Anti-human IgE antibody and application thereof

Abstract

The invention relates to the field of antibodies, in particular to an anti-human IgE antibody and application thereof. The CDR of a heavy chain variable region in the anti-human IgE antibody provided by the invention comprises a CDR-H1 of which the amino acid sequence is shown as SEQ ID No. 1 or SEQ ID No. 7, a CDR-H2 of which the amino acid sequence is shown as SEQ ID No. 2 or SEQ ID No. 8, and a CDR-H3 of which the amino acid sequence is shown as SEQ ID No. 3 or SEQ ID No. 9; the CDR of a light chain variable region in the anti-human IgE antibody comprises a CDR-L1 of which the amino acid sequence is as shown in SEQ ID No. 4 or SEQ ID No. 10, a CDR-L2 of which the amino acid sequence is as shown in SEQ ID No. 5 or SEQ ID No. 11, and a CDR-L3 of which the amino acid sequence is as shown in SEQ ID No. 6 or SEQ ID No. 12. The antibody provided by the invention is high in affinity and high in sensitivity, can detect human IgE with the concentration of 0.02 IU / mL, and has clinical diagnostic significance for identifying anaphylactic reaction.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to an anti-human IgE antibody and its use. Background Art

[0002] Immunoglobulin E (IgE) is a type of antibody that exists only in mammals. IgE is composed of two identical heavy chains and light chains, and each heavy chain contains four constant ε domains (Cε1-4). The main function of IgE is to produce immunity to parasites. In addition, IgE plays a key role in type I hypersensitivity reactions, such as sinusitis, allergic rhinitis, food allergies, chronic urticaria, and atopic dermatitis. IgE also plays an important role in responding to allergens, such as allergy drugs, bee stings, and antigens used in desensitization immunotherapy. Although the IgE content is small (the IgE content in normal human serum accounts for only 0.05% of all Ig classes), it can trigger a strong inflammatory response. The IgE content in the blood of patients with hereditary allergies is as high as 10 times that of normal people. In many autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), IgE levels are elevated, and it is speculated that it plays an important pathogenic role in RA and SLE by inducing hypersensitivity reactions. Summary of the invention

[0003] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide an anti-human IgE antibody and use thereof, so as to solve the problems in the prior art.

[0004] To achieve the above objectives and other related objectives, the present invention provides an anti-human IgE antibody, wherein the anti-human IgE antibody comprises a heavy chain variable region and a light chain variable region, and the anti-human IgE antibody has one or more of the following technical features:

[0005] 1) the complementarity determining region of the heavy chain variable region includes CDR-H1 with the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 7;

[0006] 2) the complementarity determining region of the heavy chain variable region includes CDR-H2 with an amino acid sequence as shown in SEQ ID No. 2 or SEQ ID No. 8;

[0007] 3) the complementarity determining region of the heavy chain variable region includes CDR-H3 with an amino acid sequence as shown in SEQ ID No. 3 or SEQ ID No. 9;

[0008] 4) the complementarity determining region of the light chain variable region includes CDR-L1 with the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 10;

[0009] 5) the complementarity determining region of the light chain variable region includes CDR-L2 with an amino acid sequence as shown in SEQ ID No. 5 or SEQ ID No. 11;

[0010] 6) The complementarity determining region of the light chain variable region includes CDR-L3 having an amino acid sequence as shown in SEQ ID No.6 or SEQ ID No.12.

[0011] Preferably, the complementary determining region of the heavy chain variable region of the anti-human IgE antibody includes CDR-H1 with an amino acid sequence as shown in SEQ ID No.1, CDR-H2 with an amino acid sequence as shown in SEQ ID No.2, and CDR-H3 with an amino acid sequence as shown in SEQ ID No.3; or, the complementary determining region of the heavy chain variable region includes CDR-H1 with an amino acid sequence as shown in SEQ ID No.7, CDR-H2 with an amino acid sequence as shown in SEQ ID No.8, and CDR-H3 with an amino acid sequence as shown in SEQ ID No.9.

[0012] The present invention also provides an isolated polynucleotide encoding one or more of the following peptide segments:

[0013] 1) the heavy chain variable region of the aforementioned anti-human IgE antibody;

[0014] 2) the light chain variable region of the aforementioned anti-human IgE antibody;

[0015] 3) The aforementioned anti-human IgE antibody.

[0016] The present invention also provides a nucleic acid construct, which contains the aforementioned isolated polynucleotide.

[0017] The present invention also provides an isolated engineered cell, wherein the engineered cell contains the aforementioned nucleic acid construct or the aforementioned polynucleotide.

[0018] The present invention also provides the use of the aforementioned anti-human IgE antibody, the aforementioned polynucleotide or the aforementioned nucleic acid construct in preparing a product for treating a disease caused by type I hypersensitivity reaction, a product for treating an autoimmune disease or a product for detecting an allergen.

[0019] The present invention also provides a diagnostic kit, which contains the aforementioned anti-human IgE antibody.

[0020] As described above, an anti-human IgE antibody and its use of the present invention have the following beneficial effects:

[0021] The present invention successfully expresses human IgE Fc protein through a mammalian cell expression system. After immunizing New Zealand rabbits, a group of monoclonal antibodies with high affinity and high sensitivity to human IgE are screened out. The monoclonal antibody combination is applied to an anti-human IgE magnetic particle chemiluminescent detection reagent, which can detect human IgE with a concentration of 0.02 IU / mL, and has clinical diagnostic significance for identifying allergic reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown is the SDS-PAGE identification diagram of the purified human IgE Fc protein of the present invention.

[0023] Figure 2 The figure shows the SDS-PAGE identification diagram after purification of the anti-human IgE monoclonal antibody of the present invention.

[0024] Figure 3 Shown is a standard curve diagram of the anti-human IgE magnetic microparticle chemiluminescence detection reagent of the present invention.

