Anti-mpo human igg monoclonal antibody and application thereof

Abstract

The application discloses an anti-MPO human IgG monoclonal antibody and application thereof. Amino acid sequences of CDR1, CDR2 and CDR3 in a heavy chain variable region of the anti-MPO human IgG monoclonal antibody are sequentially shown as SEQ ID No. 1 26-33, 51-58 and 97-111, and amino acid sequences of CDR1, CDR2 and CDR3 in a light chain variable region are sequentially shown as SEQ ID No. 2 26-34, 52-54 and 91-102. Experiments prove that the monoclonal antibody not only has good binding activity with MPO, with a Kd of 0.0088 nM and an EC50 of 1.33 ng / mL, but also has good universality and stability, and can be used as MPO-ANCA quality control instead of a clinical positive sample, and then used for AAV diagnosis, activity evaluation and treatment effect monitoring.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-MPO human IgG monoclonal antibody and its applications. Background Technology

[0002] Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by necrotizing small vessel vasculitis, with myeloperoxidase (MPO) being one of the most important autoantigens. Circulating anti-MPO antibodies (MPO-ANCA) have been listed as a core serological marker for AAV diagnosis, activity assessment, and treatment monitoring in international guidelines. With the widespread use of multi-platform detection systems such as chemiluminescence, flow cytometry, and enzyme-linked immunosorbent assay (ELISA) in clinical laboratories, third-party medical examination centers, and regional clinical testing centers, higher requirements have been placed on the consistency, comparability, and traceability of MPO-ANCA testing quality.

[0003] However, existing MPO-ANCA quality control materials rely almost entirely on the collection of serum from MPO-ANCA-positive patients, which has the following inherent drawbacks: First, the source is scarce and ethical collection is restricted, making it difficult to achieve large-scale, continuous production; second, antibody titers, subtypes, and matrix components fluctuate with individual, disease course, and storage conditions, resulting in large batch-to-batch differences, large assignment uncertainty, discrete interlaboratory quality assessment (EQA) results, and low interlaboratory recognition of results.

[0004] The aforementioned issues directly impact the reliability, accuracy, and clinical interpretability of MPO-ANCA test results, becoming a bottleneck restricting precision diagnosis and treatment of AAV and large-scale multi-center research. Therefore, developing a large-scale, batch-to-batch controllable, and traceable MPO-ANCA quality control product is of urgent practical significance for improving the testing quality of medical laboratories in my country and globally, achieving regional mutual recognition, and ensuring result comparability. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a large-scale, traceable, versatile, and stable anti-MPO human IgG monoclonal antibody, which can be used as an MPO-ANCA quality control material. The technical problem to be solved is not limited to the technical subject matter described herein; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0006] To address the aforementioned technical problems, the present invention first provides an anti-MPO monoclonal antibody.

[0007] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the anti-MPO monoclonal antibody provided by the present invention are shown in positions 26-33, 51-58, and 97-111 of SEQ ID No. 1, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the anti-MPO monoclonal antibody are shown in positions 26-34, 52-54, and 91-102 of SEQ ID No. 2, respectively.

[0008] Among them, CDR1, CDR2 and CDR3 are three complementary determinant regions in the heavy chain variable region or the light chain variable region, and the sequence of complementary determinant regions is defined according to Kabat.

[0009] Furthermore, the heavy chain variable region and the light chain variable region also include a frame region.

[0010] The structure of the heavy chain variable region is as follows: frame region FR1 - complementarity-determining region CDR1 - frame region FR2 - complementarity-determining region CDR2 - frame region FR3 - complementarity-determining region CDR3 - frame region FR4. The amino acid sequence of frame region FR1 is shown as positions 1-25 of SEQ ID No. 1; the amino acid sequence of frame region FR2 is shown as positions 34-50 of SEQ ID No. 1; the amino acid sequence of frame region FR3 is shown as positions 59-96 of SEQ ID No. 1; and the amino acid sequence of frame region FR4 is shown as positions 112-122 of SEQ ID No. 1.

