An antibody against Der p1 and its uses

Abstract

This invention relates to the field of antibodies, and particularly to an anti-Der p 1 antibody and its uses. The invention provides an anti-Der p 1 antibody comprising a heavy chain variable region and a light chain variable region. The amino acid sequence of the complementarity-determining region (CDR1) of the heavy chain variable region is as shown in SEQ ID No. 1, CDR2 of SEQ ID No. 2, and CDR3 of SEQ ID No. 3; or, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 8. The anti-Der p 1 antibody provided by this invention is a high-affinity, high-sensitivity monoclonal antibody. After humanizing this monoclonal antibody with IgE, it is applied to a magnetic particle chemiluminescent quantitative detection reagent for Der p 1-specific IgE of house dust mite allergens. This allows for the generation of a separately calibrated standard curve for Der p 1, achieving truly accurate and reproducible absolute quantification. It can detect Der p 1-specific IgE at a concentration of 0.1 IU / mL, and has clinical diagnostic significance for identifying allergic reactions.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and in particular to an antibody against Der p 1 and its uses. Background Technology

[0002] House dust mites are among the most important indoor allergens worldwide, closely associated with the development of allergic asthma, allergic rhinitis, and atopic dermatitis. Group 1 allergens (Der p 1 and Der f 1) are the most abundant and potent sensitizing components in house dust mite extracts. Der p 1 is a glycoprotein protease derived from the excrement of house dust mites (Dermatophagoides pteronyssinus), belonging to the cysteine ​​protease family. Der p 1-specific IgE is detectable in up to 80-95% of patients with house dust mite allergies, making it the most valuable single indicator molecule for diagnosing house dust mite allergies.

[0003] A key challenge currently facing the field of allergen-specific IgE testing is the lack of international standards and allergen-specific standard curves, leading to variability in test results and a standardization dilemma. Traditional allergen diagnostics use whole-allergen extracts containing mixed proteins for skin prick tests or specific IgE detection.

[0004] Chemiluminescence immunoassay is a rapidly developing immunoassay method in recent years. Its application in human IgE antibody detection offers advantages such as high automation, rapid detection, high sensitivity, accuracy, and specificity, safety, and lower serum consumption. Chemiluminescence methods can be classified into direct chemiluminescence and enzyme-catalyzed chemiluminescence based on reaction type. Direct chemiluminescence uses luminescent agents that do not require enzyme catalysis and directly participate in the luminescence reaction. These agents possess specific luminescent groups in their chemical structure, allowing for direct labeling of antigens or antibodies. Commonly used direct chemiluminescence labels include acridinium esters and ruthenium tripyridine. Enzyme-catalyzed chemiluminescence is an indirect luminescence method. The luminescent agents used do not possess luminescent groups themselves and require enzyme catalysis for luminescence. While it offers some sensitivity, the luminescence rate is slower, and enzyme activity is easily affected by external factors. Representative reagents include luminol and its derivatives, isoluminol, etc., with activating enzymes such as horseradish peroxidase or alkaline phosphatase.

[0005] Currently, commercially available specific IgE immunoassay platforms (such as ImmunoCAP and Immulite) generally use a universal standard curve for total IgE (rather than heterologous IgE) when quantitatively detecting specific IgE. This total IgE standard curve is calibrated with anti-IgE antibodies and is used to measure the concentration of all allergen-specific IgE. This approach has several drawbacks: 1. Doubtful quantitative accuracy: Different allergen-specific IgE antibodies may have different spatial conformations and binding efficiencies with allergens on solid-phase carriers due to the different specific antigens bound to their Fab fragments. Using the same "one-size-fits-all" standard curve to calculate the concentration of all specific IgEs cannot accurately reflect the actual absolute concentration of different specific IgEs; 2. Poor comparability of results between laboratories: Because there is no standard for Der p1 specific IgE, accurate calibration and traceability cannot be performed between test reagents from different manufacturers, different testing platforms, or even different batches. This leads to significant differences in the Der p1 specific IgE values ​​of the same serum sample detected in different laboratories using different methods. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an antibody against Der p 1 and its use, in order to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention provides an anti-Der p1 antibody, the anti-Der p1 antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the complementarity-determining region of the heavy chain variable region is as shown in SEQ ID No. 1, CDR1, CDR2, and CDR3; or, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 8.

[0008] Preferably, the amino acid sequence of the light chain variable region containing the complementarity-determining region is: CDR1 as shown in SEQ ID No. 4, CDR2 sequence is WAS, and CDR3 as shown in SEQ ID No. 5, wherein WAS in the CDR2 sequence represents tryptophan, alanine, and serine, respectively; or, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 9.

[0009] The present invention also provides an isolated nucleic acid molecule that encodes the aforementioned anti-Derp1 antibody.

[0010] The present invention also provides a nucleic acid construct comprising the aforementioned nucleic acid molecule and expression vector.

[0011] The present invention also provides an isolated engineered cell containing the aforementioned nucleic acid construct or the aforementioned nucleic acid molecule.

[0012] The present invention also provides a detection kit comprising the aforementioned anti-Der p 1 antibody.

[0013] Preferably, the test kit further comprises multiple of the following: anti-human IgE antibody, magnetic beads, buffer solution, stabilizer, protectant, preservative, chelating agent, or surfactant.

[0014] The present invention also provides the use of the aforementioned anti-Derp1 antibody, the aforementioned nucleic acid molecule, the aforementioned nucleic acid construct or the aforementioned engineered cell in the preparation of detection products.

[0015] Preferably, the detection product is a detection product for hypersensitivity diseases.

[0016] As described above, the anti-Der p 1 antibody of the present invention and its use have the following beneficial effects:

[0017] This invention successfully expressed the Derp1 protein, a component of the house dust mite allergen, using a mammalian cell expression system. After immunizing New Zealand rabbits, a group of monoclonal antibodies with high affinity and high sensitivity to Derp1 were screened. These monoclonal antibodies were then modified into humanized IgE and applied to a magnetic particle chemiluminescent quantitative detection reagent for Derp1-specific IgE. This allows for the generation of a standard curve for Derp1, achieving truly accurate and reproducible absolute quantification. It can detect Derp1-specific IgE at a concentration of 0.1 IU / mL, and has clinical diagnostic significance for identifying allergic reactions. Attached Figure Description

[0018] Figure 1 The image shown is an SDS-PAGE diagram of the purified Der p1 protein, a component of the house dust mite allergen, in this invention.

[0019] Figure 2 The image shown is an SDS-PAGE image of the purified Der p 1 monoclonal antibody 1A3, which is an anti-house dust mite allergen component, as described in this invention.

[0020] Figure 3 The results shown are the affinity assay results for the anti-Derp1 monoclonal antibody in this invention.

[0021] Figure 4 The graph shown is the total IgE standard curve in Embodiment 6 of the present invention.