[0025] Figure 4 Shown are the affinity results of the anti-human IgE monoclonal antibodies of the present invention. DETAILED DESCRIPTION

[0026] The present invention provides an anti-human IgE antibody, wherein the anti-human IgE antibody comprises a heavy chain variable region and a light chain variable region, and the anti-human IgE antibody has one or more of the following technical features:

[0027] 1) the complementarity determining region of the heavy chain variable region includes CDR-H1 with the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 7;

[0028] 2) the complementarity determining region of the heavy chain variable region includes CDR-H2 with an amino acid sequence as shown in SEQ ID No. 2 or SEQ ID No. 8;

[0029] 3) the complementarity determining region of the heavy chain variable region includes CDR-H3 with an amino acid sequence as shown in SEQ ID No. 3 or SEQ ID No. 9;

[0030] 4) the complementarity determining region of the light chain variable region includes CDR-L1 with the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 10;

[0031] 5) the complementarity determining region of the light chain variable region includes CDR-L2 with an amino acid sequence as shown in SEQ ID No. 5 or SEQ ID No. 11;

[0032] 6) The complementarity determining region of the light chain variable region includes CDR-L3 having an amino acid sequence as shown in SEQ ID No.6 or SEQ ID No.12.

[0033] In some specific embodiments, the complementary determining region of the heavy chain variable region of the anti-human IgE antibody includes CDR-H1 as shown in SEQ ID No.1, CDR-H2 as shown in SEQ ID No.2, and CDR-H3 as shown in SEQ ID No.3; or, the complementary determining region of the heavy chain variable region includes CDR-H1 as shown in SEQ ID No.7, CDR-H2 as shown in SEQ ID No.8, and CDR-H3 as shown in SEQ ID No.9.

[0034] In some specific embodiments, the complementary determining region of the light chain variable region of the anti-human IgE antibody includes a CDR-L1 with an amino acid sequence as shown in SEQ ID No.4, a CDR-L2 with an amino acid sequence as shown in SEQ ID No.5, and a CDR-L3 with an amino acid sequence as shown in SEQ ID No.6; or, the complementary determining region of the light chain variable region includes a CDR-L1 with an amino acid sequence as shown in SEQ ID No.10, a CDR-L2 with an amino acid sequence as shown in SEQ ID No.11, and a CDR-L3 with an amino acid sequence as shown in SEQ ID No.12.

[0035] In some specific embodiments, the complementary determining region of the heavy chain variable region in the anti-human IgE antibody includes CDR-H1 as shown in SEQ ID No.1, CDR-H2 as shown in SEQ ID No.2, and CDR-H3 as shown in SEQ ID No.3, and the complementary determining region of the light chain variable region includes CDR-L1 as shown in SEQ ID No.4, CDR-L2 as shown in SEQ ID No.5, and CDR-L3 as shown in SEQ ID No.6; the complementary determining region of the heavy chain variable region in the anti-human IgE antibody includes CDR-H1 as shown in SEQ ID No.7, CDR-H2 as shown in SEQ ID No.8, and CDR-H3 as shown in SEQ ID No.9, and the complementary determining region of the light chain variable region includes CDR-L1 as shown in SEQ ID No.10, CDR-L2 as shown in SEQ ID No.11, and CDR-L3 as shown in SEQ ID No.12.

[0036] In some embodiments, the anti-human IgE antibody is a monoclonal antibody.

[0037] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-human IgE antibody comprises:

[0038] a) an amino acid sequence as shown in one of SEQ ID No. 13 and 17; or

[0039] b) an amino acid sequence having 80% or more identity with the amino acid sequence shown in one of SEQ ID No. 13 and 17 and having the function of the amino acid sequence defined in a).

[0040] Specifically, the amino acid sequence in b) specifically refers to an amino acid sequence as shown in one of SEQ ID No. 13 and 17 obtained by substitution, deletion or addition of one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, or an amino acid sequence as shown in one of SEQ ID No. 13 and 17 obtained by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having the function of the amino acid sequence as shown in one of SEQ ID No. 13 and 17. The amino acid sequence in b) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more identity with one of SEQ ID No. 13 and 17.

[0041] In some embodiments, the amino acid sequence of the light chain variable region of the anti-human IgE antibody comprises:

[0042] c) an amino acid sequence as shown in one of SEQ ID No. 14 and 18; or

[0043] d) an amino acid sequence having 80% or more identity with the amino acid sequence shown in one of SEQ ID No. 14 and 18 and having the function of the amino acid sequence defined in c).

[0044] Specifically, the amino acid sequence in d) specifically refers to an amino acid sequence as shown in one of SEQ ID No. 14 and 18 obtained by substitution, deletion or addition of one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, or an amino acid sequence as shown in one of SEQ ID No. 14 and 18 obtained by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having the function of the amino acid sequence as shown in one of SEQ ID No. 14 and 18. The amino acid sequence in d) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more identity with one of SEQ ID No. 14 and 18.

[0045] In some specific embodiments, the anti-human IgE antibody comprises a peptide segment having an amino acid sequence of a heavy chain variable region such as SEQ ID No. 13 and an amino acid sequence of a light chain variable region such as SEQ ID No. 14; or, a peptide segment having an amino acid sequence of a heavy chain variable region such as SEQ ID No. 17 and an amino acid sequence of a light chain variable region such as SEQ ID No. 18.

[0046] In some embodiments, the anti-human IgE antibody further comprises a secretion signal peptide. Specifically, the secretion signal peptide is derived from DsbA, PelB, OmpA, TolB, MalE, lpp, TorA or HylA signal peptide. Wherein, the secretion signal peptide directs the anti-human IgE antibody to the periplasmic space of the host cell.

[0047] In some embodiments, the anti-human IgE antibody comprises a histidine tag. A histidine tag or a polyhistidine tag is a sequence of 2 to 20 histidine residues attached to an anti-human IgE antibody. The histidine tag comprises 2 to 20 histidine residues, 5 to 15 histidine residues, 5 to 18 histidine residues, 5 to 16 histidine residues, 5 to 15 histidine residues, 5 to 14 histidine residues, 5 to 13 histidine residues, 5 to 12 histidine residues, 5 to 11 histidine residues, 5 to 10 histidine residues, 6 to 12 histidine residues, 6 to 11 histidine residues or 7 to 10 histidine residues. The present invention also provides an isolated polynucleotide encoding one or more of the following peptide segments:

[0048] 1) the heavy chain variable region of the aforementioned anti-human IgE antibody;

[0049] 2) the light chain variable region of the aforementioned anti-human IgE antibody;

[0050] 3) The aforementioned anti-human IgE antibody.

[0051] The present invention also provides a nucleic acid construct, which contains the aforementioned isolated polynucleotide.

