Monoclonal antibodies against wheat allergen IgE, their preparation and use

Abstract

The present application relates to the technical field of biology, and particularly relates to an IgE monoclonal antibody against wheat allergen, a preparation method and application thereof. The present application adopts F4 natural antigen, and uses phage screening technology to screen a human F4 IgE monoclonal antibody with strong reactivity, the monoclonal antibody can specifically recognize wheat F4 antigen, can have significant enzyme-linked immunoassay with the antigen, and 37 DEG C acceleration stability meets the kit use requirement, and the monoclonal antibody can play an important role in wheat allergen F4 project detection as a quality control product, and has a wide market application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an IgE monoclonal antibody against wheat allergen, a preparation method and application thereof. BACKGROUND

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

[0003] Wheat is one of the important foods that can cause IgE-mediated allergic reactions in children. IgE-mediated allergic reactions to ingested wheat proteins include symptoms of the gastrointestinal tract, respiratory tract and skin. The reaction typically occurs within one hour after ingesting wheat. Sensitized patients can be 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 needed as a quality control. However, it is difficult to collect and obtain high-concentration wheat IgE samples; and most of the wheat allergen-specific IgE antibody detection kits on the market evaluate the mode as an indirect method, i.e. coating the antigen, using anti-human IgE antibody as the secondary antibody, and detecting IgE antibody. In view of this, it is necessary to develop a wheat F4 IgE human monoclonal antibody. SUMMARY

[0004] Therefore, the present application aims to provide an IgE monoclonal antibody against wheat allergen, a preparation method and application thereof.

[0005] The present application provides a monoclonal antibody of wheat F4 allergen,

[0006] the amino acid sequence of CDR1 of the 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 of 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 of 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 the heavy chain variable region is set forth in SEQ ID NO: 1, the amino acid sequence of CDR2 of the heavy chain variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of CDR3 of the heavy chain variable region is set forth in SEQ ID NO: 3 (antibody 1H7); and / or

[0010] the amino acid sequence of CDR2 of the heavy chain variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of CDR3 of the heavy chain variable region is set forth in SEQ ID NO: 3 (antibody 1H7); and / or

[0011] the amino acid sequence of CDR2 of the heavy chain variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of CDR3 of the heavy chain variable region is set forth in SEQ ID NO: 3 (antibody 1H7); and / or

[0012] Further, the monoclonal antibody according to the present application,

[0013] the amino acid sequence of CDR1 of the heavy chain variable region is set forth in SEQ ID NO: 1, the amino acid sequence of CDR2 of the heavy chain variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of CDR3 of the heavy chain variable region is set forth in SEQ ID NO: 3 (antibody 1H7); and / or

[0014] the amino acid sequence of CDR2 of the heavy chain variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of CDR3 of the heavy chain variable region is set forth in SEQ ID NO: 3 (antibody 1H7); and / or

[0015] the amino acid sequence of CDR2 of the heavy chain variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of CDR3 of the heavy chain variable region is set forth in SEQ ID NO: 3 (antibody 1H7); and / or

[0016] the amino acid sequence of CDR1 of the heavy chain variable region is set forth in SEQ ID NO: 1, the amino acid sequence of CDR2 of the heavy chain variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of CDR3 of the heavy chain variable region is set forth in SEQ ID NO: 3 (antibody 1H7); and / or

[0017] the amino acid sequence of CDR2 of the heavy chain variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of CDR3 of the heavy chain variable region is set forth in SEQ ID NO: 3 (antibody 1H7); and / or

[0018] the amino acid sequence of CDR2 of the heavy chain variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of CDR3 of the heavy chain variable region is set forth in SEQ ID NO: 3 (antibody 1H7); and / or

[0019] Further, the monoclonal antibody of the present application,

[0020] 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;

[0021] 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.

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

[0023] The amino acid sequence of the heavy chain variable region of the 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 the 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 the 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 application, the constant region of the monoclonal antibody is IgE subtype;

[0027] The antibody of the present application can be derived from human, rat, mouse, rabbit, cow, sheep, etc., and the present application does not limit this; in a specific embodiment of the present application, the antibody is derived from human, and the constant region is human IgE subtype.