[0022] Figure 5The figure shown is a standard curve of the chemiluminescent quantitative detection reagent for Der p 1 specific IgE magnetic microparticles of house dust mite allergen components in this invention. Detailed Implementation

[0023] The present invention provides an anti-Der p 1 antibody, the anti-Der p 1 antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the complementarity-determining region of the heavy chain variable region is as shown in SEQ ID No. 1, CDR1, CDR2, and CDR3; or, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 8.

[0024] In some specific embodiments, the amino acid sequence of the antigen Der p 1 protein is shown in SEQ ID No. 14.

[0025] In some specific embodiments, the amino acid sequence of the light chain variable region containing the complementarity-determining region is: CDR1 as shown in SEQ ID No. 4, WAS as shown in CDR2, and CDR3 as shown in SEQ ID No. 5, wherein WAS in the CDR2 sequence represents tryptophan, alanine, and serine, respectively; or, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 9.

[0026] In some specific embodiments, the anti-Derp1 antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of the complementarity-determining region in the heavy chain variable region is as follows: CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3. The amino acid sequence of the complementarity-determining region in the light chain variable region is as follows: CDR1 shown in SEQ ID No. 4, CDR2 sequence is WAS, and CDR3 shown in SEQ ID No. 5; or, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 9.

[0027] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-Der p 1 antibody is shown in SEQ ID No. 6; and / or, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 7.

[0028] In some specific embodiments, the anti-Derp1 antibody further comprises a human IgE heavy chain constant region or a human Kappa chain constant region.

[0029] Further, the amino acid sequence of the human IgE heavy chain constant region is shown in SEQ ID No. 10; and / or, the amino acid sequence of the human Kappa chain constant region is shown in SEQ ID No. 11.

[0030] In some specific embodiments, the amino acid sequence of the heavy chain region of the anti-Der p 1 antibody is shown in SEQ ID No. 12; and / or, the amino acid sequence of the light chain region of the anti-Der p 1 antibody is shown in SEQ ID No. 13.

[0031] In some specific embodiments, the anti-Der p1 antibody is a monoclonal antibody. Specifically, the anti-Der p1 antibody is a monoclonal IgE antibody.

[0032] The present invention also provides an isolated nucleic acid molecule that encodes the aforementioned anti-Derp1 antibody.

[0033] In some specific embodiments, the nucleic acid molecule comprises a DNA fragment with a nucleotide sequence as shown in SEQ ID No. 15; and / or, the nucleic acid molecule comprises a DNA fragment with a nucleotide sequence as shown in SEQ ID No. 16.

[0034] In some specific embodiments, the nucleic acid molecule also comprises multiple DNA fragments with nucleotide sequences as shown in SEQ ID No. 17-20.

[0035] The present invention also provides a nucleic acid construct comprising the aforementioned nucleic acid molecule and expression vector.

[0036] In some specific embodiments, the nucleic acid construct is constructed by inserting the aforementioned isolated nucleic acid molecules into the multiple cloning site of an expression vector. The expression vector can be transformed, transduced, or transfected into host cells, allowing its carried genetic material elements to be expressed within the host cells. The construct can be a viral vector or a non-viral vector. For example, non-viral vectors include: plasmids, phage particles, Cos plasmids, 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, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). The vector may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site. The carrier may also include components that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein coats.

[0037] In some specific embodiments, the backbone plasmid of the expression vector may be selected from PCDNA3.1, PIRES, PCDNA3.4, 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.

[0038] The present invention also provides an isolated engineered cell containing the aforementioned nucleic acid construct or the aforementioned isolated nucleic acid molecule.

[0039] In this invention, the engineered cells can be obtained by introducing the aforementioned nucleic acid constructs into host cells, or the engineered cells are cells whose genomes integrate the aforementioned isolated exogenous nucleic acid molecules.

[0040] Any cell suitable for expression via the expression vector can serve as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; 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 like *Escherichia coli* or *Bacillus subtilis*, fungal cells like yeast or *Aspergillus*, insect cells like S2 ​​*Drosophila* or Sf9, or animal cells like fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0041] The present invention also provides a detection kit comprising the aforementioned antibody.

[0042] In some specific embodiments, the test kit further includes multiple of the following: anti-human IgE antibody, magnetic beads, buffer solution, stabilizer, protectant, preservative, chelating agent, or surfactant.

[0043] Furthermore, the anti-human IgE antibody also contains a detectable marker, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0044] The present invention also provides the use of the aforementioned antibodies, the aforementioned isolated nucleic acid molecules, the aforementioned nucleic acid constructs or the aforementioned engineered cells in the preparation of detection products.

[0045] In some specific embodiments, the detection product is a detection product for hypersensitivity diseases.

[0046] In some specific embodiments, the hypersensitivity disease is selected from one or more of the following: allergic reaction, allergic rhinitis, allergic cough, anaphylactic shock, food allergy, hypersensitivity reaction, asthma (such as moderate to severe persistent allergic asthma), chronic urticaria (such as chronic spontaneous urticaria), acute urticaria, acute bronchospasm, atopic dermatitis, inflammatory skin disease, eczema, laryngeal edema, angioedema, nasal polyps, or sinusitis.

[0047] Furthermore, the hypersensitivity disease detection product is a Der p 1 specific IgE antibody detection product for dust mite allergen components.

[0048] In this invention, the term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen, said immunoglobulin-derived molecule binding to said target antigen through at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, it includes not only the complete antibody but also antigen-binding fragments capable of specifically binding to a target antigen. A "complete antibody" typically consists of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). The antibody light chain can be classified as κ (kappa) and λ (lambda) light chains. The heavy chain can be classified as μ, δ, γ, α, or ε, and the isotypes of the antibody are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one CL domain. The constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs).

[0049] In this invention, the term "complementarity determining region" or "CDR" generally refers to a region in an antibody that is spatially complementary to the antigenic determinant. The variability in an antibody is usually not uniformly distributed throughout the variable region. Both the heavy chain and light chain variable regions of a monoclonal antibody typically have three hypervariable regions (HVRs). These regions are spatially complementary to the antigenic determinant, so they are also called complementarity determining regions (CDRs). Specifically, the heavy chain variable region typically includes three CDRs: HCDR1, HCDR2, and HCDR3, while the light chain variable region typically includes three CDRs: LCDR1, LCDR2, and LCDR3.

[0050] In this invention, the term "monoclonal antibody" generally refers to a group of antibodies that are substantially identical (except for a few possible naturally occurring mutations). Monoclonal antibodies typically target specific determinants on an antigen.