[0052] In some specific embodiments, the nucleic acid construct is constructed by inserting the isolated polynucleotide into the multiple cloning site of the expression vector. The expression vector can be transformed, transduced or transfected into the host cell so that the genetic material elements it carries are expressed in the host cell. The construct is a viral vector or a non-viral vector. For example, non-viral vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Viral vectors include: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including but not limited to viral particles, liposomes or protein shells.

[0053] In some specific embodiments, the backbone plasmid of the expression vector can be selected from pET expression vector, pCW expression vector, pUC expression vector, pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pGAPZαB, pGAPZαC, pPICZ A, pPICZ B, pPICZ C, pGAPZ A, pGAPZ B or pGAPZ C.

[0054] The present invention also provides an isolated engineered cell, wherein the engineered cell contains the aforementioned nucleic acid construct or the aforementioned polynucleotide.

[0055] In the present invention, the engineered cell can be obtained by introducing the aforementioned nucleic acid construct into a host cell, or a cell having the aforementioned exogenous polynucleotide integrated into its genome is the engineered cell.

[0056] Any cell suitable for expression by an expression vector can be used as a host cell, for example, the host cell can be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. The host cell includes many cell types, such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0057] The present invention also provides the use of the aforementioned anti-human IgE antibody, the aforementioned polynucleotide or the aforementioned nucleic acid construct in preparing a product for treating a disease caused by type I hypersensitivity reaction, a product for treating an autoimmune disease or a product for detecting an allergen.

[0058] In some embodiments, the disease caused by the type I hypersensitivity reaction is selected from one or more of sinusitis, allergic rhinitis, food allergy, chronic urticaria and atopic dermatitis.

[0059] In some specific embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, systemic lupus erythematosus nephritis, AIDS, rheumatoid arthritis, inflammatory bowel disease, psoriasis, vasculitis, bronchial asthma, chronic obstructive pulmonary disease, and one or more combinations thereof.

[0060] In some embodiments, the allergen is IgE.

[0061] The present invention also provides a diagnostic kit, which contains the aforementioned anti-human IgE antibody.

[0062] In the present invention, the term "complementarity determining region" or "CDR" generally refers to a region in an antibody that can form complementarity with an antigenic determinant in terms of spatial structure. The variability in an antibody is generally not evenly distributed throughout the variable region of the antibody. The heavy chain variable region and the light chain variable region of a monoclonal antibody generally have three hypervariable regions (HVRs), which can generally form complementarity with an antigenic determinant in terms of spatial structure, so the hypervariable region is also called a complementarity determining region (CDR), i.e., the heavy chain variable region generally includes three complementarity determining regions, i.e., HCDR1, HCDR2 and HCDR3, and the light chain variable region generally includes three complementarity determining regions, i.e., LCDR1, LCDR2 and LCDR3.

[0063] In the present invention, the term "monoclonal antibody" generally refers to a population of antibodies, wherein the antibodies included in the population are substantially identical (except for a few possible naturally occurring mutations). Monoclonal antibodies are generally directed against specific determinants on antigens.

[0064] In the present invention, the term "identity" means that if two or more than two sequences have the same length and order of nucleotides or amino acids, the sequences are identical. The percentage of identity usually describes the degree to which two sequences are identical, that is, it usually describes the percentage of nucleotides corresponding to the same nucleotides of the reference sequence at the sequence position. In order to determine the degree of identity (identity%), the sequences to be compared are usually of the same length, that is, the length of the longest sequence in the sequences to be compared. For example, a first sequence consisting of 8 nucleotides is 80% identical to a second sequence consisting of 10 nucleotides comprising the first sequence. It can therefore be understood that, in the context of the present invention, the identity of the sequence preferably relates to the percentage of nucleotides or amino acids in the sequence having the same position in two or more sequences of the same length. Specifically, the "identity%" of two amino acid sequences or two nucleotide sequences can be determined by aligning the sequences for optimal comparison purposes and comparing the amino acids or nucleotides at corresponding positions, for example, a gap can be introduced in either sequence to best align with the other sequence. A gap is usually regarded as a non-identical position, regardless of its actual position in the pair. The "best alignment" is generally the alignment of two sequences that produces the highest percent identity. The percent identity is determined by the number of identical nucleotides in the compared sequences, i.e., % identity = number of identical positions / total number of positions x 100. Other known mathematical algorithms can also be used by those skilled in the art to determine the percent identity between two sequences.

[0065] In the present invention, the term "plasmid backbone" is generally a circular DNA molecule or a linear DNA molecule that can autonomously replicate and express the inserted target gene in the cell. The backbone plasmid may contain regulatory sequences such as promoters, replicons, transcription and translation start and stop codons. The backbone plasmid is usually connected to the target gene to form a complete expression vector that can express a specific output in the cell.

[0066] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0067] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0068] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0069] The nucleotide or amino acid sequence information used in the present invention is as follows:

[0070] SEQ ID No.1: GFSLSSNA

[0071] SEQ ID No.2:ITNTGNT

[0072] SEQ ID No.3: ARFRGAGIDFYGYAMNRLDL

[0073] SEQ ID No.4: QSVYNNNY

[0074] SEQ ID No.5:EASTLAS

[0075] SEQ ID No.6: AGGYKSSSVDND

[0076] SEQ ID No.7: GFSLSSYA

[0077] SEQ ID No.8: IHSRGST

[0078] SEQ ID No.9:ARESLDYNTGNMIDI

[0079] SEQ ID No.10: QSISTY

[0080] SEQ ID No.11: RASTLAS

[0081] SEQ ID No.12: QQTASSTNLGNI

[0082] SEQ ID No.13:

[0083] QSLEESGGRLVTPGTPLTLTCTASGFSLSSNAISWVRQAPGKGLEYIGIITNTGNTYYATWAKGRFTLSKTSSTTVDLKITSPTTEDMATYFCARFRGAGIDFYGYAMNRLDLWGQGTLVTVSS

[0084] SEQ ID No.14:

[0085] AAVLTQTPSPVSAAVGGTVTISCQASQSVYNNNYLAWYQQKPGQPPKLLIYEASTLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGGYKSSSVDNDFGGGTEVVVK

[0086] SEQ ID No.15:

[0087] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAGCAATGCAATTAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTACTAATACTGGTAACACATACTACGCGACCTGGGCGAAAGGCCGATTCACCCTCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCAACAACCGAGGACATGGCCACCTATTTCTGTGCCAGATTCCGTGGTGCTGGTATTGATTTTTATGGTTATGCTATGAATCGATTGGATCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA

[0088] SEQ ID No.16:

[0089] GCCGCCGTGCTGACCCAGACACCATCCCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGGCCAGTCAGAGTGTTTATAATAACAACTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGAAGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAAAAGTAGTAGTGTTGATAATGATTTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0090] SEQ ID No.17:

[0091] QSMGESGGRLVTPGTPLTLTCTVSGFSLSSYAMNWVRQAPGKGLEWIGIIHSRGSTDYANWAKGRFTISKTSTTVDLRITSPTTEDTATYFCARESLDYNTGNMIDIWGPGTLVTVSL

[0092] SEQ ID No.18:

[0093] DVVMTQTPASVEAVVGGTVTIKCQASQSISTYLAWYQQKPGQPPKLLIYRASTLASGVSARFRGSGSGTQFTLTISGVECADAATYYCQQTASSTNLGNIFGGGTEVVVK

[0094] SEQ ID No.19:

[0095] CAGTCGATGGGGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGCAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTCATAGTCGTGGTAGTACCGACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAGAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGAGTCTCTTGATTATAATACTGGTAATATGATTGACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTTA

[0096] SEQ ID No.20:

[0097] GATGTTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGTTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTACCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCTCGGCGCGGTTCAGAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGACTGCAAGTAGTACTAACCTTGGGAATATTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0098] Expression and Purification of Human IgE Fc Protein in Example 1

[0099] 1. Gene Synthesis

[0100] The amino acid sequence of human IgE Fc (P01854-1, amino acid C105-K428) was downloaded from the uniprot database, and the rabbit IgG1 heavy chain signal peptide (METGLRWLLLVAVLKGVQC) was added to the N-terminus of the sequence, and a 6×His tag was added to the C-terminus. After translation and codon optimization, it was handed over to Sesophy (Jiangsu Sesophy Biotechnology Co., Ltd.) for gene synthesis and synthesized into the pCDNA3.1 vector. The synthesized recombinant plasmid was transformed into DH5α competent cells, spread on Amp+ medium plates, single colonies were screened, and the single colonies were shaken to obtain the pCDNA3.1-IgE Fc recombinant strain.

[0101] 2. Protein Expression

[0102] The pCDNA3.1-IgE Fc recombinant strain was activated and cultured, and the pCDNA3.1-IgE Fc recombinant plasmid was extracted according to the instructions of the endotoxin-free plasmid extraction kit. The pCDNA3.1-IgE Fc recombinant plasmid was transfected into Expi293F suspension cells (purchased from Gibco) for protein expression.

[0103] The transfection steps are as follows:

[0104] (1) Count the cells on the day of transfection, and the viability should be above 95% and the density should be 3×106 cells / mL. Take the transfection of 25mL cells as an example: take 20μg of plasmid, dilute with 1.5ml Opti-MEM reduced serum medium, mix gently and let stand for 5min; then take ExpiFectamine TM 80 μl of 293 reagent was diluted with 1.5 mL of Opti-MEM reduced serum medium, mixed thoroughly and allowed to stand for 5 min; the plasmid and transfection reagent were mixed after standing, mixed gently and allowed to stand for another 10 min.

[0105] (2) After standing, add the plasmid-transfection reagent mixture dropwise to 25 mL of cells, gently shake the shaker while adding, and finally place in a shaker for incubation (37°C, 8% CO2, 125 rpm).

[0106] (3) 18-24h after transfection, add 150μl ExpiFectamine TM 293Transfection Enhancer 1 and 1.5ml ExpiFectamine TM 293Transfection Enhancer 2, and continue to culture on a shaker.

[0107] (4) The expression product can be collected 5-7 days after transfection.

[0108] 3. Protein purification

[0109] The IgE Fc recombinant protein was purified using a Ni column (Sanggong).

[0110] (1) Collect the cell supernatant after transfection and expression by centrifugation, and filter the cell supernatant with a 0.45 μm filter membrane;

[0111] (2) First, wash the resin with 5 to 10 column volumes of pure water at 50 to 150 cm / h to remove ethanol, and then equilibrate the medium with 5 to 10 column volumes of binding buffer (20 mM PB, 0.5 M NaCl, pH 7.4) at 150 to 600 cm / h to ensure that the composition and pH of the solution in the medium are consistent with those of the sample;

[0112] (3) Start loading sample at a flow rate of 150 cm / h;

[0113] (4) After loading, wash with 10 to 20 column volumes of washing solution (20 mM PB, 0.5 M NaCl, 1 to 30 mM imidazole, pH 7.4) at 150 cm / h to clean non-specifically adsorbed impurities, and collect the washing solution for subsequent analysis;

[0114] (5) Elute the target protein with 5-10 volumes of elution buffer (20 mM PB, 0.5 M NaCl, 500 mM imidazole, pH 7.4) and collect the eluate;

[0115] (6) The collected target protein eluate was ultrafiltered with 1× PBS to remove imidazole, concentrated to a concentration of 1 mg / ml after ultrafiltration, and stored at -20°C for future use.

[0116] like Figure 1 As shown, after the purification, the protein was subjected to SDS-PAGE electrophoresis analysis, and the molecular weight of the IgE Fc protein was below 80KD, which was in line with expectations (the IgE Fc monomer was around 36KD and was in a dimerized form under a non-reduced state), and the purity was more than 90%.

[0117] Example 2 Preparation of human IgE monoclonal antibody

[0118] 1. Rabbit immunization

[0119] Two New Zealand rabbits aged 6-8 weeks, weighing about 2 kg, were taken, and each rabbit was immunized with 30 μg of the human IgE Fc protein prepared in Example 1. In the first immunization, the IgE Fc protein was emulsified with Freund's complete adjuvant in equal volumes, and the New Zealand rabbits were immunized by multiple subcutaneous injections. A total of three immunizations were performed, with the second immunization being performed two weeks after the first immunization, and the third immunization being performed one week later. The antigen was emulsified with Freund's incomplete adjuvant for the second and third immunizations, and the immunization dose and method remained unchanged.

[0120] After the third immunization, a small amount of blood was collected from behind the ear and the serum titer was determined by indirect ELISA.

[0121] (1) Antigen coating: Dilute the antigen to 2ug / ml with PBS buffer, add 100ul per well to a 96-well ELISA plate, and incubate overnight at 4°C. Wash the coated ELISA plate three times with a plate washer and tap dry, add 100ul of blocking solution per well, incubate in a 37°C incubator for 2h, and then wash the blocked ELISA plate three times with a plate washer and tap dry.