[0028] The present application screens 190 positive phage clones on human antibody library after screening and washing with F4 natural antigen, and then obtains phage containing 1H7, 3B6 and 6A8 monoclonal antibodies with strong reactivity through phage monoclonal Elisa detection. Through construction and expression of complete monoclonal antibodies, it is found that 1H7 and 6A8 IgE antibodies have obvious reactivity, and 3B6 IgE reactivity is weak. The expression amount of 1H7 and 6A8 full-length IgE antibodies after purification is 11.2 mg / L and 15.4 mg / L respectively. Through titer detection of purified antibodies, the titers of 1H7 and 6A8 IgE antibodies are 365210 IU / mL and 234667 IU / mL respectively, and the reactivity is strong, which meets the reactivity requirements as quality control.

[0029] The present application provides nucleic acids comprising nucleic acids encoding the monoclonal antibodies of the present application.

[0030] In the present application,

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

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

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

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

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

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

[0037] The nucleic acids of the present application can be DNA, RNA, cDNA or PNA. In the embodiments of the present application, 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 acids 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 acids can be linear or circular in topology. The nucleic acids can be part of a vector (e.g. an expression or cloning vector) or a fragment. The nucleic acids can be obtained directly from natural sources, or can be prepared with the aid of recombination, enzymatic or chemical techniques. The RNA form is mRNA and the like obtained by transcription of a gene.

[0038] Further, the nucleic acids of the present application can be optimized or unoptimized, and the optimization includes but is not limited to: codon usage bias, elimination of secondary structures (such as hairpin structures) that are not conducive to expression, alteration of GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA instability regions, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that can affect cloning.

[0039] The present application provides recombinant vectors containing the nucleic acids of the present application.

[0040] The recombinant vector of the present application refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule comprising a desired coding sequence and suitable nucleic acid sequences or elements necessary for the expression of the operably linked coding gene in a specific host organism. In the present specification, "plasmid" and "vector" can sometimes be used interchangeably because plasmid is the most commonly used form of vector at present. However, the present application is intended to include such other forms of expression vectors which perform equivalent functions, which are known in the art or will become known, including but not limited to: plasmid, phage particle, viral vector and / or only potential genomic insert.

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

[0042] Further, the transformation method includes chemical transformation and electroporation; the transfection method includes calcium phosphate coprecipitation, artificial liposome method, viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0043] The host cell provided by the present application includes plant, animal, bacteria, fungi, bacteriophage or virus, which is not limited by the present application. The host cell is transformed or transfected by the vector constructed by the recombinant DNA technology of the present application, so that the transformed host cell has the ability to replicate the vector encoding protein or express the desired protein, or perform the corresponding function.

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

[0045] The present application provides a solid object marked, fixed, connected or attached, adhered with the antibody of the present application;

[0046] The antibody of the present application can be marked, fixed, connected or attached, adhered on a solid object, which can be a well plate, magnetic beads, nanomaterials and other solid phase carriers; the connection can be direct or indirect coupling, such as covalent coupling, which is not limited by the present application.

[0047] The present application provides the antibody of the present application which is labeled or chemically labeled; in the present application, 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 application provides a reagent containing monoclonal antibody, which includes at least one of the monoclonal antibody of the present application or the monoclonal antibody obtained by culturing the host cell of the present application and adjuvant;

[0049] Further, the adjuvant includes a buffer, an antioxidant, a stabilizer, and / or a preservative, etc. for preserving the monoclonal antibody, or maintaining the activity of the monoclonal antibody, or assisting the monoclonal antibody to play a role.

[0050] The present application provides a kit for detecting wheat allergen immunity, characterized in comprising reagents for detecting wheat allergen immunity and at least one of a) to c) as follows:

[0051] a) the monoclonal antibody according to the present application;

[0052] b) the monoclonal antibody obtained by culturing the host cell according to the present application;

[0053] c) the reagent according to the present application.

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

[0055] The present application provides at least one of I) to VII) as follows for use in detecting wheat allergen immunity:

[0056] I) the monoclonal antibody according to the present application;

[0057] II) the nucleic acid according to the present application;

[0058] III) the recombinant vector according to the present application;

[0059] IV) the host cell according to the present application;

[0060] V) the monoclonal antibody obtained by culturing the host cell according to the present application;

[0061] VI) the reagent according to the present application;

[0062] VII) the kit according to the present application.