[0051] In this invention, the term "nucleic acid molecule" can refer to either a natural nucleic acid molecule or an artificial nucleic acid molecule, such as DNA or RNA that does not naturally exist. In other words, an artificial nucleic acid molecule can be understood as a non-natural nucleic acid molecule. Such nucleic acid molecules may be non-natural due to their individual sequence (which is not naturally occurring) and / or due to other modifications that are not naturally occurring, such as structural modifications of nucleotides. An artificial nucleic acid molecule can be a DNA molecule, an RNA molecule, or a hybrid molecule containing both DNA and RNA portions. Typically, artificial nucleic acid molecules can be designed and / or generated by genetic engineering methods to correspond to a desired artificial nucleotide sequence (heterologous sequence). In this case, the artificial sequence is usually a sequence that may not naturally exist, i.e., it differs from the wild-type sequence by at least one nucleotide. The term "wild-type" can be understood as a naturally occurring sequence. Furthermore, the term "artificial nucleic acid molecule" is not limited to meaning "a single molecule," but is generally understood to include the whole of the same molecule. Therefore, it can refer to multiple identical molecules contained in a sample.

[0052] In this invention, the term "DNA" is a common abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, that is, a polymer composed of nucleotides. These nucleotides are typically monomers of deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate, which themselves consist of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, and are polymerized through a characteristic backbone structure. Typically, the backbone structure is formed by a phosphodiester bond between the sugar moiety, i.e., the deoxyribose, of the first nucleotide and the phosphate moiety of the second adjacent monomer. The specific sequence of the monomers, i.e., the sequence of bases linked to the sugar / phosphate backbone, is called the DNA sequence. DNA can be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, for example, through A / T base pairing and G / C base pairing.

[0053] The following specific examples illustrate the implementation of the present invention. 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.

[0054] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0055] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0056] The amino acid and nucleotide sequences used in this invention are as follows:

[0057] SEQ ID No.1GFSLSNYY

[0058] SEQ ID No.2IGGGGST

[0059] SEQ ID No.3ATYFCATGIGASLI

[0060] SEQ ID No.4QSVYNNKN

[0061] SEQ ID No.5QGEFSCSSADCNA

[0062] SEQ ID No.6

[0063] QSLEESGGRLVTPGTPLTLTCTASGFSLSNYYMSWVRQAPGKGLEWIGGIGGGGSTWYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCATGIGASLIWGPGTLVTVSS

[0064] SEQ ID No.7

[0065] ALVMTQTPASVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYWASILKSGVSSRFSGSGSGTQFTLTISGVQCADAATYYCQGEFSCSSADCNAFGGGTEVVVK

[0066] SEQ ID No.8

[0067] QSVEESGGRLVTPGTPLTLTCTVSGFSLRRYGVNWVRQAPGKGLEWIGIIDISGRTSYAKWAKGRFTISKTSTTVDLKITSPTTEDTAAYFCTRNPYGGESTLWGPGTLVTVSS

[0068] SEQ ID No.9

[0069] AIVMTQTPSSKSVAVGDTVTINCQASESVYAYNRLAWFQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTEFTLTISDVVCDDAATYYCAGYKSDSDGSTFGGGTEVVVK

[0070] SEQ ID No.10

[0071] ASTQSPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMTLPATTLTLSGHYATISLLTVSGAWAKQMFTCRVAHTPSSTDWVDNKTFSVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK

[0072] SEQ ID No.11

[0073] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0074] SEQ ID No.12

[0075] QSLEESGGRLVTPGTPLTLTCTASGFSLSNYYMSWVRQAPGKGLEWIGGIGGGGSTWYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCATGIGASLIWGPGTLVTVSSASTQSPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMTLPATTLTLSGHYATISLLTVSGAWAKQMFTCRVAHTPSSTDWVDNKTFSVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK

[0076] SEQ ID No.13

[0077] ALVMTQTPASVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYWASILKSGVSSRFSGSGSGTQFTLTISGVQCADAATYYCQGEFSCSSADCNAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0078] SEQ ID No.14

[0079] RPSSIKTFEEYKKAFNKSYATFEDEEAARKNFLESVKYVQSNGGAINHLSDLSLDEFKNRFLMSAEAFEHLKTQFDLNAETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTHSAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANIDLMMIEEYPYVVIL

[0080] SEQ ID No.15

[0081] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAACTACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGGCATTGGTGGTGGTGGTAGTACATGGTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCACAGGCATTGGTGCTAGTCTCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0082] SEQ ID No.16

[0083] GCCCTTGTGATGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAACTGCCAGGCCAGTCAGAGTGTTTATAATAACAAAAATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACTGGGCATCCATTCTGAAATCTGGGGTCTCATCACGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGTGGCGTGCAGTGTGCCGATGCTGCCACTTACTACTGTCAAGGCGAATTTAGTTGTAGTAGTGCTGATTGCAATGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAA

[0084] SEQ ID No.17

[0085] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGAAGGTATGGAGTGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATTATTGATATTAGTGGACGCACATCCTACGCGAAGTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCGCCTATTTCTGTACCAGAAATCCTTATGGTGGTGAGAGTACTTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0086] SEQ ID No.18

[0087] GCCATCGTGATGACCCAGACTCCATCTTCCAAGTCTGTCGCTGTGGGAGACACAGTCACCATCAATTGCCAGGCCAGTGAGAGTGTTTATGCGTATAACCGCTTAGCCTGGTTTCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGATGTGGTGTGCGACGATGCTGCCACTTACTACTGTGCAGGATATAAAAGTGATAGTGATGGTAGTACTTTCGGCGGAGGGACCGAGGTGGTCGTCAAA

[0088] SEQ ID No.19

[0089]

[0090] SEQ ID No.20

[0091] AGGACCGTGGCCGCTCCAAGCGTGTTCATCTTCCCCCCCTCTGACGAGCAGCTGAAGTCCGGCACCGCTAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAAGCCAAAGTGCAGTGGAAGGTCGACAATGCCCTGCAAAGCGGCAACAGCCAGGAGAGCGTTACAGAGCAGGACAGCAAGGACTCCACCTATAGCCTGAGCTCTACACTGACACTGAGCAAAGCTGATTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCAGTGACCAAGAGCTTCAACAGAGGCGAATGT

[0092] SEQ ID No.21

[0093] SKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0094] Expression and Purification of Dermatophagoides pteronyssinus Allergen Component Protein Der p 1 in Example 1

[0095] 1. Gene Synthesis

[0096] The amino acid sequence (P08716, amino acid R19-L320) of the human house dust mite allergen component protein Der p1 was downloaded from the uniprot database. A rabbit IgG1 heavy chain signal peptide (METGLRWLLLVAVLKGVQC) was added to the N-terminus of this sequence, and a 6×His tag was added to the C-terminus. After codon optimization, the sequence was sent to Sassofi Biotechnology Co., Ltd. (Jiangsu Sassofi Biotechnology Co., Ltd.) for gene synthesis, and the resulting recombinant plasmid was injected into the pCDNA3.1 vector. The synthesized recombinant plasmid was transformed into DH5α competent cells, plated on Amp+ medium plates, and single colonies were screened. These single colonies were then cultured by shaking to obtain the pCDNA3.1-Der p1 recombinant bacterial strain.