[0122] (2) Incubation with primary antibody: dilute alpaca serum with PBS buffer, add 100 μl of the diluted serum to the corresponding wells, and incubate at 37°C for 1 h. After incubation, wash the ELISA plate three times in a plate washer and dry.

[0123] (3) Incubation with secondary antibody: Add 100 μl of HRP-labeled goat anti-rabbit secondary antibody to the corresponding wells and incubate at 37°C for 30 min. Wash the plate three times in a plate washer and dry.

[0124] (4) Color development: Add 100 μl of the mixed color development solution to each well of a 96-well plate and incubate at 37°C for 15 min.

[0125] (5) Stopping and reading: Add stop solution to each well of the 96-well plate, 100 μl, and then place the plate in an ELISA reader for reading. Set the detection wavelength to 450 nm and read the test results. An OD value greater than 2.1 times that of the negative control is considered positive.

[0126] Table 1. IgE-Fc antigen protein coated plate, rabbit serum titer test

[0127]

[0128] The ELISA titer test results showed that the titers of R0166 and R0167 sera were both greater than 512K, and the titers met the standards.

[0129] (6) The same amount of antigen was emulsified with Freund's incomplete adjuvant and injected intraperitoneally into R0166 rabbits as the last impact immunization before sorting.

[0130] 2. B cell sorting

[0131] (1) Preparation of single cell suspension

[0132] Rabbit peripheral blood was collected to obtain cell suspension. Ficoll-Paque PLUS cell separation solution and cell suspension were added to a test tube at a volume ratio of 1:2. The tube was centrifuged at 1500rpm for 15 minutes. The middle layer of the test tube after centrifugation is generally a mononuclear cell layer. The mononuclear cell layer was aspirated and SOL024 solution was added. The tube was centrifuged at 1500rpm for 10 minutes. The supernatant was removed after centrifugation and 500μL SOL024 solution was added to resuspend the cells.

[0133] (2) Flow cytometry sorting of MBC

[0134] The target MBCs can be sorted out from the cell suspension one by one by flow cytometry and placed in a 96-well plate. The antigen protein is fluorescently labeled by taking advantage of the fact that the BCR on the surface of MBC can specifically bind to the antigen protein. The MBCs that specifically bind to the antigen protein can be sorted out from the cell suspension one by one by flow cytometry into a 96-well PCR plate and resuspended in 5 μl of nuclease-free water.

[0135] 3. Cell lysis and heavy and light chain gene amplification

[0136] A single-cell antibody gene amplification system was used to obtain the variable region fragments of the heavy chain and light chain.

[0137] (1) One-step reverse transcription amplification of Kappa chain and Heavy chain variable region fragments

[0138] The 50 single cell suspensions sorted were amplified using the Single Cell Sequence Specific Amplification Kit.

[0139] Some of the reverse transcription amplification primers are as follows:

[0140] Fκ1:CCCACTCAGCTGCTGGGGCTC;

[0141] Rκ1: GCGGGAAGATGAGGACAGTAGGTGC;

[0142] FH1:GCTGGCTTCTCCTGGTCGCTGTGC;

[0143] RH1:GCAGCAGGGGGCCAGTGGGAAGACTG.

[0144] The reaction system is as follows:

[0145] 2×Reaction Mix 7.5μl, 2×Reaction Mix 2.5μl, Kappa chain and Heavy chain F+R primer combination 0.5μl (the final concentration of each primer is 0.1μM), 2×Reaction Mix 2.5μl, RT / Taq enzyme 0.2μl, nuclease-free water 1.8μl, a total of 10μl, was added to the PCR plate wells and mixed with the cell suspension.

[0146] Add single cells, cover the tube tightly, and immediately place in a -70°C refrigerator for 2 minutes. Centrifuge at 3,000 rpm (1,000 × g) for 2 minutes, and immediately place in a PCR instrument to perform the following reaction:

[0147]

[0148] 4. Construction of expression plasmid and plasmid extraction

[0149] (1) Amplification of target gene fragment

[0150] According to the constructed Kappa chain and Heavy chain backbone vectors and the Kappa chain and Heavy chain variable region templates obtained by one-step reverse transcription amplification, the downstream primers and upstream primers of the Kappa chain and Heavy chain were designed to amplify the variable region fragments.

[0151] Primers:

[0152] Fκ2: GGCTCCCAGGTGCCAGATGT;

[0153] Rκ2: GTAGGTGCAACTGGATCACC;

[0154] FH2:GCTCAAAGGTGTCCAGTGT;

[0155] RH2:GATGGAGCCTTAGGTTGCCC.

[0156] Phanta polymerase (purchased from Novazon) was used to perform PCR amplification using the one-step reverse transcription amplification product as a template, and the reaction system was 50 μL. Template 3 μL, upstream primer (10 μM) 2.5 μL, downstream primer (10 μM) 2.5 μL, dNTPs 1 μL, phanta polymerase 1 μL, 2× Reaction Buffer 25 μL, and sterile water 15 μL.

[0157] The PCR reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s; 30 cycles; and 72°C for 5 min to end the program.

[0158] (2) Cloning of PCR amplification products

[0159] The PCR products were subjected to 1% agarose gel electrophoresis, and the Kappa chain and Heavy chain amplified fragments were recovered using a PCR product recovery kit (purchased from Tiangen), and a total of 24 groups of Kappa chain and Heavy chain amplified fragments were obtained.

[0160] The 24 groups of amplified paired antibody light and heavy chain Fab base sequences were connected to the pcDNA3.4 vector containing rabbit IgG1 CL and rabbit IgG1 CH1+Fc fragments using the ClonExpress II One Step Cloning Kit to construct light and heavy chain recombinant expression plasmids.

[0161] After the positive strains were identified by colony PCR, the target bacteria were cultured and plasmids were extracted using a plasmid extraction kit. For specific steps, refer to the plasmid extraction instructions in the kit.

[0162] 5. Transient expression

[0163] 24 sets of Kappa chain and Heavy chain plasmid combinations were transfected into Expi293F cells for expression, referring to the method of IgE Fc in Example 1.