[0011] The structure of the light chain variable region is as follows: frame region FR1 - complementarity-determining region CDR1 - frame region FR2 - complementarity-determining region CDR2 - frame region FR3 - complementarity-determining region CDR3 - frame region FR4. The amino acid sequence of frame region FR1 is shown as positions 1-25 of SEQ ID No. 2; the amino acid sequence of frame region FR2 is shown as positions 35-51 of SEQ ID No. 2; the amino acid sequence of frame region FR3 is shown as positions 55-90 of SEQ ID No. 2; and the amino acid sequence of frame region FR4 is shown as positions 103-112 of SEQ ID No. 2.

[0012] Furthermore, the amino acid sequence of the heavy chain variable region is any one of the following A1)-A4):

[0013] A1) The amino acid sequence is that of the protein shown in SEQ ID No. 1; A2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 1; A3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1; A4) Proteins that have 90% or more of the same amino acid sequence as shown in SEQ ID No. 1 and have the same function.

[0014] The amino acid sequence of the light chain variable region is any one of the following B1)-B4): B1) The amino acid sequence is that of the protein shown in SEQ ID No. 2; B2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 2; B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2; B4) is a protein that has 80% or more of the same amino acid sequence as the one shown in SEQ ID No. 2 and has the same function.

[0015] In the protein described in A2) or B2) above, the tag refers to a polypeptide or protein fused with a target antibody using in vitro DNA recombination technology, to facilitate the expression, detection, tracing, and / or purification of the target antibody. The tag includes, but is not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0016] In the proteins described in A3) or B2) above, the substitution and / or deletion and / or addition of one or more amino acid residues constitutes a substitution and / or deletion and / or addition of no more than 10 amino acid residues. In some embodiments, substitution and / or deletion and / or addition of one or more amino acid residues may occur within one or more complementarity-determining regions and / or frame regions of the antibody described in this invention, provided that such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conservative changes (e.g., conservative substitutions, as is well known to those skilled in the art, where conservative substitution of amino acids does not alter the properties and function of the protein) may be made to the complementarity-determining regions and / or frame regions without materially reducing binding affinity.

[0017] In the proteins described in A4) or B4) above, the identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained. The identity includes amino acid sequences that have 80% or more, or 85% or more, or 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 of the present invention.

[0018] The proteins described in A1), A2), A3), A4), B1), B2), B3), or B4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0019] Furthermore, the antibody also includes a heavy chain constant region and a light chain constant region.

[0020] The heavy chain constant region of the anti-MPO monoclonal antibody is the heavy chain constant region of human IgG1.

[0021] The light chain constant region of the anti-MPO monoclonal antibody is the human Lambda-type light chain constant region.

[0022] In some embodiments, the amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 3.

[0023] In some embodiments, the amino acid sequence of the light chain constant region is shown in SEQ ID No. 4.

[0024] To address the aforementioned technical problems, the present invention also provides any of the following biomaterials: C1) The active fragment of the antibody is derived from any of the following from the above-mentioned anti-MPO monoclonal antibody: antigen-binding fragment, Fab fragment, Fab′ fragment, F(ab′)2 fragment, single-chain antibody, single-domain antibody, bispecific antibody, or smallest recognition unit; C2) A nucleic acid molecule, wherein the nucleic acid molecule encodes the above-described anti-MPO monoclonal antibody or the active fragment described in C1); C3) Expression cassettes, recombinant vectors, recombinant cells or recombinant microorganisms containing the nucleic acid molecules described in C2).

[0025] In the nucleic acid molecule described in C2), the nucleotide sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region encoding the anti-MPO monoclonal antibody are shown in positions 76-99, 151-174 and 289-333 of SEQ ID No. 5, respectively.

[0026] In the nucleic acid molecule described in C2), the nucleotide sequences of CDR1, CDR2 and CDR3 in the light chain variable region encoding the anti-MPO monoclonal antibody are shown in positions 76-102, 154-162 and 271-306 of SEQ ID No. 6, respectively.

[0027] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the anti-MPO monoclonal antibody is SEQ ID No. 5, or a sequence having at least 75% identity with SEQ ID No. 5.

[0028] The nucleotide sequence encoding the light chain variable region of the anti-MPO monoclonal antibody is SEQ ID No. 6, or a sequence having at least 75% identity with SEQ ID No. 6.

[0029] Furthermore, the nucleotide sequence encoding the heavy chain constant region of the antibody is SEQ ID No. 7, or a sequence having at least 75% identity with SEQ ID No. 7.