[0097] The amino acid sequence of Der p 1 (P08716, amino acids R19-L320) is shown in SEQ ID No. 14.

[0098] 2. Protein expression

[0099] The pCDNA3.1-Der p1 recombinant bacterial strain was activated and cultured. Following the instructions of the endotoxin-free plasmid extraction kit, the pCDNA3.1-Der p1 recombinant plasmid was extracted and transfected into Expi293F suspension cells (purchased from Gibco) for protein expression.

[0100] The transfection steps are as follows:

[0101] (1) Cell counting was performed on the day of transfection. The viability was above 95%, and the density was 3 × 10⁶ cells / year. 6 Cells / mL. For example, to transfect 25 mL of cells: take 20 μg of plasmid, dilute with 1.5 mL of Opti-MEM serum-depleted medium, gently mix, and let stand for 5 min; then take ExpiFectamine... TM Add 80 μl of reagent 293 to 1.5 mL of Opti-MEM serum-depleted medium, mix thoroughly, and let stand for 5 min. Mix the plasmid and transfection reagent after standing, mix gently, and let stand for another 10 min.

[0102] (2) After standing, add the plasmid-transfection reagent mixture dropwise to 25 mL of cells while gently shaking the flask. Finally, place it in a shaker for culture (37℃, 8% CO2, 125 rpm).

[0103] (3) Add 150 μl of ExpiFectamine 18-24 h after transfection. TM 293Transfection Enhancer 1 and 1.5ml ExpiFectamine TM293 Transfection Enhancer 2, continue incubation on a shaker.

[0104] (4) The expression product can be collected 5-7 days after transfection.

[0105] 3. Protein purification

[0106] The Der p 1 recombinant protein was purified using a Ni column (Sangon Biotech).

[0107] (1) Centrifuge to collect the cell supernatant after transfection and expression, and filter the cell supernatant through a 0.45 μm filter membrane;

[0108] (2) First, wash the resin with 5-10 column volumes of pure water at 50-150 cm / h to remove ethanol. Then, use 5-10 column volumes of binding buffer (20 mM PB, 0.5 M NaCl, pH 7.4) at 150-600 cm / h to equilibrate the medium and ensure that the composition and pH of the solution in the medium are consistent with the sample.

[0109] (3) Start loading the sample at a flow rate of 150 cm / h;

[0110] (4) After loading the sample, wash the non-specifically adsorbed proteins with 10 to 20 column volumes of washing buffer (20 mM PB, 0.5 M NaCl, 1 to 30 mM imidazole, pH 7.4) at 150 cm / h, and collect the washing buffer for subsequent analysis.

[0111] (5) Elute the target protein with 5-10 times the volume of elution buffer (20mM PB, 0.5M NaCl, 500mM imidazole, pH 7.4) and collect the eluent;

[0112] (6) The collected target protein eluent was ultrafiltered with 1×PBS to remove imidazole, and then concentrated to a concentration of 1 mg / ml after ultrafiltration and stored at -20℃ for later use.

[0113] like Figure 1 As shown, the purified protein was analyzed by SDS-PAGE electrophoresis. The molecular weight of Der p 1 protein was around 35 KD, which is in line with expectations (Der p 1 is around 34.3 KD), and the purity can reach more than 90%.

[0114] Example 2: Preparation of rabbit monoclonal antibody against house dust mite allergen component Der p1

[0115] Two 6-8 week old New Zealand rabbits, approximately 2 kg each, were immunized with 30 μg of prepared Der p 1 protein. For the first immunization, the Der p 1 protein was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites. A total of three immunizations were administered: a second immunization two weeks after the first, and a third immunization one week later. For both the second and third immunizations, incomplete Freund's adjuvant was used for antigen emulsification, and the dosage and method of immunization remained unchanged. After the third immunization, a small amount of blood was collected from behind the ear, and serum titer was determined by indirect ELISA.

[0116] (1) Antigen coating: Dilute the antigen to 2ug / ml with PBS buffer and add 100ul to each well of a 96-well microplate. Incubate overnight at 4°C. Wash the coated microplate three times with a plate washer and dry it. Add 300ul of blocking buffer to each well and incubate at room temperature for 1 hour. Then wash the blocked microplate three times with a plate washer and dry it.

[0117] (2) Incubation of primary antibody: Dilute rabbit serum with PBS buffer, add 100 μL of diluted serum to each well, incubate at room temperature for 1 hour, and after incubation, wash the microplate three times in a plate washer and then dry it.

[0118] (3) Incubation of secondary antibody: Add 100 μL of prepared HRP-labeled goat anti-rabbit secondary antibody to each well and incubate at room temperature for 1 hour. Then, wash the plate three times in a plate washer and dry it.

[0119] (4) Color development: Add 200 μL of the mixed color development solution to each well of the 96-well plate and incubate at room temperature for 15 minutes.

[0120] (5) Termination and reading: Add 100 μL of stop solution to each well of the 96-well plate, and then place it in a microplate reader for reading. Set the detection wavelength to 450 nm and read the detection results as shown in Table 1. An OD value greater than 2.1 times that of the negative control is considered positive.

[0121] Table 1. Dep1 antigen protein coating and rabbit serum titer detection.

[0122] Dilution factor / K R019# R020# PBS 0.468 0.444 1 4.425 4.396 2 4.452 4.335 4 4.122 4.227 8 4.089 4.137 16 3.843 3.811 32 3.450 3.565 64 2.977 2.883 128 2.522 2.428 256 1.673 1.727 512 0.991 0.973

[0123] (6) The results of ELISA titer test showed that the serum titers of R019 and R020 were both greater than 512K, and the titers met the standard. The same amount of antigen was emulsified with Freund's incomplete adjuvant and injected intraperitoneally into R019 rabbits as the last shock immunization before sorting.

[0124] 3. Sorting of B cells

[0125] (1) Preparation of Der p 1 magnetic beads

[0126] a) Activation of magnetic beads: Take 10 mg of carboxyl magnetic beads (3 μm, JSR MS300) into 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 rack, and wait for the magnetic beads to be completely adsorbed onto the side wall. Use a pipette tip 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 at room temperature for 15 minutes;

[0127] b) Antigen coating: Place the centrifuge tube on a magnetic rack, wait for the magnetic beads to be completely adsorbed onto the side wall, remove the supernatant, resuspend the magnetic beads with 1 ml of washing buffer, add 0.1 mg of Der p 1 protein, invert and mix at room temperature for 2 hours.

