An anti-human IgE antibody and its uses

Abstract

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

Description

Technical Field

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

[0002] Immunoglobulin E (IgE) is a class of antibodies that exist only in mammals. IgE consists of two identical heavy chains and light chains, and each heavy chain contains 4 constant ε domains (Cε1-4). The main function of IgE is to generate immunity against parasites. Additionally, 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 the response to allergens, such as allergy drugs, bee stings, and antigens used in desensitization immunotherapy. Although the content of IgE is low (the IgE content in normal human serum only accounts for 0.05% of all Ig classes), it can trigger strong inflammatory reactions. The IgE content in the blood of patients with hereditary allergies is up to 10 times that of normal people. In various autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), the IgE level increases, and it is speculated that it has an important pathogenic effect on RA and SLE by inducing hypersensitivity reactions. Summary of the Invention

[0003] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide an anti-human IgE antibody and its uses to solve the problems in the prior art.

[0004] To achieve the above purpose and other related purposes, the present invention provides an anti-human IgE antibody, which includes 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 an amino acid sequence as 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 an amino acid sequence as shown in SEQ ID No.4 or SEQ ID No.10;

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

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

[0011] Preferably, the complementarity-determining regions of the heavy chain variable region of the anti-human IgE antibody include CDR-H1 having an amino acid sequence as shown in SEQ ID No. 1, CDR-H2 having an amino acid sequence as shown in SEQ ID No. 2, and CDR-H3 having an amino acid sequence as shown in SEQ ID No. 3; or, the complementarity-determining regions of the heavy chain variable region include CDR-H1 having an amino acid sequence as shown in SEQ ID No. 7, CDR-H2 having an amino acid sequence as shown in SEQ ID No. 8, and CDR-H3 having 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 containing the aforementioned isolated polynucleotide.

[0017] The present invention also provides an isolated engineered cell containing 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 the preparation of therapeutic products for diseases caused by type I hypersensitivity, therapeutic products for autoimmune diseases or allergen detection products.

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

[0020] As described above, an anti-human IgE antibody and its use according to 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. This monoclonal antibody combination is applied to an anti-human IgE magnetic particle chemiluminescence detection reagent, which can detect human IgE at 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 Shown is an SDS-PAGE identification diagram of the purified anti-human IgE monoclonal antibody of the present invention.

[0024] Figure 3 Shown is the standard curve 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, comprising a heavy chain variable region and a light chain variable region, and having 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 the amino acid sequence 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 the amino acid sequence 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 the amino acid sequence 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 complementarity-determining regions of the heavy chain variable region of the anti-human IgE antibody include CDR-H1 having an amino acid sequence as shown in SEQ ID No. 1, CDR-H2 having an amino acid sequence as shown in SEQ ID No. 2, and CDR-H3 having an amino acid sequence as shown in SEQ ID No. 3; or, the complementarity-determining regions of the heavy chain variable region include CDR-H1 having an amino acid sequence as shown in SEQ ID No. 7, CDR-H2 having an amino acid sequence as shown in SEQ ID No. 8, and CDR-H3 having an amino acid sequence as shown in SEQ ID No. 9.

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

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

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

[0037] In some specific 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 any one of SEQ ID No.13 and 17; or

[0039] b) an amino acid sequence having more than 80% identity with the amino acid sequence shown in any 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 obtained by substituting, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids to the amino acid sequence shown in any one of SEQ ID No.13 and 17, or an amino acid sequence obtained by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids at the N-terminus and / or C-terminus, and having the function of the amino acid sequence shown in any one of SEQ ID No.13 and 17. The amino acid sequence in b) may have 80%, 85%, 90%, 93%, 95%, 9 / 7% or more than 99% identity with any one of SEQ ID No.13 and 17.

[0041] In some specific 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 any one of SEQ ID No.14 and 18; or

[0043] d) an amino acid sequence having more than 80% identity with the amino acid sequence shown in any 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: the amino acid sequence shown in SEQ ID No. 14 or 18, which is obtained by substituting, deleting, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, or by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids at the N-terminus and / or C-terminus, and having the function of the amino acid sequence shown in SEQ ID No. 14 or 18. The amino acid sequence in d) may have an identity of 80%, 85%, 90%, 93%, 95%, 97%, or more than 99% with one of SEQ ID No. 14 and 18.