[0030] The nucleotide sequence encoding the light chain constant region of the antibody is SEQ ID No. 8, or a sequence having at least 75% identity with SEQ ID No. 8.

[0031] Those skilled in the art can readily mutate the nucleotide sequences encoding the variable or constant regions of the antibodies of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides having 75% or higher identity to the nucleotide sequences encoding the variable or constant regions of the antibodies, as long as they encode the variable or constant regions and have the same function, are nucleotide sequences derived from and equivalent to those of this invention. Considering codon degeneracy, the gene sequence encoding the aforementioned antibodies can be modified in its coding region without altering the amino acid sequence to obtain genes encoding the same antibodies; alternatively, genes can be artificially synthesized to improve antibody expression efficiency based on the codon preference of the host expressing the antibody. Variants of the antibodies obtained using methods known in the art are all included within the scope of this invention. Furthermore, polynucleotides containing sequences that optimize antibody specificity or neutralizing activity by applying directed evolution to any nucleic acid sequence of this invention are also within the scope of this invention.

[0032] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No. 4 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0033] In C3 above, the expression cassette refers to DNA capable of expressing the antibody in a host cell, which may include not only a promoter that initiates transcription of the antibody-encoding gene sequence, but also a terminator that terminates transcription of the antibody-encoding gene sequence.

[0034] The vectors include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpesviruses (such as herpes simplex virus), etc. The recombinant vector refers to a recombinant DNA molecule constructed by in vitro ligation of the aforementioned nucleic acid molecules or expression cassettes with the vector. This recombinant vector can directly or indirectly introduce the aforementioned nucleic acid molecules or expression cassettes into host cells for gene expression through transformation, transfection, or transduction. Specifically, the recombinant vector is a heavy chain recombinant expression plasmid or a light chain recombinant expression plasmid as described below.

[0035] The cells (host cells) include, but are not limited to, eukaryotic cells (such as yeast cells, Aspergillus), animal cells (such as mammalian cells, insect cells), or prokaryotic cells. The recombinant cells refer to cells containing the aforementioned recombinant vector. Specifically, the recombinant cells are HEK293 cells containing the aforementioned recombinant vector.

[0036] The microorganisms mentioned include, but are not limited to, yeast, bacteria, algae, or fungi. Among them, bacteria may be derived from genera such as *Escherichia*, *Erwinia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, and *Bacillus*; yeast may be *Pichia pastoris*. The recombinant microorganisms refer to microorganisms containing the aforementioned recombinant vectors.

[0037] To address the aforementioned technical problems, the present invention also provides the application of the above-mentioned anti-MPO monoclonal antibody in any of the following (X1)-X6): X1) As a quality control, calibrator, or positive sample analogue for anti-MPO antibody detection reagents; X2) Detection of anti-MPO antibodies; X3) Prepare products for detecting anti-MPO antibodies; X4) Prepare products for AAV diagnosis, AAV activity assessment, or AAV treatment efficacy monitoring; X5) Detection of MPO protein; X6) Prepare products for detecting MPO protein.

[0038] The application of the above-mentioned biomaterials in any of the following Y1)-Y5) is also within the scope of protection of this invention: Y1) Prepare the above-mentioned anti-MPO monoclonal antibody; Y2) Preparation of quality control materials, calibrators, or positive sample analogs for anti-MPO antibody detection reagents; Y3) Prepare products for detecting anti-MPO antibodies; Y4) Prepare products for AAV diagnosis, AAV activity assessment, or AAV treatment efficacy monitoring; Y5) Prepare products for detecting MPO protein.

[0039] In the above applications, the anti-MPO antibody detection reagents include commercially available anti-MPO antibody detection reagents or kits commonly used in this technical field, such as anti-MPO antibody IgG assay kits from brands like AESKU, WOFEN, and EUROIMMUN.

[0040] To address the aforementioned technical problems, the present invention also provides a product for detecting anti-MPO antibodies.

[0041] The product for detecting anti-MPO antibodies provided by this invention includes the above-mentioned anti-MPO monoclonal antibody.

[0042] In some implementations, the product may be a quality control sample, a calibrator, or a positive sample analogue.

[0043] To address the aforementioned technical problems, this invention also provides a method for preparing the anti-MPO monoclonal antibody.