[0063] The present application provides a method for detecting wheat allergen immunity, characterized in comprising detecting by using at least one of A) to G) as follows:

[0064] A) the monoclonal antibody according to the present application;

[0065] B) the nucleic acid according to the present application;

[0066] C) the recombinant vector of the present application;

[0067] D) the host cell of the present application;

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

[0069] F) the reagent of the present application;

[0070] G) the kit of the present application.

[0071] The present application adopts F4 natural antigen, and uses phage screening technology to screen human F4 IgE monoclonal antibody with strong reactivity, which can specifically recognize wheat F4 antigen, can have significant enzyme-linked immunoreaction with the antigen, and the 37℃ acceleration stability meets the kit use requirement, and the monoclonal antibody can play an important role in wheat allergen F4 project detection as a quality control product, and has wide market application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0072] Figure 1 SDS-PAGE diagram, wherein 1 is 1H7 reduced band (treated with reducing agent DTT); 2 is 1H7 purified flow-through sample; 3 is 1H7 non-reduced band; 4 is 6A8 non-reduced band; 5 is 6A8 purified flow-through sample; 6 is 6A8 reduced band (treated with reducing agent DTT). DETAILED DESCRIPTION

[0073] The present application provides an anti-wheat allergen IgE monoclonal antibody, a preparation method and application thereof, and those skilled in the art can refer to the content herein, and appropriately improve process parameters to realize. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all regarded as included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

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

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

[0076] Immunoglobulin E (IgE) antibodies only exist in mammals. IgE is synthesized by plasma cells. IgE monomer is composed of two heavy chains (ε chain) and two light chains, and the ε chain contains four Ig constant regions (Cε1-Cε4). The molecular weight of IgE antibody is 160 kD, and the important feature is the cytophilic antibody, which can bind to the high-affinity FcεR I on mast cells and basophils, and can cause type I hypersensitivity when the antigen reenters the body after binding. In addition, IgE antibodies may be related to the body's anti-parasite immunity.

[0077] Humanized anti-IgE monoclonal antibodies are currently used for the treatment of allergic asthma, and the symptoms are significantly improved after treatment, and the feasibility of treating food allergies is being 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 allergy, and its binding site with IgE is the high-affinity binding site FcεRI of IgE receptor, which exists on the membrane of mast cells and basophils. When the allergen enters the body, it binds to IgE, mast cells, and causes mast cell activation and degranulation, causing type I hypersensitivity. When Omalizumab monoclonal antibody binds to the FcεRI site of IgE, IgE cannot bind to effector cells, thereby blocking type I hypersensitivity and achieving the effect of treating food allergy. Based on this, in this study, we tried to use this antibody as a purification affinity medium to explore the purification conditions, so as to purify IgE antibodies.

[0078] Allergen detection is crucial for accurate diagnosis, treatment plan development, and avoidance strategies. The IgE monoclonal antibody developed in this study against F4 allergen has strong reactivity, meeting the requirements for reactivity as a quality control product.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0101] 3B6 heavy chain variable region amino acid sequence: EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIGWVRQMPGKGLEWMGFIYPGDSDTKYNPSFEGQVTISADTSINTAYLQWSSLRASDTAMYYCARHTGGSYSTWGRETLVTVSS (SEQ ID NO: 23);

[0102] 3B6 heavy chain variable region amino acid sequence: DIVMTQSPLSLPVTLGQPASISCRSSQSLVYGDGNTYLSWFQQRPGQSPRRLIYKVSNWDSGVPDRFSGSGSGTYFTLKISRVEAEDVGIYYCMQSTLWPPTFGLGTRLEIK (SEQ ID NO: 24);

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

[0104] 1H7 light chain variable region nucleotide sequence: 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] 6A8 heavy chain variable region nucleotide sequence: caggtacagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcagtgtctctggtggctccatcagtagcagtagttactactggggctggattcgtcagcccccagggaagggcttggagtggattggcagtatctattacactgggacaacttactacaacccgcccctcaagagtcgactcaccatatccgtggacacgtccaagaagcagttctccctgaggctgacctctgtgaccgccgcagacacggctgtgtattattgtgtgagggggactgggagctattggaagtttgggtactactttgactcctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 29);