[0128] c) Blocking of magnetic beads: Add 20 μl of blocking agent (JSR CE510:CE210 = 9:1) to the centrifuge tube, mix by inversion at room temperature and incubate for 1 hour;

[0129] d) Cleaning: Place the centrifuge tube on a magnetic rack and wait for the magnetic beads to be completely adsorbed onto the sidewall. Remove the supernatant and resuspend the magnetic beads in 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. Finally, resuspend the magnetic beads in 10 ml of magnetic bead preservation 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).

[0130] (2) Preparation of single-cell suspension

[0131] Rabbit spleen was harvested, crushed with a scalpel, ground, resuspended in RPMI 1640 medium, filtered through a 100μm cell filter, and then through a 70μm cell filter to obtain a single-cell suspension. The suspension was centrifuged at 500g for 10 min at room temperature and then resuspended in RPMI 1640 medium to a final volume of 1×10⁻⁶ cells / mL. 8 Cells / ml

[0132] (3) Magnetic bead sorting MBC

[0133] a) Magnetic bead cleaning: Place 1 ml of magnetic bead suspension on a magnetic rack. After the magnetic beads are completely adsorbed onto the side wall, remove the supernatant with a pipette tip. Resuspend the magnetic beads with 1 ml of PBS buffer. Repeat twice and then remove the supernatant.

[0134] b) Sorting MBC cells: Take 1 ml of cell suspension and gently resuspend the magnetic beads, incubate at room temperature for 20 min at 120 rpm, and wash 3 times with PBS to remove unbound cells.

[0135] c) Resuspend the cells in 1 ml of cell storage medium (PBS buffer containing 1% m / v BSA), take 10 μl of the resuspended solution and place it in a hemocytometer for cell counting under a microscope;

[0136] d) Dilute with nuclease-free water according to cell density to a single cell / μl, and take 1 μl per well in a 96-well plate for a total of 96 wells.

[0137] 4. Cell lysis and amplification of heavy and light chain genes.

[0138] A single-cell antibody gene amplification system was used to obtain variable region fragments of the heavy and light chains.

[0139] (1) One-step reverse transcription amplification of the variable region fragment of the Kappa strand and the heavy strand.

[0140] The 96 single-cell suspensions of the selected Novizan single-cell one-step reverse transcription amplification reaction solution (SingleCell Sequence Specific Amplification Kit) were used.

[0141] The primers for one-step reverse transcription amplification are as follows:

[0142] raCL1-3 ACAGTTCTTCCTACTGAAGCT

[0143] raCL1-2GCAGTCACCCCTGTTGAA

[0144] raCL1-1 ACAGTCACCCCTATTGAAG

[0145] RaCH-RTPCR CTCGGGGGTGCGTGAGAT

[0146] raLS-Lc-1F ATGGACACGAGGGCCCCCAC

[0147] raLS-Hc-1F ATGGAGACTGGGCTGCGCTGG

[0148] ra-HcG-1st-1R AGATCATGAGGGTTGTCCTTG

[0149] The reaction system is as follows:

[0150] Add 7.5 μl of 2×Reaction Mix, 2.5 μl of 2×Reaction Mix, 0.5 μl of Kappa and Heavy chain F+R primer combination (final concentration of each primer is 0.1 μM), 2.5 μl of 2×Reaction Mix, 0.2 μl of RT / Taq enzyme, and 1.8 μl of nuclease-free water, totaling 10 μl, to the wells of the PCR plate and mix with the cell suspension.

[0151] Add single cells, tighten the cap, and immediately place in a -70°C freezer for 2 minutes. Centrifuge at 3,000 rpm (1,000 × g) for 2 minutes, and immediately transfer to a PCR instrument for the following reaction:

[0152]

[0153] 5. Construction of expression plasmids and plasmid extraction

[0154] (1) Amplification of the target gene fragment

[0155] Based on the pre-constructed Kappa and Heavy chain backbone vectors and the Kappa and Heavy chain variable region templates obtained by one-step reverse transcription amplification, downstream and upstream primers for the Kappa and Heavy chains were designed to amplify the variable region fragments.

[0156] Primers:

[0157] XbaI-Rab Lsig-F

[0158] GATCCAGCCTCCGGACTCTAGATGGACACGAGGGCCCCACTC

[0159] Rab-LC-2nd-R1

[0160] CGAACCCTTATAGCGGCCGTTACCTAYTGAAGCTCTGGACGA

[0161] Rab-LC-2nd-R2

[0162] CGAACCCTTATAGCGGCCGTTAACAGTCACCCCTATTGAAGCTCTGG

[0163] Rab-LC-2nd-R3

[0164] CGAACCCTTATAGCGGCCGTTAGCAGTCACCCCTGTTGAAGCTCT

[0165] Rab-LC-2nd-R4

[0166] CGAACCCTTATAGCGGCCGTTACAGTTCTTCCTACTGAAGCTCAGG

[0167] EcoRI-Rab Hsig-F

[0168] CCCTTGGATCTCTAGCGATGGAGACTGGGCTGCGCTGGCTTC

[0169] ra-HcG-2nd-1R

[0170] TTAGTGGGCACGTGGGCTTGCTG

[0171] PCR amplification was performed using Phanta polymerase (purchased from Novizan) with the one-step reverse transcription amplification product as a template. The reaction volume was 50 μL. The contents were: 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.

[0172] The PCR reaction conditions were: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 30 s, 72℃ for 30 s, for 30 cycles; and 72℃ for 5 min to end the program.

[0173] (2) Cloning of PCR amplification products

[0174] PCR products were subjected to 1% agarose gel electrophoresis. The Kappa and Heavy chain amplified fragments were recovered using a PCR product recovery kit (purchased from Tiangen). Twenty-four combinations were selected where both the Kappa and Heavy chains amplified fragments of the correct size.

[0175] The 24 amplified pair antibody light and heavy chain DNA fragments were ligated into the pcDNA3.4 vector and the pcDNA3.4 vector containing the pre-constructed rabbit IgG1Fc fragment (amino acid sequence as shown in SEQ ID NO.21) using the ClonExpress II One Step Cloning Kit to construct light and heavy chain recombinant expression plasmids.

[0176] After identifying positive strains by colony PCR, the target bacteria are cultured and plasmids are extracted using a plasmid extraction kit. For specific steps, please refer to the plasmid extraction instructions in the kit.

[0177] 6. Instantaneous expression

[0178] Twenty-four Kappa and Heavy chain plasmid combinations were transfected into Expi293F cells for transient expression. The transfection steps were as follows: (1) Cell counts were performed on the day of transfection, with a viability of over 95% and a density of 3 × 10⁻⁶ cells. 6 Cells / mL. Dilute cells to 2 × 10⁶ cells / mL with serum-free medium. 5 Add 0.5 mL of cells per well to each well of a 24-well plate and incubate for 2 hours.