[0164] 6. Cell supernatant detection

[0165] Indirect ELISA test to see if the cell supernatant is positive

[0166] (1) Antigen coating: Dilute IgE Fc antigen to 2ug / ml with PBS buffer, add 100ul per well to a 96-well ELISA plate, and incubate overnight at 4°C. Wash the coated ELISA plate three times with a plate washer and tap dry, add 100ul of blocking solution per well, incubate in a 37°C incubator for 2h, and then wash the blocked ELISA plate three times with a plate washer and tap dry.

[0167] (2) Incubation with primary antibody: Place 100ul of cell supernatant in each well, take the same amount of positive antibody as the positive control, and blank culture cell supernatant as the negative control, and incubate at 37°C for 1 hour. After incubation, wash the ELISA plate in a plate washer three times and dry it.

[0168] (3) Incubation with secondary antibody: Add 100 μl of HRP-labeled goat anti-rabbit to each well and incubate at 37°C for 30 min. Wash the plate three times in a plate washer and dry.

[0169] (4) Color development: Add 100 μl of the mixed color development solution to each well of a 96-well plate and incubate at 37°C for 15 min.

[0170] (5) Stop and read: Add stop solution to the 96-well plate, 100 μl per well, and then place it in an ELISA reader for reading. Set the detection wavelength to 450 nm and read the test results.

[0171] Table 2 Layout of antigen protein coated plate and cell supernatant plate

[0172] 1 2 3 4 A 1M1B1 1M1E2 1M1G4 PBS B 1M1C1 1M1F2 1M1A5 PBS C 1M1D1 1M1G2 1M1C5 Immune serum D 1M1E1 1M1A3 1M1B6 Immune serum E 1M1G1 1M1B3 1M1C6 F 1M1A2 1M1C3 1M1G6 G 1M1B2 1M1G3 1M1A7 H 1M1C2 1M1B4 1M1B7

[0173] Table 3 ELISA test results

[0174] 1 2 3 4 A 0.0576 0.1987 2.2817 0.0481 B 2.3299 0.053 2.0066 0.0481 C 2.2366 2.1104 0.9359 2.4203 D 0.0564 0.2363 2.0654 2.4494 E 0.1436 0.0879 0.0482 F 0.2348 0.0861 0.0493 G 0.1854 0.0808 2.0024 H 2.0557 2.3256 0.052

[0175] The results of ELISA titer test showed that 9 positive clones were screened out from 24 clones, and the test results were >2.0.

[0176] Example 3: Selection of anti-human IgE monoclonal antibodies for detection of IgE levels in human serum (sandwich method)

[0177] 1. Expression and purification of antibodies:

[0178] The 9 monoclonal antibodies with Elisa test results greater than 2 in Example 2 were purified.

[0179] Antibody purification:

[0180] (1) Centrifuge the antibody-expressing Expi 293F cell culture, collect the supernatant, and filter it with a 0.22 μm filter membrane;

[0181] (2) The supernatant was passed through 1 ml of Protein A agarose matrix and washed with 5 column volumes of PBS buffer;

[0182] (3) Elute with 1 ml of 3 M glycine buffer (pH adjusted to 3.0 with hydrochloric acid) and collect the eluate;

[0183] (4) Neutralize with 100 μl 1 M Tris-HCl buffer (8.0);

[0184] (5) Dialyze three times against PBS buffer.

[0185] 9 antibodies were purified with a concentration of ≥1 mg / ml. Figure 2 shown.

[0186] 2. Nine anti-human IgE monoclonal antibodies were used as capture antibodies and coated on carboxyl magnetic beads and labeled with acridinium ester. In addition, IBIS anti-human IgE mouse monoclonal antibody (4C3) was used as capture antibody and coated on carboxyl magnetic beads, and IBIS anti-human IgE mouse monoclonal antibody (1A2) was used as detection antibody and labeled with acridinium ester as a control group.

[0187] (1) Preparation of capture antibody magnetic beads

[0188] 1) Activation of magnetic beads: Take 10 mg of carboxyl magnetic beads (3 μm, JSR MS300) in a 2 ml centrifuge tube, add 1 ml of washing buffer (0.1 M MES, pH 5.0), vortex and shake, place the centrifuge tube on the magnetic rack, wait for the magnetic beads to be completely adsorbed to the side wall, use a pipette to remove the supernatant, and repeat the washing 3 times; add 1 ml of binding buffer (1 mg / ml EDC, 0.1 M MES, pH 5.0), invert and mix well, and incubate at room temperature for 15 minutes;

[0189] 2) Antibody coating: Place the centrifuge tube on a magnetic rack, wait until the magnetic beads are completely adsorbed to the side wall, remove the supernatant with a pipette, resuspend the magnetic beads with 1 ml of washing buffer, add 0.1 mg of anti-human IgE antibody protein, invert and mix, and incubate at room temperature for 2 hours;

[0190] 3) Blocking of magnetic beads: Add 20 μl of blocking agent (JSR CE510:CE210=9:1) to the centrifuge tube, mix by inversion and incubate at room temperature for 1 hour;

[0191] 4) Cleaning: Place the centrifuge tube on the magnetic rack, wait until the magnetic beads are completely adsorbed to the side wall, remove the supernatant with a pipette tip, resuspend the magnetic beads with 1 ml TBST buffer (25 mM Tris-HCl, 140 mM NaCl; 3 mM KCl, 0.5‰ v / v Tween 20, pH 7.4), repeat 3 times, and finally use 10 ml magnetic bead storage solution (25 mM Tris-HCl, 140 mM NaCl; 3 mM KCl, 0.5‰ v / v Tween 20, 0.1% m / v BSA, 0.1% Proclin 300, pH 7.4).

[0192] (2) Acridinium ester labeled anti-human IgE antibody

[0193] 1) Take 100 μg of anti-human IgE antibody, add 5 μl of 5 mg / ml acridinium ester (NSP-DMAE-NHS), and mix by inversion at room temperature for 30 minutes in the dark;

[0194] 2) Blocking: Add 100 μl of blocking solution (10 mg / ml DL-lysine, 0.1 M carbonate buffer, pH 9.0), mix by inversion at room temperature for 30 minutes in the dark;

[0195] 3) Desalting: Prepare G-25 one day in advance and swell it in excess deionized water, take 10 ml to load the column, balance it with 5 times the volume of the column bed, pH 6.8 0.1M PB buffer, load the labeled antibody (volume ≤ 1 ml), collect the effluent, mark it as the first tube, elute it with 1 ml PB buffer, collect the effluent and mark it as the second tube; repeat the elution with 1 ml PB buffer and collect it in the sixth tube; take a sample of the collected liquid and measure the protein concentration using the BCA protein concentration detection kit, and take the tube with the highest concentration for subsequent experiments.