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

[0109] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market. The present application is further described below in combination with examples:

[0110] Example 1 Phage human antibody library panning of target antibodies

[0111] The F4 natural antigen was used to screen the previously constructed human antibody library in the laboratory. The evaluation mode was indirect, i.e. 1 mL of F4 natural antigen was coated in an immunization tube, after blocking and washing, 1 mL of natural antibody phage supernatant was added, and after 2 h of incubation at room temperature, 10 times of PBST washing was performed, Gly-HCl (pH 2.2) was used for acid dissociation, and the dissociated phage was neutralized with 2M Tris base solution. Subsequently, logarithmic growth phase TG1 bacteria solution was added, and after 30 min of incubation at 37°C, 10 μL was plated (containing Amp resistance), and the plate was cultured at 37°C overnight to detect the output. The remaining bacteria solution was added with helper phage M13K07, and after 30 min of incubation at 37°C, the solution was centrifuged and replaced with 2YT medium containing 1 μM IPTG and 100 μg / mL AMP, and cultured at 30°C overnight. The above screening process was repeated for 3 rounds, i.e. 3 rounds of panning, to enrich phage binding to F4 natural antigen.

[0112] Example 2 Induced expression of positive phage clones

[0113] After 3 rounds of enrichment, 190 single colonies were selected and added to 2YT / GA medium (containing 2wt% glucose and 50 μg / mL Amp) for overnight culture. The next day, when the bacteria were activated to an OD value of about 2, 2YT+helper phage (MOI=5) mixed solution was added and incubated at 37°C for 30 min. After 1.5 h of shaking culture at 37°C, 3 times the volume of 2YT-AK medium (containing 100 μg / mL Amp and 50 μg / mL Kan+) was added per well, and the culture was incubated overnight for 16-18 h. The next day, the supernatant was centrifuged and used for detection.

[0114] Example 3 Phage monoclonal Elisa detection

[0115] F4 wheat natural antigen was coated on an enzyme-labeled plate and incubated at 4°C overnight; after Casion blocking, monoclonal phage was added, and after 30 min of incubation at 37°C, 6 times of PBST washing was performed, HRP-M13 was added, and after 30 min of incubation at 37°C, 5 times of PBST washing was performed, color development was performed for 15 min, 2M H2SO4 was added to terminate the reaction, and the absorbance value at 450 nm was detected by an enzyme-labeled instrument. As shown in Table 1, 3 clones (1H7, 3B6, and 6A8) with strong reactivity were selected from Table 1 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 an amino acid sequence as shown in SEQ ID NO: 7 (nucleotide sequence as shown in SEQ ID NO: 25); its light chain variable region has an amino acid sequence as shown in SEQ ID NO: 8 (nucleotide sequence as shown in SEQ ID NO: 26).

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

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

[0122] Taking the selected three positive monoclonals (1H7, 3B6, 6A8) as templates, the variable region gene sequences were amplified by PCR technology and spliced with IgE constant region (overlap PCR). The spliced full-length sequence was constructed into pTT5 vector (Fenghui Biotechnology) through EcoRI / HindIII enzyme cutting sites. After the recombinant plasmid was transformed 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] The day before transfection, the density of HEK 293F cells was adjusted to 2×10 6 cells / mL~2.5×10 6 cells / mL, and on the day of transfection, the cells were diluted to 3×10 6 cells / mL with SMM 293-TII medium (Yiqiao, M293TII). The plasmid was diluted according to the PEI transfection reagent instructions, and the plasmid DNA and PEI were diluted to transfect the cells at a ratio of 1 µg:5 µL. After 24 h, 20% of the feed SMM 293-TII medium was added, and the culture was continued until the cell viability decreased to about 70%~75%, and the supernatant was harvested. After the supernatant was identified by Elisa, it was shown that the full-length IgE antibody expression, 1H7 and 6A8 IgE antibodies had obvious reactivity, and 3B6 IgE reactivity was weak, as shown in Table 2.

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

[0126]

[0127] Example 6 F4 IgE antibody purification

[0128] First, the purchased Omalizumab mAb (MCE, 242138-07-4) was coupled to NHS activated magnetic sepharose chromatography filler (5 mg of antibody for 1 mL of filler, refer to the standard operation procedure of NHS-activated sepharose filler coupling). Then the Omalizumab antibody coupled to the gravity column was loaded for affinity purification.