[0179] (2) Take 0.5 μg of plasmid (heavy chain: light chain = 1:2), dilute with 25 μl of Opti-MEM serum-reduced medium, mix gently and let stand for 5 min; then take 1.5 μl of PEI transfection reagent, add 25 μl of Opti-MEM serum-reduced medium to dilute, mix thoroughly and let stand for 5 min; mix the plasmid and transfection reagent after standing, mix gently and let stand for another 10 min.

[0180] (3) After standing, add the plasmid-transfection reagent mixture dropwise into the wells of the cell culture plate while gently shaking the culture plate. Finally, place it in a shaker for static culture (37℃, 8% CO2).

[0181] (4) The supernatant can be collected 48 hours after transfection. The cell supernatant is tested for positivity using indirect ELISA. The detection steps are as follows: (a) Antigen coating: Dilute Der p 1 antigen to 2ug / ml with PBS buffer and add 100ul to each well of a 96-well microplate. Incubate overnight at 4°C. Wash the coated microplate three times and dry it. Add 300ul of blocking buffer to each well and incubate at room temperature for 1 hour. Then wash the blocked microplate three times and dry it.

[0182] (b) Incubation with primary antibody: Following the layout in Table 2, add 100 μL of cell supernatant to each well. Use an equal amount of positive serum as a positive control and blank culture cell supernatant as a negative control. Incubate at room temperature for 1 hour. After incubation, wash the ELISA plate three times and dry it.

[0183] Table 2. Layout of antigen protein coating and cell supernatant plates.

[0184] 1 2 3 4 A 1A1 1B7 1D8 negative control B 1A2 1B8 1E4 negative control C 1A3 1C2 1E5 Immune serum D 1A5 1C3 1E8 Immune serum E 1A8 1C4 1F3 F 1B2 1C8 1F4 G 1B4 1D1 1F6 H 1B5 1D7 1F7

[0185] (c) Incubation of secondary antibody: Add 200 μL of HRP-labeled goat anti-rabbit antibody to each well and incubate at room temperature for 1 hour. After washing the plate three times and drying it, add the chromogenic solution and incubate. After incubation, add the stop solution and place it in a microplate reader for reading. Set the detection wavelength to 450 nm and read the detection results.

[0186] Table 3 Results of ELISA test for anti-Derp1 monoclonal antibody

[0187]

[0188]

[0189] The ELISA titer results in Table 3 show that 4 positive clones with titers > 4.0 were selected from 24 clones.

[0190] Example 3: Determination of Affinity for Der p 1 Monoclonal Antibody

[0191] 3.1 Antibody Expression and Purification:

[0192] The four monoclonal antibodies (1A3, 1B8, 1E4, 1F3) with ELISA results greater than 4.0 in Example 2 were expressed:

[0193] The transfection steps are as follows:

[0194] (1) Cell counting was performed on the day of transfection. The viability was above 95%, and the density was 3 × 10⁶ cells / year. 6 Cells / mL. For example, to transfect 25 mL of cells: take 20 μg of plasmid (heavy chain: light chain = 1:2), dilute with 1.5 mL of Opti-MEM serum-depleted medium, gently mix, and let stand for 5 min; then take ExpiFectamine... TM Add 80 μl of reagent 293 to 1.5 mL of Opti-MEM serum-depleted medium, mix thoroughly, and let stand for 5 min. Mix the plasmid and transfection reagent after standing, mix gently, and let stand for another 10 min.

[0195] (2) After standing, add the plasmid-transfection reagent mixture dropwise to 25 mL of cells while gently shaking the flask. Finally, place it in a shaker for culture (37℃, 8% CO2, 125 rpm).

[0196] (3) Add 150 μl of ExpiFectamine 18-24 h after transfection. TM 293Transfection Enhancer 1 and 1.5ml ExpiFectamine TM 293 Transfection Enhancer 2, continue incubation on a shaker.

[0197] (4) The expression products were collected and purified 5-7 days after transfection. The concentration of the purified antibodies was ≥1 mg / ml. The SDS-PAGE electrophoresis results of the anti-Der p1 monoclonal antibody (1A3, 1B8, 1E4, 1F3) proteins are as follows: Figure 2 As shown.

[0198] 3.2. Affinity assay for anti-Derp1 monoclonal antibody

[0199] (1) Coating: Dilute the receptor protein Der p 1 to 0.5 μg / ml with coating buffer, add 100 μl / well, and incubate overnight at 2-8℃;

[0200] (2) Blocking: Wash the plate three times with washing solution, add 300 μl / well blocking solution, and block at room temperature for 1 hour;

[0201] (3) Washing: Wash the plate 3 times with washing solution, pat dry and set aside;

[0202] (4) Add the antibody to a microplate at a starting concentration of 4000 ng / ml, and perform a 5-fold serial dilution to obtain 7 concentration gradients. Add 100 μl / well to the microplate and incubate at room temperature for 1 hour.

[0203] (5) Washing: Wash the plate 3 times with detergent and pat dry;

[0204] (6) Add detection secondary antibody: Dilute enzyme-linked antibody to working concentration, mix well, 100 μl / well, incubate at room temperature for 1 hour;

[0205] (7) Detection: Wash the plate three times with washing solution and pat dry; add 200 μl / well of chromogenic solution and incubate at room temperature in the dark for 15 minutes; stop the reaction by adding 100 μl / well of stop solution, and measure the signal value at 450 nm using an ELISA reader. The results are shown in Table 4. Figure 3 As shown.

[0206] Table 4. Results of OD450 determination of Der p 1 monoclonal antibody using the ELISA saturation concentration method.

[0207]

[0208] Plot the curves and calculate the EC50 values ​​as follows:

[0209] Der p 1 antibody 1A3 1B8 1E4 1F3 EC50 (ng / ml) 8.600 9.575 10.26 13.07

[0210] The results above show that the EC50 value of antibody 1A3 against antigen Der p 1 is lower than that of antibodies 1B8, 1E4 and 1F3, indicating that antibody 1A3 has higher affinity. The amino acid sequence of the heavy chain variable region of antibody 1A3 is shown in SEQ ID No. 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 7; the amino acid sequence of the heavy chain variable region of antibody 1B8 is shown in SEQ ID No. 8, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 9.

[0211] The CDR regions of the 1A3 heavy chain variable region and light chain variable region sequences were analyzed using the IMGT scheme.

[0212] Among them, the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of 1A3 heavy chain are at positions 25-32, 50-56 and 88-101, respectively, and are GFSLSNYY, IGGGGST and ATYFCATGIGASLI;

[0213] The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the 1A3 light chain are at positions 27-34, 52-54, and 91-103, respectively, and are QSVYNNKN, WAS, and QGEFSCSSADCNA.

[0214] Example 4: Preparation of humanized IgE chimeric antibody against Derp1 monoclonal antibody 1A3

[0215] 4.1 Humanized IgE chimeric antibody modification of anti-Derp1 monoclonal antibody 1A3.