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

[0046] In some specific 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. Among them, the secretion signal peptide directs the anti-human IgE antibody to the periplasmic space of the host cell.

[0047] In some specific embodiments, the anti-human IgE antibody comprises a histidine tag. The histidine tag or polyhistidine tag is a sequence of 2 to 20 histidine residues attached to the anti-human IgE antibody. The histidine tag contains 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 an expression vector. The expression vector can be transformed, transduced or transfected into a host cell, enabling the genetic elements it carries to be 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 (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Viral vectors include: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). The vector can contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. Additionally, the vector can contain a replication origin. The vector can also include components to assist its entry into cells, including but not limited to, virus particles, liposomes or protein coats.

[0053] In some specific embodiments, the backbone plasmid of the expression vector can be selected from pET expression vectors, pCW expression vectors, pUC expression vectors, 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, which contains the aforementioned nucleic acid construct or the aforementioned polynucleotide.

[0055] In the present invention, the aforementioned engineered cell can be obtained by introducing the aforementioned nucleic acid construct into a host cell, or a cell with the exogenous aforementioned polynucleotide integrated into its genome is the aforementioned 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 cells include many cell types as follows, such as prokaryotic cells like Escherichia coli or Bacillus subtilis, fungal cells like yeast cells or Aspergillus, insect cells like Drosophila S2 cells or Sf9 cells, or animal cells like fibroblast cells, 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 the preparation of therapeutic products for diseases caused by type I hypersensitivity, therapeutic products for autoimmune diseases or allergen detection products.

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

[0059] In some specific embodiments, the autoimmune diseases are selected from one or more combinations of rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, AIDS, rheumatoid arthritis, inflammatory bowel disease, psoriasis, vasculitis, bronchial asthma, chronic obstructive pulmonary disease, eosinophilic sinusitis.

[0060] In some specific 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 "complementary determining region" or "CDR" generally refers to the region in an antibody that can form complementarity with an antigenic determinant in terms of spatial structure. The variability in an antibody is usually 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 usually both have 3 hypervariable regions (HVRs), and these regions can usually form complementarity with an antigenic determinant in terms of spatial structure, so the hypervariable region is also called the complementary determining region (CDR), that is, the heavy chain variable region usually includes three complementary determining regions, namely HCDR1, HCDR2 and HCDR3, and the light chain variable region usually includes three complementary determining regions, namely LCDR1, LCDR2 and LCDR3.

[0063] In the present invention, the term "monoclonal antibody" generally refers to a population of antibodies that are substantially identical (except for a few naturally occurring mutations that may be present). Monoclonal antibodies typically target specific determinants on an antigen.

[0064] In the present invention, the term "identity" means that if two or more sequences have the same nucleotide or amino acid length and order, then the sequences are identical. The percentage of identity generally describes the degree of similarity between two sequences, that is, it generally describes the percentage of nucleotides corresponding to the same nucleotides in the reference sequence at sequence positions. To determine the degree of identity (identity %), the sequences to be compared are usually made to have the same length, that is, the length of the longest sequence among 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 that contains the first sequence. Thus, it can be understood that in the context of the present invention, the identity of sequences preferably relates to the percentage of nucleotides or amino acids of sequences at the same positions in two or more sequences having 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, gaps can be introduced in either sequence to achieve optimal alignment with the other sequence. Gaps are generally considered as non-identical positions, regardless of their actual position in the alignment. "Optimal alignment" is generally the alignment of two sequences that results in the highest percentage of identity. The percentage of identity is determined by the number of identical nucleotides in the sequences being compared, that is, identity % = number of identical positions / total number of positions × 100. Those skilled in the art can also use other known mathematical algorithms to determine the percentage of 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 a cell. The backbone plasmid may contain regulatory sequences such as promoters, replicons, transcription and translation start and stop codons. The backbone plasmid is usually ligated with the target gene to form a complete expression vector that can express a specific product in a cell.

[0066] The following illustrates the embodiments of the present invention through specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed 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 protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than for limiting the protection scope of the present invention; in the description and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an" and "the" include the plural forms.