[0129] Purification process: First, the antibody supernatant pretreatment was performed, i.e. overnight dialysis to buffer 20 mM PBS pH 7.4, the affinity column was equilibrated with 20 mM PBS pH 7.4 buffer, after the filtered antibody supernatant was loaded, the dissociation buffer (0.2M Gly glycine + 0.15M NaCl, pH 2.7) was used to elute the target protein antibody, the dissociation peak was collected, and the final storage buffer was 10 mM PBS + 1 vt% P300. The protein content was detected by spectrophotometer NanoDrop One, and the protein expression level was detected by SDS-PAGE.

[0130] The results show that after the purification of 1H7 and 6A8 full-length IgE antibodies, the expression amounts are 11.2 mg / L and 15.4 mg / L respectively, the heavy chain molecular weight is about 90 KD, and the light chain molecular weight is about 25 KD, which is consistent with the expectation (the heavy chain is larger than the theoretical molecular weight due to glycosylation); the antibody purity reaches more than 98% (about 150 KD) Figure 1 ).

[0131] Example 7 Whole human F4 IgE antibody titer detection

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

[0133] Table 3. 1H7 and 6A8 IgE antibody reactivity detection results

[0134]

[0135] The above merely is the preferred embodiment of the present application, it should be pointed out that, for ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A monoclonal antibody to a wheat F4 allergen, characterized in that, comprising: a monoclonal antibody 1H7, a monoclonal antibody 6A8 or a monoclonal antibody 3B6; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody 1H7 are respectively shown as SEQ ID NO: 1, 2 and 3; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of the monoclonal antibody 1H7 are respectively shown as SEQ ID NO: 4, 5 and 6; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody 6A8 are respectively shown as SEQ ID NO: 9, 10 and 11; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of the monoclonal antibody 6A8 are respectively shown as SEQ ID NO: 12, 13 and 14; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody 3B6 are respectively shown as SEQ ID NO: 17, 18 and 19; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of the monoclonal antibody 3B6 are respectively shown as SEQ ID NO: 20, 21 and 22.

2. The monoclonal antibody according to claim 1, wherein: the variable region of the heavy chain of the monoclonal antibody 1H7 has an amino acid sequence shown as SEQ ID NO: 7; the variable region of the light chain of the monoclonal antibody 1H7 has an amino acid sequence shown as SEQ ID NO: 8; the variable region of the heavy chain of the monoclonal antibody 6A8 has an amino acid sequence shown as SEQ ID NO: 15; the variable region of the light chain of the monoclonal antibody 6A8 has an amino acid sequence shown as SEQ ID NO: 16; the variable region of the heavy chain of the monoclonal antibody 3B6 has an amino acid sequence shown as SEQ ID NO: 23; the variable region of the light chain of the monoclonal antibody 3B6 has an amino acid sequence shown as SEQ ID NO:

24.

3. The monoclonal antibody according to claim 2, characterized in that, the constant region thereof is IgE subtype.

4. A nucleic acid, characterized in that, a nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that, the nucleic acid according to claim 4.

6. A host cell characterized in that, the recombinant vector according to claim 5.

7. A reagent comprising a monoclonal antibody, characterized in that, the monoclonal antibody according to any one of claims 1 to 3 or the monoclonal antibody obtained by culturing the host cell according to claim 6 and an excipient.

8. A kit for the immunological detection of a wheat allergen, characterized in that, a wheat allergen immunoassay reagent and at least one of a) to c) as follows: a) the monoclonal antibody according to any one of claims 1 to 3; b) the monoclonal antibody obtained by culturing the host cell according to claim 6; c) the reagent according to claim 7.

9. Use of at least one of I) to VII) as follows in the preparation of a wheat allergen immunoassay product: I) the monoclonal antibody according to any one of claims 1 to 3; II) the nucleic acid according to claim 4; III) the recombinant vector according to claim 5; IV) the host cell according to claim 6; V) the monoclonal antibody obtained by culturing the host cell according to claim 6; VI) the reagent according to claim 7; VII) a kit according to claim 8; The wheat allergen is F4.

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