[0196] 4) Preparation of chemiluminescent detection antibody working solution: The labeled antibody was prepared with antibody diluent (25 mM Tris-HCl, 140 mM NaCl; 3 mM KCl, 0.5‰ v / v Tween 20, 0.1% m / v BSA, 0.1% Proclin 300, pH 7.4) to prepare a 1 μg / ml working solution and stored at 4°C.

[0197] 3. Chemiluminescent immunoassay for total human IgE

[0198] Take the international standard total IgE diluent (PBS, 0.1% m / v BSA) to dilute to 50 IU / ml standard, and the diluent as a negative control, put the detection kit (including anti-human IgE antibody magnetic beads and labeled acridinium ester anti-human IgE antibody) into the Yingkai Shine i2910 fully automatic chemiluminescence to perform sandwich chemiluminescence detection of the luminescence value of 50 IU / ml total IgE standard and negative control (diluent), the reaction mode is as follows:

[0199] Step 1: Pipette 5 μl sample, 20 μl anti-human IgE antibody magnetic beads, mix and incubate at 37°C for 20 minutes;

[0200] Step 2: Cleaning;

[0201] Step 3: Pipette 100 μl of labeled acridinium ester anti-human IgE antibody working solution, mix and incubate at 37°C for 20 minutes;

[0202] Step 4: Cleaning;

[0203] Step 5: Add 100 μl pre-excitation solution and 100 μl excitation solution, and detect the luminescence value.

[0204] The signal-to-noise ratio was calculated as shown in Table 4.

[0205] Table 4

[0206]

[0207] The results showed that the signal-to-noise ratio was the highest when 1M1C2 was selected as the capture antibody and 1M1G2 was selected as the detection antibody. This combination was selected to test the total IgE standard curve. The amino acid sequences of the heavy chain variable region and the light chain variable region of 1M1C2 were SEQ ID No.13 and SEQ ID No.14, respectively; the amino acid sequences of the heavy chain variable region and the light chain variable region of 1M1G2 were SEQ ID No.18 and SEQ ID No.19, respectively; the above two antibodies can be respectively detected by nucleotide sequences such as SEQ ID No.15-16, SEQ ID After the nucleic acid fragments of No.19-20 are combined with the vector plasmid and introduced into engineering cells such as HEK293T, the obtained nucleic acid fragments are expressed in the introduced engineering cells. The combination of IBIX 4C3+1A2 is selected as a control. The control kit includes IBIX anti-human IgE antibody (4C3) magnetic beads and labeled acridinium ester IBIX anti-human IgE antibody (1A2). The concentrations of the standard products are set to: 0, 0.01, 0.02, 0.05, 0.1, 0.35, 0.7, 3.5, 17.5, 50, 100 IU / ml to test the luminescence value and calculate the signal-to-noise ratio. As shown in Table 5.

[0208] Table 5

[0209]

[0210]

[0211] The results showed that when the total IgE level was 0.02IU / ml, the signal-to-noise ratio of the luminescence value of the 1M1C2+1M1G2 combination test was greater than 2, while when the total IgE level of the Aibixin 4C3+1A2 combination was 0.1IU / ml, the signal-to-noise ratio of the luminescence value of the test was greater than 2. Therefore, the sensitivity of the total IgE test of 1M1C2+1M1G2 was ≤0.02IU / ml.

[0212] The test results of 1M1C2+1M1G2 were used to prepare a standard curve with total IgE (IU / ml) as the horizontal axis and the luminescence value as the vertical axis. Figure 3 As shown, 4PLC calibration algorithm formula: y = (ad) / (1 + (x / c) b )+d; a=788.87005615, b=0.99495542, c=237.00404358, d=7503388; main curve correlation coefficient: 0.999967270863;

[0213] The standard curve was prepared by taking the IBIX 4C3+1A2 test results, with total IgE (IU / ml) as the horizontal axis and the luminescence value as the vertical axis. Figure 3 As shown, 4PLC calibration algorithm formula: y = (ad) / (1 + (x / c) b)+d; a=1127.23403855, b=1.00555051, c=961.27647106, d=18187526.7767; Main curve correlation coefficient: 0.999855290844.

[0214] Example 4 Binding ability test of anti-human IgE antibodies

[0215] Anti-IgE monoclonal antibody affinity determination

[0216] (1) Coating: Dilute the receptor protein IgE Fc to 0.5 μg / ml with coating solution, add 100 μl / well, and incubate at 2-8°C overnight;

[0217] (2) Blocking: Wash the plate three times with washing solution, add 300 μl / well blocking solution, and block at room temperature for 1 hour;

[0218] (3) Washing: Wash the plate 3 times with detergent and pat dry for later use;

[0219] (4) Sample loading: dilute the antibody 5-fold into 7 concentration gradients with a starting concentration of 4000 ng / μl, add 100 μl / well into the microplate, and incubate at room temperature for 1 hour;

[0220] (5) Washing: Wash the plate three times with detergent and pat dry;

[0221] (6) Add secondary detection antibody: enzyme-linked antibody (1C2 and 1G2 are HRP-labeled goat anti-rabbit antibodies, IBIS 4C3 and 1A2 are HRP-labeled goat anti-mouse antibodies) diluted to the working concentration, mixed, 100 μl / well, incubated at room temperature for 1 hour;

[0222] (7) Washing: Wash the plate three times with detergent and pat dry;

[0223] (8) Color development: Use an 8-channel gun to add 200 μl / well of color development solution and place at room temperature in the dark for 15 min;

[0224] (9) Termination: Use an 8-channel gun to add 100 μl / well of stop solution to terminate the reaction;

[0225] (10) Measure the signal value at 450 nm using an ELISA reader.