[0068] When an embodiment gives a numerical range, it should be understood that unless otherwise specified in the present invention, any value at both ends of each numerical range and any value between the two ends can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement 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] Download the amino acid sequence of human IgE Fc (P01854-1, amino acids C105-K428) from the UniProt database. Add the rabbit IgG1 heavy chain signal peptide (METGLRWLLLVAVLKGVQC) to the N-terminus of this sequence and add a 6×His tag to the C-terminus. After codon optimization of the translation, send it to Saisuofei Co., Ltd. (Jiangsu Saisuofei Biotechnology Co., Ltd.) for gene synthesis, and synthesize it onto the pCDNA3.1 vector. Transform the synthesized recombinant plasmid into DH5α competent cells, spread it on an Amp+ medium plate, screen out single colonies, and shake-culture this single colony to obtain the pCDNA3.1-IgE Fc recombinant strain.

[0101] 2. Protein expression

[0102] Activate and culture the pCDNA3.1-IgE Fc recombinant strain. According to the instructions of the endotoxin-free plasmid large-scale extraction kit, extract the pCDNA3.1-IgE Fc recombinant plasmid, and transfect the pCDNA3.1-IgE Fc recombinant plasmid into Expi293F suspension cells (purchased from Gibco) for protein expression.

[0103] The transfection steps are as follows:

[0104] (1) Perform cell counting on the day of transfection. The viability is above 95% and the density is 3×106 cells / mL. Taking 25 mL of transfected cells as an example: Take 20 μg of plasmid, dilute it with 1.5 ml of Opti-MEM serum-free medium, gently mix it evenly and then let it stand for 5 min; then take 80 μl of ExpiFectamine TM 293 reagent, add it to 1.5 mL of Opti-MEM serum-free medium for dilution, fully mix it evenly and then let it stand for ⑤ min; Mix the standing plasmid and transfection reagent, gently mix it evenly and then let it stand for 10 min.

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

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

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

[0108] 3. Protein Purification

[0109] Purify the IgE Fc recombinant protein using a Ni column (Sangon Biotech).

[0110] (1) Centrifuge to collect the cell supernatant after transfection and expression, and filter the cell supernatant through a 0.45 μm filter membrane;

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

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

[0113] (4) After loading the sample, wash away non - specifically adsorbed miscellaneous proteins with 10 - 20 column volumes of washing buffer (20 mM PB, 0.5 M NaCl, 1 - 30 mM imidazole, pH 7.4) at a flow rate of 150 cm / h, 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) Remove imidazole from the collected target protein eluate by ultrafiltration with 1×PBS, concentrate it to a concentration of 1 mg / ml after ultrafiltration, and store it at - 20 °C for later use.

[0116] As Figure 1 shown, the purified protein was analyzed by SDS - PAGE electrophoresis. The molecular weight of the IgE Fc protein is below 80 KD, meeting the expectation (the IgE Fc monomer is around 36 KD and exists as a dimer in the non - reduced state), and the purity can reach over 90%.

[0117] Example 2 Preparation of Human IgE Monoclonal Antibody

[0118] 1. Rabbit Immunization

[0119] Take 2 New Zealand rabbits at � - 8 weeks old, about 2 kg each, and immunize each rabbit with 30 μg of the human IgE Fc protein prepared in Example 1. At the first immunization, emulsify the IgE Fc protein with an equal volume of Freund's complete adjuvant and inject it subcutaneously at multiple points to immunize the New Zealand rabbits. Immunize three times in total. Immunize the second time two weeks after the first immunization, and then immunize the third time one week later. For the second and third immunizations, emulsify the antigen with Freund's incomplete adjuvant, and keep the immunization dose and method unchanged.

[0120] After three immunizations, a small amount of blood was collected from the posterior ear, and the serum titer was determined by indirect ELISA.

[0121] (1) Antigen coating: Dilute the antigen with PBS buffer to 2 μg / ml, add 100 μl per well to a 96-well ELISA plate, and incubate overnight at 4°C. Wash the coated ELISA plate 3 times with a plate washer and dry it. Add blocking solution at 100 μl per well, incubate in a 37°C incubator for 2 h, and then wash the blocked ELISA plate 3 times with a plate washer and dry it.

[0122] (2) Incubation with primary antibody: Dilute the serum of alpaca with PBS buffer, add the diluted serum to the corresponding wells at 100 μl per well, incubate at 37°C for 1 h. After incubation, place the ELISA plate in a plate washer, wash 3 times and dry it.

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

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

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

[0126] Table 1. Detection of rabbit serum titer by IgE-Fc antigen protein coating

[0127]

[0128] From the ELISA titer detection results, the serum titers of R0166 and R0167 are both greater than 512K, and the titer meets the standard.