[0044] The method for preparing the anti-MPO monoclonal antibody provided by the present invention includes the following steps: expressing the encoding gene of the anti-MPO monoclonal antibody in a host cell to obtain the anti-MPO monoclonal antibody.

[0045] Furthermore, the method for expressing the encoding gene of the antibody in the host cell is to introduce the encoding gene of the antibody into the host cell.

[0046] The introduction can be performed by transforming host cells with a vector carrying the antibody-encoding gene using any known transfection method, such as calcium phosphate co-precipitation, liposome-mediated transfection, electroporation, or viral vector transfection.

[0047] Furthermore, the cells (host cells) include, but are not limited to: eukaryotic cells (such as yeast cells, Aspergillus), animal cells (such as mammalian cells, insect cells) or prokaryotic cells.

[0048] Furthermore, the host cell is a HEK293 cell.

[0049] In some embodiments, the antibody-encoding gene is introduced into a host cell via a heavy chain recombinant expression plasmid or a light chain recombinant expression plasmid, as described below.

[0050] The amino acid sequence of any of the MPO proteins described above is shown in SEQ ID No. 9.

[0051] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding affinity to the target. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target.

[0052] The term "Fab fragment" refers to a heterodimer composed of a heavy chain Fd and a complete light chain linked by disulfide bonds, containing only one antigen-binding site. By linking the coding genes for the heavy chain Fd and the complete light chain and fusing them with a bacterial protein signal peptide gene, Fab antibodies (Fab fragments) can be secreted and expressed in *E. coli*, exhibiting complete stereofolding and intra- and inter-chain disulfide bonds. The heavy chain Fd refers to approximately half of the H chain portion of the Fab fragment (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0053] The term "Fab′ fragment" contains a portion of a light chain and a heavy chain containing the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of two Fab′ fragments to form the F(ab′)2 molecule.

[0054] The term "F(ab′)2 segment" contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab′)2 segment consists of two Fab′ segments held together by the disulfide bond between the two heavy chains.

[0055] The term "single-chain antibody (ScFv)" refers to an antibody that expresses a single polypeptide chain by linking the light and heavy chain variable regions of a gene with an appropriate oligonucleotide linker. The polypeptide chain can spontaneously fold into its native conformation, maintaining the specificity and affinity of the Fv.

[0056] The term "single-domain antibody (nanobody)" refers to an antibody containing only the VH fragment, obtained by expressing the V region of the antibody heavy chain through genetic engineering. The binding ability and stability of single-domain antibodies to antigens are essentially the same as those of complete antibodies.

[0057] The term "bispecific antibody" refers to a high-yield, uniform, and pure bispecific antibody obtained by introducing two sets of light chain and heavy chain genes into myeloma cells and selecting appropriate antibody constant regions and Ig types. Alternatively, bispecific antibodies can also be obtained using chemical cross-linking techniques or hybridization-hybridoma techniques.

[0058] The term "minimum recognition unit (MRU)" refers to a single CDR structure containing only the variable region, with a molecular weight of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.

[0059] This invention uses MPO protein as an antigen and obtains a human MPO monoclonal antibody through phage display technology. Experiments demonstrate that this anti-MPO monoclonal antibody exhibits good binding activity to MPO, with an affinity dissociation constant (Kd) of 0.0088 nM and an EC50 of 1.33 ng / mL. Furthermore, this invention uses three mainstream commercial anti-MPO antibody IgG assay kits to detect the anti-MPO monoclonal antibody, finding that it reacts on all mainstream commercial kits, indicating that the anti-MPO monoclonal antibody prepared in this invention has good versatility. Next, this invention also tests the stability of the anti-MPO monoclonal antibody. The results show that after 10 freeze-thaw cycles at -20℃, the titer decrease is less than 10%, and after accelerated freezing at 37℃ for 7 days, the titer decrease is less than 20%, indicating good stability. The above results indicate that the anti-MPO monoclonal antibody prepared in this invention has good versatility and stability, and can be used as a quality control product (or calibrator or positive sample analog) for anti-MPO antibody detection reagents (or third-party testing laboratories) to replace clinical positive samples, and thus be used for AAV diagnosis, activity assessment and treatment effect monitoring. Attached Figure Description

[0060] Figure 1 This is the SDS-PAGE analysis result of the anti-MPO monoclonal antibody. A is the reduced band; B is the non-reduced band.

