122] Using the selected 3 positive monoclonal antibodies as templates (1H7, 3B6, 6A8), the variable region gene sequences were amplified by PCR technology and spliced with the IgE constant region (overlap PCR). The full-length sequence after splicing was constructed into the pTT5 vector (Fenghui Biology) through the EcoRI / HindIII restriction enzyme sites. After the recombinant plasmid was transferred into competent DH5α cells, positive clones were selected for sequencing and plasmid extraction (Tiangen, DP118).

[0123] Example 5 Expression of full-length IgE antibody

[0124] One day before transfection, adjust the density of HEK 293F cells to 2×10 6 cells / mL~2.5×10 6 cells / mL. On the day of transfection, dilute the cells to 3×10 6 cells / mL with SMM 293-TII medium (Sino Biological, M293TII). Dilute the plasmid DNA and PEI according to the PEI transfection reagent instruction manual, and transfect the cells at a ratio of 1µg:5µL. Add 20% fed-batch SMM293-TII medium 24 hours later, and harvest the supernatant when the cell viability drops to about 70% - 75%. After the supernatant was identified by Elisa and showed the expression of full-length IgE antibody, the 1H7 and 6A8 IgE antibodies had obvious reactivity, and the 3B6 IgE reactivity was weak, as shown in Table 2.

[0125] Table 2. Elisa detection of full-length IgE antibody

[0126]

[0127] Example 6 Purification of F4 IgE Antibody

[0128] First, the purchased Omalizumab monoclonal antibody (MCE, 242138 - 07 - 4) was conjugated to NHS-activated magnetic agarose chromatography packing material (5 mg of antibody was used for 1 mL of packing material, referring to the standard operating procedure for conjugating NHS-activated sephrose packing material). Subsequently, the packing material conjugated with Omalizumab antibody was loaded into a gravity column for affinity purification.

[0129] Purification process: First, perform pretreatment on the antibody supernatant, that is, dialyze overnight to 20 mM PBS with a pH of 7.4. The affinity column was equilibrated with 20 mM PBS with a pH of 7.4 buffer. After loading the filtered antibody supernatant, the target protein antibody was eluted using dissociation buffer (0.2 M Gly glycine + 0.15 M NaCl, pH 2.7), and the dissociation peak was collected. The final storage buffer was 10 mM PBS + 1 vt‰ P300. A spectrophotometer NanoDrop One was used to detect the protein content and SDS-PAGE was used to detect the protein expression level.

[0130] The results showed that after purification, the expression levels of the full-length IgE antibodies 1H7 and 6A8 were 11.2 mg / L and 15.4 mg / L respectively. The molecular weight of the heavy chain was approximately 90 KD, and the molecular weight of the light chain was approximately 25 KD, which was consistent with the expectation (the heavy chain is affected by glycosylation and is larger than the theoretical molecular weight); the antibody purity reached over 98% (around 150 KD) ( Figure 1 )

[0131] Example 7 Detection of the Titer of Fully Human F4 IgE Antibody

[0132] The purified 1H7 and 6A8 IgE antibodies in Example 6 were adjusted to a protein concentration of 2 mg / mL and detected using a detection kit for wheat allergen-specific IgE according to the instructions. According to the judgment standard of the kit: the titer of the quality control product is not less than 1000 IU / mL. The detection results of the 1H7 and 6A8 IgE antibodies prepared in this experiment are shown in Table 3. The titers of the 1H7 and 6A8 IgE antibodies were 365210 IU / mL and 234667 IU / mL respectively, with strong reactivity, and both met the reactivity requirements for quality control products.

[0133] Table 3. Detection Results of the Reactivity of 1H7 and 6A8 IgE Antibodies

[0134]

[0135] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A monoclonal antibody to wheat F4 allergen, characterized in that The amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO: 1, SEQ ID NO: 9 and / or SEQ ID NO: 17; The amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 2, SEQ ID NO: 10 and / or SEQ ID NO: 18; The amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO:3, SEQ ID NO:11 and / or SEQ ID NO:19; The amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO:4, SEQ ID NO:12 and / or SEQ ID NO:20; The amino acid sequence of CDR2 of the light chain variable region is shown in SEQ ID NO:5, SEQ ID NO:13 and / or SEQ ID NO:21; The amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID NO:6, SEQ ID NO:14 and / or SEQ ID NO:

22.

2. The monoclonal antibody according to claim 1, characterized in that The heavy chain variable region comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 3; and / or The heavy chain variable region comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO: 9, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 10, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 11; and / or The heavy chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO: 17, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 18, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 19; The light chain variable region comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO: 4, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 6; and / or The light chain variable region comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO: 12, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 13, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 14; and / or The light chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO:20, a CDR2 with an amino acid sequence as shown in SEQ ID NO:21, and a CDR3 with an amino acid sequence as shown in SEQ ID NO:

22.

3. The monoclonal antibody according to claim 2, characterized in that The amino acid sequence of the heavy chain variable region is at least one or more of SEQ ID NO: 7, SEQ ID NO: 15 and / or SEQ ID NO: 23; The amino acid sequence of the light chain variable region is at least one or more of SEQ ID NO:8, SEQ ID NO:16 and / or SEQ ID NO:

24.

4. The monoclonal antibody according to claim 3, characterized in that Its constant region is IgE subtype.

5. A nucleic acid, characterized in that Comprising a nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 4.

6. A recombinant vector, characterized in that Containing the nucleic acid according to claim 5.

7. A host cell, characterized in that Transfect or transform the recombinant vector as described in claim 6.

8. A reagent containing a monoclonal antibody, characterized in that: The method comprises at least one of the monoclonal antibodies according to any one of claims 1 to 4 or the monoclonal antibodies obtained by culturing the host cell according to claim 7 and auxiliary materials.

9. A kit for wheat allergen immunoassay, characterized in that: The invention comprises a wheat allergen immunoassay reagent and at least one of the following a) to c): a), the monoclonal antibody according to any one of claims 1 to 4; b), culturing the monoclonal antibody obtained by the host cell according to claim 7; c) The reagent according to claim 8.

10. Use of at least one of the following I) to VII) in wheat allergen immunoassay: I), the monoclonal antibody according to any one of claims 1 to 4; II), the nucleic acid according to claim 5; III), the recombinant vector according to claim 6; IV), the host cell according to claim 7; V), culturing the monoclonal antibody obtained by the host cell according to claim 7; VI), the reagent according to claim 8; VII), the kit according to claim 9.

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