[0129] (6) Emulsify the same amount of antigen with Freund's incomplete adjuvant and inject it intraperitoneally into rabbit R0166 as the last boost immunization before sorting.

[0130] 2. Sorting of B cells

[0131] (1) Preparation of single-cell suspension

[0132] Collect peripheral blood from rabbits to obtain cell suspension. Take Ficoll-Paque PLUS cell separation solution and cell suspension according to a volume ratio of 1:2 and add them to a test tube. Centrifuge at 1500 rpm for 15 minutes. Generally, the middle layer of the centrifuged test tube is the mononuclear cell layer. Aspirate the mononuclear cell layer, add SOL024 solution, centrifuge at 1500 rpm for 10 minutes, remove the supernatant after centrifugation, and resuspend the cells with 500 μL of SOL024 solution.

[0133] (2) Sort MBC by flow cytometry

[0134] Target MBC can be sorted out one by one from the cell suspension by flow cytometry and placed in a 96-well plate. Utilizing the property that the BCR on the surface of MBC can specifically bind to antigen protein, fluorescently label the antigen protein. The MBC that specifically binds to the antigen protein can be sorted out one by one from the cell suspension by flow cytometry into a 96-well PCR plate and resuspended in 5 μl of nuclease-free water.

[0135] 3. Lyse cells and amplify heavy and light chain genes

[0136] Use a single-cell antibody gene amplification system to obtain variable region fragments of heavy and light chains.

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

[0138] Add the sorted 50 single-cell suspensions to the Novizan single-cell one-step reverse transcription amplification reaction solution (SingleCell Sequence Specific Amplification Kit)

[0139] Some 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 7.5 μl, 2×Reaction Mix 2.5 μl, Kappa chain, Heavy chain F+R primer combination 0.5 μl (final concentration of each primer is 0.1 μM), 2×Reaction Mix 2.5 μl, RT / Taq enzyme 0.2 μl, nuclease-free water 1.8 μl, total 10 μl, added to the wells of the PCR plate and mixed with the cell suspension.

[0146] Add single cells, tighten the tube cap, immediately place in a -70 °C refrigerator for 2 min, centrifuge at 3,000 rpm (1,000 × g) for 2 min, and immediately put into a PCR instrument for the following reaction:

[0147]

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

[0149] (1) Amplification of target gene fragment

[0150] Design the downstream and upstream primers for the Kappa chain and Heavy chain according to the already 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, and amplify the variable region fragments.

[0151] Primers:

[0152] Fκ2: GGCTCCCAGGTGCCAGATGT;

[0153] Rκ2: GTAGGTGCAACTGGATCACC;

[0154] FH2: GCTCAAAGGTGTCCAGTGT;

[0155] RH2: GATGGAGCCTTAGGTTGCCC.

[0156] Use Phanta polymerase (purchased from Novoprotein) to perform PCR amplification with the one-step reverse transcription amplification product as the template. The reaction system is 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, sterile water 15 μL.

[0157] PCR reaction conditions are: 95 °C for 3 min; 95 °C for 15 s, 58 °C for 30 s, 72 °C for 30 s; cycle 30 times; end the program at 72 °C for 5 min.

[0158] (2) Cloning of PCR amplification products

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

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

[0161] After identifying positive strains by colony PCR, the target bacteria were cultured, and plasmid extraction was performed using a plasmid large extraction kit. The specific steps refer to the plasmid extraction instruction manual in the kit.

[0162] 5. Transient transfection and expression

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

[0164] 6. Detection of cell supernatant

[0165] Indirect ELISA was used to detect whether the cell supernatant was positive

[0166] (1) Antigen coating: Dilute the IgE Fc antigen to 2 μg / ml with PBS buffer, add 100 μl per well to a 96-well enzyme-labeled plate, and incubate overnight at 4°C. Wash the coated enzyme-labeled plate 3 times with a plate washer and drain. Add 100 μl of blocking solution per well, incubate in a 37°C incubator for 2 h, and then wash the blocked enzyme-labeled plate 3 times with a plate washer and drain.

[0167] (2) Incubation with primary antibody: Add 100 μl of cell supernatant to each well, take an equal amount of positive antibody as a positive control, and the cell supernatant from blank culture as a negative control, and incubate at 37°C for 1 h. After incubation, place the enzyme-labeled plate in a plate washer and wash 3 times and drain.