[0226] Table 5, ELISA saturation concentration method to measure the OD450 results of anti-human IgE monoclonal antibodies:

[0227]

[0228]

[0229] Table 6, EC50 values ​​of anti-human IgE monoclonal antibodies determined by ELISA saturation concentration method:

[0230] 1M1C2 1M1G2 Aibixin 4C3 Aibixin 1A2 EC50(ng / ml) 8.448 8.086 14.93 15.78

[0231] like Figure 4 As shown, the EC50 values ​​of 1M1C2 (comprising CDRs shown in amino acid sequences such as SEQ ID No.1-6) and 1M1G2 (comprising CDRs shown in amino acid sequences such as SEQ ID No.7-12) (the above two antibodies can be obtained by respectively combining nucleic acid fragments with nucleotide sequences such as SEQ ID No.15-16 and SEQ ID No.19-20 with vector plasmids and then introducing them into engineering cells such as yeast, and expressing them in the introduced engineering cells) are both lower than those of Aibixin mouse monoclonal antibodies 4C3 and 1A2, indicating that the affinity is also higher than that of Aibixin mouse monoclonal antibodies 4C3 and 1A2.

[0232] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. An anti-human IgE antibody, characterized in that The anti-human IgE antibody comprises a heavy chain variable region and a light chain variable region, and the anti-human IgE antibody has one or more of the following technical features: 1) the complementarity determining region of the heavy chain variable region includes CDR-H1 with the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 7; 2) the complementarity determining region of the heavy chain variable region includes CDR-H2 with an amino acid sequence as shown in SEQ ID No. 2 or SEQ ID No. 8; 3) the complementarity determining region of the heavy chain variable region includes CDR-H3 with an amino acid sequence as shown in SEQ ID No. 3 or SEQ ID No. 9; 4) the complementarity determining region of the light chain variable region includes CDR-L1 with the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 10; 5) the complementarity determining region of the light chain variable region includes CDR-L2 with an amino acid sequence as shown in SEQ ID No. 5 or SEQ ID No. 11; 6) The complementarity determining region of the light chain variable region includes CDR-L3 having an amino acid sequence as shown in SEQ ID No.6 or SEQ ID No.

12.

2. The anti-human IgE antibody according to claim 1, characterized in that , the complementary determining region of the heavy chain variable region of the anti-human IgE antibody includes CDR-H1 as shown in SEQ ID No.1, CDR-H2 as shown in SEQ ID No.2, and CDR-H3 as shown in SEQ ID No.3; or, the complementary determining region of the heavy chain variable region includes CDR-H1 as shown in SEQ ID No.7, CDR-H2 as shown in SEQ ID No.8, and CDR-H3 as shown in SEQ ID No.

9.

3. The anti-human IgE antibody according to claim 1, characterized in that , the complementary determining region of the light chain variable region of the anti-human IgE antibody includes a CDR-L1 with an amino acid sequence as shown in SEQ ID No.4, a CDR-L2 with an amino acid sequence as shown in SEQ ID No.5, and a CDR-L3 with an amino acid sequence as shown in SEQ ID No.6; or, the complementary determining region of the light chain variable region includes a CDR-L1 with an amino acid sequence as shown in SEQ ID No.10, a CDR-L2 with an amino acid sequence as shown in SEQ ID No.11, and a CDR-L3 with an amino acid sequence as shown in SEQ ID No.

12.

4. The anti-human IgE antibody according to claim 1, characterized in that The complementary determining region of the heavy chain variable region in the anti-human IgE antibody includes CDR-H1 as shown in SEQ ID No.1, CDR-H2 as shown in SEQ ID No.2, and CDR-H3 as shown in SEQ ID No.3, and the complementary determining region of the light chain variable region includes CDR-L1 as shown in SEQ ID No.4, CDR-L2 as shown in SEQ ID No.5, and CDR-L3 as shown in SEQ ID No.6; or, the complementary determining region of the heavy chain variable region in the anti-human IgE antibody includes CDR-H1 as shown in SEQ ID No.7, CDR-H2 as shown in SEQ ID No.8, and CDR-H3 as shown in SEQ ID No.9, and the complementary determining region of the light chain variable region includes CDR-L1 as shown in SEQ ID No.10, CDR-L2 as shown in SEQ ID No.11, and CDR-L3 as shown in SEQ ID No.

12.

5. The anti-human IgE antibody according to claim 1, characterized in that The anti-human IgE antibody further comprises one or more of the following features: a) the amino acid sequence of the heavy chain variable region of the anti-human IgE antibody is shown in one of SEQ ID No. 13 and 17; or b) the amino acid sequence of the heavy chain variable region of the anti-human IgE antibody has more than 80% identity with the amino acid sequence shown in one of SEQ ID No. 13 and 17, and has the amino acid sequence function defined in a); c) the amino acid sequence of the light chain variable region of the anti-human IgE antibody is shown in one of SEQ ID No. 14 and 18; d) The amino acid sequence of the light chain variable region of the anti-human IgE antibody has more than 80% identity with the amino acid sequence shown in one of SEQ ID No. 14 and 18, and has the amino acid sequence function defined in c).

6. The anti-human IgE antibody according to claim 5, characterized in that The anti-human IgE antibody comprises a peptide segment having an amino acid sequence of a heavy chain variable region such as SEQ ID No.13 and an amino acid sequence of a light chain variable region such as SEQ ID No.14; or a peptide segment having an amino acid sequence of a heavy chain variable region such as SEQ ID No.17 and an amino acid sequence of a light chain variable region such as SEQ ID No.

18.

7. An isolated polynucleotide, characterized in that The polynucleotide encodes one or more of the following peptide segments: 1) The heavy chain variable region of the anti-human IgE antibody according to any one of claims 1 to 6; 2) The light chain variable region of the anti-human IgE antibody according to any one of claims 1 to 6; 3) The anti-human IgE antibody according to any one of claims 1 to 6.

8. A nucleic acid construct, characterized in that The nucleic acid construct contains the polynucleotide according to claim 7.

9. An isolated engineered cell, characterized in that The engineered cell contains the nucleic acid construct of claim 8 or the polynucleotide of claim 7.

10. Use of the anti-human IgE antibody according to any one of claims 1 to 6, the polynucleotide according to claim 7 or the nucleic acid construct according to claim 8 in the preparation of a product for treating a disease caused by type I hypersensitivity reaction, a product for treating an autoimmune disease or a product for detecting an allergen.

11. The use according to claim 10, characterized in that The disease caused by the type I hypersensitivity reaction is selected from one or more of sinusitis, allergic rhinitis, food allergy, chronic urticaria and atopic dermatitis.

12. A diagnostic kit, characterized in that: The diagnostic kit contains the anti-human IgE antibody according to any one of claims 1-6.

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