[0216] The variable region of the 1A3 heavy chain was fused with the constant region of the human IgE heavy chain (amino acid sequence as shown in SEQ ID No. 10) (CH1+CH2+CH3+CH4) to form the complete heavy chain region of the chimeric antibody (the amino acid sequence of the chimeric antibody heavy chain is shown in SEQ ID No. 12). The variable region of the 1A3 light chain was fused with the constant region of the human Kappa chain (amino acid sequence as shown in SEQ ID No. 11) CL to form the complete light chain region of the chimeric antibody (the amino acid sequence of the chimeric antibody light chain is shown in SEQ ID No. 13). The gene was then synthesized into the pCDNA3.4 vector by Jiangsu Saisofe Biotechnology Co., Ltd.

[0217] 4.2 Expression and purification of anti-Derp1 humanized IgE antibody:

[0218] (1) Cell counting was performed on the day of transfection. The viability was above 95%, and the density was 3 × 10⁶ cells / year. 6 Cells / mL. For example, to transfect 25 mL of cells: take 20 μg of plasmid (heavy chain: light chain = 1:2), dilute with 1.5 mL of Opti-MEM serum-depleted medium, gently mix, and let stand for 5 min; then take ExpiFectamine... TM Add 80 μl of reagent 293 to 1.5 mL of Opti-MEM serum-depleted medium, mix thoroughly, and let stand for 5 min. Mix the plasmid and transfection reagent after standing, mix gently, and let stand for another 10 min.

[0219] (2) After standing, add the plasmid-transfection reagent mixture dropwise to 25 mL of cells while gently shaking the flask. Finally, place it in a shaker for culture (37℃, 8% CO2, 125 rpm).

[0220] (3) Add 150 μl of ExpiFectamine 18-24 h after transfection. TM 293Transfection Enhancer 1 and 1.5ml ExpiFectamine TM 293 Transfection Enhancer 2, continue incubation on a shaker.

[0221] (4) The expression product can be collected 5-7 days after transfection.

[0222] (5) Centrifuge the culture of Expi 293F cells expressing the antibody, collect the supernatant, and filter it through a 0.22 μm filter membrane;

[0223] (6) Flow through 1 ml of Protein L agarose chromatography matrix (Shanghai Sangon Biotech Order No. C600697) with the supernatant and wash with PBS buffer for 5 column volumes;

[0224] (7) Elute with 1 ml of 0.1 M glycine buffer (adjust pH to 3.0 with hydrochloric acid) and collect the eluent;

[0225] (8) Neutralize with 100 μl of 1M Tris-HCl buffer (pH 8.0);

[0226] (9) PBS buffer was used for ultrafiltration dialyzing three times, and finally the concentration was concentrated to ≥1 mg / ml.

[0227] 0.3 mg of humanized IgE antibody against Der p 1 monoclonal antibody was obtained after purification.

[0228] Example 5: Assignment of humanized IgE antibody against Der p1

[0229] Assignment of anti-Derp1 humanized IgE antibody using total IgE standard curve:

[0230] The anti-Der p 1 humanized IgE antibody was serially diluted to 100 ng / ml (international standard IgE 1 IU ≈ 2.4 ng). The concentration of the anti-Der p 1 humanized IgE antibody was detected using a total IgE assay kit (EUROIMMUN, catalog number: EV 3840-9601E). The result was 38.90 IU / ml (1 IU = 2.57 ng). The anti-Der p 1 humanized IgE antibody was diluted to a standard according to the table below, with 1 IU = 2.57 ng.

[0231] Der p 1 humanized IgE dilution concentration

[0232]

[0233] Example 6: Detection of specific IgE in samples using anti-Derp1 humanized IgE antibody

[0234] In this embodiment, a Derp1-specific IgE standard curve was constructed using an anti-Derp1 humanized IgE antibody, and a total IgE standard curve was also constructed. Clinical samples were tested, and the luminescence values ​​were substituted into the two standard curves to obtain the concentration of specific IgE in the samples. The specific experimental procedure is as follows:

[0235] 6.1 Constructing a total IgE standard curve

[0236] (1) Magnetic beads coated with anti-human IgE monoclonal antibody magnetic beads

[0237] a) Activation of magnetic beads: Take 10 mg of carboxyl magnetic beads (3 μm, JSR MS300) into 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 rack, and wait for the magnetic beads to be completely adsorbed onto the side wall. Use a pipette tip 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 at room temperature for 15 minutes;

[0238] b) Antibody coating: Place the centrifuge tube on a magnetic rack and wait for the magnetic beads to be completely adsorbed onto the side wall. Use a pipette tip to remove the supernatant, resuspend the magnetic beads with 1 ml of washing buffer, and add 0.1 mg of anti-human IgE antibody 1A2 (Absin ABS159965). Incubate at room temperature for 2 hours after inverting to mix.

[0239] c) Blocking of magnetic beads: Add 20 μl of blocking agent (JSR CE510:CE210 = 9:1) to the centrifuge tube, mix by inversion at room temperature and incubate for 1 hour;

[0240] d) Cleaning: Place the centrifuge tube on a magnetic rack and wait for the magnetic beads to be completely adsorbed onto the sidewall. Remove the supernatant with a pipette tip. Resuspend the magnetic beads in 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. Finally, use 10 ml of magnetic bead preservation 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).

[0241] (2) Acridinium ester-labeled anti-human IgE antibody 4C3 (Absin ABS159966):

[0242] (a) Take 100 μg of anti-human IgE antibody 4C3, add 5 μl of acridine ester (NSP-DMAE-NHS) with a concentration of 5 mg / ml, and mix by inversion at room temperature in the dark for 30 minutes.

[0243] (b) 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 in the dark for 30 minutes;

[0244] (c) Desalting: One day in advance, prepare G-25 by swelling it in excess deionized water. Take 10 ml (column bed volume) and pack it into the column. Equilibrate with 5 column bed volumes of 0.1 M PB buffer at pH 6.8. Load the labeled antibody (volume ≤ 1 ml) onto the column, collect the eluent and label it as tube 1. Elute with 1 ml PB buffer and collect the eluent and label it as tube 2. Repeat the elution with 1 ml PB buffer and collect the eluent until tube 6. Take a sample of the collected solution and measure the protein concentration using a BCA protein concentration assay kit. Use the tube with the highest concentration for subsequent experiments.

[0245] (d) Preparation of working solution for chemiluminescence detection antibody: The labeled antibody was prepared into a 1 μg / ml working solution using antibody dilution buffer (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) and stored at 4℃.