[0061] Figure 2 The binding activity of anti-MPO monoclonal antibody to MPO was detected by ELISA. Detailed Implementation

[0062] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0063] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the experimental methods in the following embodiments are performed at least three times.

[0064] Example 1: Preparation of anti-MPO monoclonal antibody I. Construction of Human Phage Library Peripheral blood mononuclear cells (PBMCs) were collected from more than 10 subjects who were strongly positive for anti-MPO antibodies. Total RNA was extracted from PBMCs using TRIzol reagent according to the reagent instructions, and cDNA was synthesized by reverse transcription. The cDNA obtained by reverse transcription was used as a template to amplify VH and VL fragments by PCR. The VH and VL fragments were spliced ​​into a nucleotide sequence encoding scFv using overlap extension splicing PCR. The fragments were then digested with a self-made restriction endonuclease SfiI and ligated into the phage vector pComb3x. The phage display scFv antibody library (ScFV phage library) was constructed by electroporation of X-Blue competent cells, and the library capacity was calculated.

[0065] II. Human Phage Bank Screening Immunotubes were coated with MPO antigen (Sino Biological, 11917-H08H), blocked with PBS containing 4% skim milk, and 1 mL of the ScFV phage library prepared in step one was added. After incubation at room temperature with shaking for 1 h, the tubes were washed with PBST, and 1 mL of arginine-acetate buffer (pH 3.0, 0.15 mol / L Arg-HAc, containing 1.6 g / L peptone and 1 g / L yeast extract) was added. The tubes were shaken at 25°C for 10 min, and the eluent was collected and immediately neutralized with 1 mol / L Tris-HCl (pH 7.4). 20 mL of TG1 bacterial culture in logarithmic growth phase was added, and the tubes were incubated at 37°C for 30 min. 10 μL of the incubator was plated onto a 2 YT / GA plate and incubated overnight at 37°C. The yield was then measured. The remaining bacterial culture was expanded, and the OD of the bacterial culture was measured. 600 When the value is 0.5, helper phage M13K07 is added, and the culture is incubated overnight. Phages are collected by precipitation. This completes the first round of selection. The above selection process is repeated three times to enrich phages that bind to the MPO antigen. ELISA plates are coated with MPO antigen, blocked with PBS containing 4% skim milk, and 50 μL of phage is added. After washing, diluted HRP antibody is added, and TMB is used for color development. Phages that show strong positive color development by ELISA are sequenced to obtain the antibody variable region gene sequence. Among them, the coding gene sequence of one antibody heavy chain variable region is shown in SEQ ID No:5, and the coding gene sequence of the light chain variable region is shown in SEQ ID No:6.

[0066] III. Expression and Purification of Anti-MPO Monoclonal Antibodies Based on the antibody variable region sequence obtained in step two, this invention provides an anti-MPO monoclonal antibody, named H341. The heavy chain of the anti-MPO monoclonal antibody H341 consists of a heavy chain variable region and a heavy chain constant region, while the light chain consists of a light chain variable region and a light chain constant region.

[0067] The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1, wherein the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown at positions 26-33, 51-58, and 97-111, respectively, in SEQ ID No. 1. The coding gene sequence of the heavy chain variable region is shown in SEQ ID No. 5, wherein the coding gene sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown at positions 76-99, 151-174, and 289-333, respectively, in SEQ ID No. 5.

[0068] The amino acid sequence of the light chain variable region is shown in SEQ ID No. 2, wherein the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown at positions 26-34, 52-54, and 91-102, respectively, in SEQ ID No. 2. The coding gene sequence of the light chain variable region is shown in SEQ ID No. 6, wherein the coding gene sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown at positions 76-102, 154-162, and 271-306, respectively, in SEQ ID No. 6.

[0069] The amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 3. The gene sequence encoding the heavy chain constant region is shown in SEQ ID No. 7.

[0070] The amino acid sequence of the light chain constant region is shown in SEQ ID No. 4. The gene sequence encoding the light chain constant region is shown in SEQ ID No. 8.