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

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

[0170] (5) Termination and reading: Add 100 μl of termination solution to each well of the 96-well plate, and then place it in an ELISA reader for reading. Set the detection wavelength to 450 nm and read the detection 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] From the ELISA titer detection results, it was found that the detection results of 9 positive clones screened from 24 clones were > 2.0.

[0176] Example 3 Selection of anti-human IgE monoclonal antibody for detecting IgE level in human serum (sandwich method)

[0177] 1. Expression and purification of antibodies:

[0178] Purify the 9 monoclonal antibodies with ELISA detection results greater than 2 in Example 2.

[0179] Purification of antibodies:

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

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

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

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

[0184] (5) Dialyze 3 times with PBS buffer.

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

[0186] 2. Coat 9 anti-human IgE monoclonal antibodies as capture antibodies on carboxyl magnetic beads and label them with acridinium ester respectively; in addition, coat the Absin anti-human IgE mouse monoclonal antibody (4C3) on carboxyl magnetic beads and label the Absin anti-human IgE mouse monoclonal antibody (1A2) 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, place the centrifuge tube on a magnetic stand. Wait until the magnetic beads are completely adsorbed on the side wall, aspirate the supernatant with a pipette tip, and repeat the washing 3 times; Add 1 ml of coupling buffer (1 mg / ml EDC, 0.1 M MES, pH 5.0), invert and mix well at room temperature for 15 minutes.

[0189] 2) Coating of antibody: Place the centrifuge tube on a magnetic stand. Wait until the magnetic beads are completely adsorbed on the side wall, aspirate the supernatant with a pipette tip, resuspend the magnetic beads with 1 ml of washing buffer, and add 0.1 mg of anti-human IgE antibody protein. Invert and mix well 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, invert and mix well at room temperature for 1 hour.

[0191] 4) Washing: Place the centrifuge tube on a magnetic stand. Wait until the magnetic beads are completely adsorbed on the side wall, aspirate the supernatant with a pipette tip, resuspend the magnetic beads with 1 ml of 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 of 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), invert and mix well in the dark at room temperature for 30 minutes.

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

[0195] 3) Desalting: Prepare G-25 one day in advance and swell it in an excess of deionized water. Take 10 ml and pack it into a column, equilibrate it with 5 column volumes of 0.1 M PB buffer at pH 6.8. Load the labeled antibody (volume ≤ 1 ml), collect the effluent, label it as tube 1, elute it with 1 ml of PB buffer, and collect the effluent and label it as tube 2; repeat the elution with 1 ml of PB buffer and collect up to tube 6; take samples of the collected solution and measure the protein concentration using a BCA protein concentration detection kit, and take the tube with the highest concentration for subsequent experiments.

[0196] 4) Prepare the chemiluminescence detection antibody working solution: Dilute the above-labeled antibody with an 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 store it at 4°C.

[0197] 3. Chemiluminescence immunoassay for detecting human total IgE

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

[0199] Step1: Pipette 5 μl of the sample, 20 μl of anti-human IgE antibody magnetic beads, and mix and incubate at 37°C for 20 minutes;

[0200] Step2: Wash;

[0201] Step3: Pipette 100 μl of the acridinium ester-labeled anti-human IgE antibody working solution, and mix and incubate at 37°C for 20 minutes;

[0202] Step4: Wash;

[0203] Step5: Add 100 μl of pre-excitation solution and 100 μl of excitation solution, and detect the luminescence value.

[0204] Calculate the signal-to-noise ratio. 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 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 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 light chain variable region of 1M1G2 were SEQ ID No.18 and SEQ ID No.19 respectively. The above two antibodies can be detected by nucleotide sequences such as SEQ ID No.15-16 and SEQ ID No.17. Nucleic acid fragments No. 19-20 were combined with a vector plasmid and introduced into engineered cells such as HEK293T, where they were expressed. The control kit, consisting of IBIXIN 4C3 magnetic beads and acridinium ester-labeled IBIXIN anti-human IgE antibody (1A2), was used. Luminescence values were measured and the signal-to-noise ratio was calculated, as shown in Table 5.