[0246] (3) Total IgE was detected by chemiluminescent immunoassay, and a standard curve for total IgE was prepared:

[0247] Total IgE (WHO International Standard No.: 11 / 234) was diluted with diluent (PBS, 0.1% m / v BSA) to concentrations of 100, 50, 17.5, 3.5, 0.7, 0.35, and 0.1 IU / ml. The diluent served as a negative control. AcriBest anti-human IgE antibody (1A2) magnetic beads and acridinium ester AcriBest anti-human IgE antibody (4C3) were placed in a Shine i2910 fully automated chemiluminescence analyzer for sandwich chemiluminescence detection of the total IgE standard and the negative control (diluent). The reaction mode is as follows:

[0248] Step 1: Take 10 μl of serially diluted international standard total IgE at different concentrations, add 20 μl of anti-human IgE antibody magnetic beads, mix and incubate at 37°C for 20 minutes;

[0249] Step 2: Cleaning;

[0250] Step 3: Take 100 μl of the working solution of acridine ester-labeled anti-human IgE antibody, mix and incubate at 37°C for 20 minutes;

[0251] Step 4: Cleaning;

[0252] Step 5: Add 100 μl of pre-excitation solution and 100 μl of excitation solution, and detect the luminescence value. The results are shown in the table below. The standard curve for the 4PLC calibration algorithm is shown below. Figure 4 y = (ad) / (1 + (x / c)) b )+d, a=1261.39830112; b=1.04241368; c=83.33895104; d=5351985.61254, correlation coefficient of the main curve: 0.999669.

[0253] Total IgE concentration (IU / ml) Luminous value 100 2887466 50 2007884 17.5 901884 3.5 188484 0.7 36484 0.35 19003 0.1 6284 0 1255

[0254] 6.2 Constructing a standard curve for anti-Der p1 humanized IgE:

[0255] Using the anti-Derp1 humanized IgE antibody already assigned in Example 5 as a standard and the diluent as a negative control, Derp1 magnetic beads (prepared in Example 1) and labeled acrid ester erbisimide anti-human IgE antibody (4C3) were placed in a Shine i2910 fully automated chemiluminescence analyzer for sandwich chemiluminescence detection. The reaction mode was basically the same as the reaction mode for the total IgE standard curve determination in 6.1 of Example 6, except that the international standard total IgE was replaced with the anti-Derp1 humanized IgE antibody already assigned in Example 5, and the anti-human IgE antibody magnetic beads were replaced with Derp1 magnetic beads. The luminescence values ​​of the Derp1 specific IgE standard curve prepared using the Derp1 humanized IgE antibody standard are shown in the table below. 4. PLC calibration algorithm formula: y=(ad) / (1+(x / c)) b )+d, where a=1255.09173763; b=1.02184142; c=122.67393791; d=5232109.98073, and the correlation coefficient of the main curve is 0.999980.

[0256]

[0257]

[0258] According to the standard curve Figure 5The results show that the signal-to-noise ratio of the luminescence value of the 0.1 U / ml sample is 4971 / 1253 = 3.97, which is more than 3 times the signal-to-noise ratio. Therefore, the sensitivity of this reagent can reach 0.1 IU / ml.

[0259] 6.3 Purification of Der p 1-specific IgE antibody from house dust mite-positive patient samples:

[0260] (1) Purify Der p 1-specific IgE antibody from serum samples

[0261] a) Take 5 mg of Der p 1 magnetic beads prepared in Example 1 and mix with 10 ml of clinical house dust mite-specific IgE positive serum, invert and incubate at room temperature for 2 h to allow Der p 1-specific IgE in the sample to form a complex with Der p 1; wash 3 times with TBST.

[0262] b) Elute with 2 ml of pH 3.0 0.1 M Glycin solution to remove Der p 1-specific IgE from the complex, and then neutralize with 200 μl of 1 M Tris-HCl (pH = 8.0) buffer.

[0263] c) Add 100 μl of antibody protection solution (PBS, 0.1% m / v BSA), dialyze three times with PBS ultrafiltration, and concentrate to 200 μl;

[0264] (2) The concentration of purified Der p1-specific IgE was detected using a total IgE detection kit (EUROIMMUN, catalog number: EV 3840-9601E).

[0265] Detection results: The concentration of Der p 1-specific IgE was 15.42 IU / ml.

[0266] 6.4 The concentration of Derp1-specific IgE was determined using total IgE standard curves and anti-Derp1 humanized IgE standard curves, respectively:

[0267] The Der p 1 specific IgE standard purified in step 6.3 at a concentration of 15.42 IU / ml was tested for luminescence using a kit consisting of Der p 1 magnetic beads and acridinium ester Erbisin anti-human IgE antibody (4C3). The concentration of Der p 1 specific IgE, a component of the allergen protein, was calculated using the total IgE standard curve and the anti-Der p 1 humanized IgE antibody standard curve prepared in the examples. The results are shown in the table below:

[0268]

[0269] The results showed that the curve calculated using anti-Derp1 humanized IgE antibody was closer to the true concentration of the sample (15.42 IU / ml), making the results more accurate.

[0270] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the 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 this invention.

Claims

1. An anti-Derp1 antibody, characterized in that, The anti-Derp1 antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequences of the complementarity-determining regions in the heavy chain variable region are: CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No.

3. The amino acid sequences of the complementarity-determining regions in the light chain variable region are: CDR1 shown in SEQ ID No. 4, CDR2 sequence is WAS, and CDR3 shown in SEQ ID No. 5, wherein WAS in the CDR2 sequence represents tryptophan, alanine, and serine, respectively.

2. The anti-Derp1 antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-Der p 1 antibody is shown in SEQ ID No. 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

7.

3. The anti-Derp1 antibody according to claim 1, characterized in that, The anti-Derp1 antibody also contains a human IgE heavy chain constant region or a human Kappa chain constant region.

4. The anti-Derp1 antibody according to claim 3, characterized in that, The amino acid sequence of the constant region of the human IgE heavy chain is shown in SEQ ID No. 10; or, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID No.

11.

5. The anti-Derp1 antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain region of the anti-Der p 1 antibody is shown in SEQ ID No. 12, and the amino acid sequence of the light chain region of the anti-Der p 1 antibody is shown in SEQ ID No.

13.

6. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-Der p1 antibody as described in any one of claims 1-5.

7. A nucleic acid construct, characterized in that, The nucleic acid construct comprises an expression vector and the nucleic acid molecule as described in claim 6.

8. An isolated engineered cell, characterized in that, The engineered cells contain the nucleic acid construct of claim 7 or the nucleic acid molecule of claim 6.

9. A test kit, characterized in that, The test kit contains the anti-Derp 1 antibody as described in any one of claims 1-5.

10. Use of the anti-Der p 1 antibody as described in any one of claims 1-5, the nucleic acid molecule as described in claim 6, the nucleic acid construct as described in claim 7, or the engineered cell as described in claim 8 in the preparation of a detection product.

11. The use according to claim 10, characterized in that, The testing product is a test product for hypersensitivity diseases.

12. The use according to claim 11, characterized in that, The hypersensitivity disease detection product is a Der p 1 specific IgE antibody detection product for dust mite allergen components.

Images