[0208] Table 5

[0209]

[0210]

[0211] The results showed that when the total IgE level was 0.02 IU / ml, the luminescence signal-to-noise ratio of the 1M1C2+1M1G2 combination test was greater than 2. However, when the total IgE level of the IBIS 4C3+1A2 combination was 0.1 IU / ml, the luminescence signal-to-noise ratio was greater than 2. Therefore, the total IgE sensitivity of the 1M1C2+1M1G2 test was ≤ 0.02 IU / 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 luminescence value as the vertical axis. Figure 3 As shown, the 4PLC calibration algorithm formula is: 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 test results of IBIXIN 4C3+1A2, with total IgE (IU / ml) as the horizontal axis and luminescence value as the vertical axis. Figure 3 As shown, the 4PLC calibration algorithm formula is: y = (ad) / (1 + (x / c) b) + d; a = 1127.23403855, b = 1.00555051, c = 961.27647106, d = 18187526.7767; Correlation coefficient of the main curve: 0.999855290844.

[0214] Example 4 Binding Ability Test of Anti-human IgE Antibody

[0215] Determination of Affinity of Anti-IgE Monoclonal Antibody

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

[0217] (2) Blocking: Wash the plate 3 times with washing buffer, add 300 μl / well of blocking solution, and block at room temperature for 1 hour;

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

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

[0220] (5) Washing: Wash the plate 3 times with washing buffer and pat dry;

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

[0222] (7) Washing: Wash the plate 3 times with washing buffer and pat dry;

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

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

[0225] (10) Measure the signal value at 450 nm using an enzyme-labeled instrument.

[0226] Table 5 Results of OD450 of Anti-human IgE Monoclonal Antibody Determined by ELISA Saturation Concentration Method:

[0227]

[0228]

[0229] Table 6 EC50 Value of Anti-human IgE Monoclonal Antibody Determined by ELISA Saturation Concentration Method:

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

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

[0232] The above embodiments are for illustrating the embodiments disclosed in the present invention and should not be construed as limitations to the present invention. In addition, various modifications listed herein and changes in the methods of 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, all obvious modifications to those skilled in the art as described above 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 has the following technical features: The complementarity determining region of the heavy chain variable region of the anti-human IgE antibody includes the CDR-H1 shown in the amino acid sequence of SEQ ID No.1, the CDR-H2 shown in the amino acid sequence of SEQ ID No.2, and the CDR-H3 shown in the amino acid sequence of SEQ ID No.3, and the complementarity determining region of the light chain variable region includes the CDR-L1 shown in the amino acid sequence of SEQ ID No.4, the CDR-L2 shown in the amino acid sequence of SEQ ID No.5, and the CDR-L3 shown in the amino acid sequence of SEQ ID No.6; or, the complementarity determining region of the heavy chain variable region of the anti-human IgE antibody includes the CDR-H1 shown in the amino acid sequence of SEQ ID No.7, the CDR-H2 shown in the amino acid sequence of SEQ ID No.8, and the CDR-H3 shown in the amino acid sequence of SEQ ID No.9, and the complementarity determining region of the light chain variable region includes the CDR-L1 shown in the amino acid sequence of SEQ ID No.10, the CDR-L2 shown in the amino acid sequence of SEQ ID No.11, and the CDR-L3 shown in the amino acid sequence of SEQ ID No.

12.

2. 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 as one of SEQ ID Nos. 13 and 17; b) 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.

3. The anti-human IgE antibody according to claim 2, characterized in that The anti-human IgE antibody comprises a peptide segment having an amino acid sequence of a heavy chain variable region as shown in SEQ ID No. 13 and an amino acid sequence of a light chain variable region as shown in SEQ ID No. 14; or a peptide segment having an amino acid sequence of a heavy chain variable region as shown in SEQ ID No. 17 and an amino acid sequence of a light chain variable region as shown in SEQ ID No.

18.

4. An isolated polynucleotide, characterized in that The polynucleotide encodes the anti-human IgE antibody according to any one of claims 1 to 3.

5. A nucleic acid construct, characterized in that The nucleic acid construct contains the polynucleotide according to claim 4.

6. An isolated engineered cell, characterized in that The engineered cell contains the nucleic acid construct of claim 5 or the polynucleotide of claim 4.

7. Use of the anti-human IgE antibody according to any one of claims 1 to 3, the polynucleotide according to claim 4, or the nucleic acid construct according to claim 5 in the preparation of an allergen detection product.

8. A diagnostic kit, characterized in that The diagnostic kit contains the anti-human IgE antibody according to any one of claims 1 to 3.

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