[0071] The specific preparation method of anti-MPO monoclonal antibody H341 is as follows: 1. Construction of recombinant expression plasmids The coding gene sequences of the variable region of the heavy chain (SEQ ID No. 5) and the coding gene sequences of the constant region of the heavy chain (SEQ ID No. 7) were ligated into the pCMV3 vector (Yiqiao Shenzhou) to obtain the heavy chain recombinant expression plasmid.

[0072] The coding gene sequence of the light chain variable region (SEQ ID No. 6) and the coding gene sequence of the light chain constant region (SEQ ID No. 8) were ligated into the pCMV3 vector (Yiqiao Shenzhou) to obtain the light chain recombinant expression plasmid.

[0073] 2. Preparation of anti-MPO monoclonal antibody H341 Anti-MPO monoclonal antibodies were prepared using a eukaryotic HEK 293 expression system. The specific preparation method includes the following steps: (1) HEK293 instantaneous rotation culture HEK293 cells were passaged in serum-free CD medium (293). Heavy chain recombinant expression plasmids and light chain recombinant expression plasmids were mixed with transfection reagent TF2 and added to the HEK293 cells. Serum-free CD medium (293) was added on days 1, 3, and 5 post-transfection. Shake flask culture conditions: 5% CO2, 37°C, shaker speed 175 rpm.

[0074] (2) Antibody purification (2-1) Sample preparation: Centrifuge at 4000 g for 30 min using a benchtop centrifuge and collect the supernatant.

[0075] (2-2) Filtration: Select different filtration methods according to the volume of the liquid, and use a 0.45 μm filter membrane for filtration.

[0076] (2-3) Connection system: Select a protein A column of appropriate specifications according to the expression level and connect it to the purification system.

[0077] (2-4) Water balance: Rinse 2CV with ultrapure water and replace the 25% ethanol preservation solution.

[0078] (2-5) Equilibrium chromatography column: AC Binding equilibrium 3CV until UV baseline is stable.

[0079] (2-6) Sample loading: Adjust the appropriate flow rate for sample loading.

[0080] (2-7) Rinsing: Rinse with rinsing solution for 5-10 CV until the UV baseline is stable, at the same flow rate as the sample loading flow rate.

[0081] (2-8) Elution: AC Elutin elution, collected according to UV peak.

[0082] (2-9) Neutralization: Add Tris and neutralize the eluted antibody at pH 8.0.

[0083] (2-10) Balance: AC Binding balances 3CV to neutral.

[0084] (2-11) CIP cleaning: CIP cleaning 5CV or more.

[0085] (2-12) Rinse with alkali: Rinse with alkali using AC Binding until the pH at the outlet is neutral, then rinse with ultrapure water for 3CV.

[0086] (2-13) Preservation: Equilibrate with 25% ethanol at 2CV and preserve the column.

[0087] 3. SDS-PAGE and purity identification of anti-MPO monoclonal antibody H341 The purified anti-MPO monoclonal antibody H341 was stored in 10 mM PBS pH 7.2 buffer, adjusted to a concentration of 0.5 mg / mL, and its purity was determined by SDS-PAGE and Coomassie brilliant blue staining. The SDS-PAGE results are shown below. Figure 1 As shown, the purified anti-MPO monoclonal antibody H341 has a purity of over 90%.

[0088] Example 2: Affinity detection of anti-MPO monoclonal antibody H341 The binding activity of the anti-MPO monoclonal antibody to MPO was detected by ELISA.

[0089] I. Affinity Testing 1. Coating: Coat with antigen protein at 0.1 μg / mL or 1 μg / mL, 100 μL / well, overnight at 4℃.

[0090] 2. Sealing: Shake off the liquid inside the plate and pat dry. Add 2% BSA, 300 μL / well, seal, and incubate at room temperature for 1 h.

[0091] 3. Washing the plate: Use 300 μL of washing solution per well to wash the plate twice, and pat dry the plate after the last wash.

[0092] 4. Antibody dilution: Dilute the anti-MPO monoclonal antibody H341 prepared in Example 1 to 1 μg / mL, add 100 μL to each well of the corresponding plate, mix well, and react at room temperature for 2 h.

[0093] 5. Washing the plate: Use 300 μL of washing solution per well to wash the plate 3 times, and pat dry after the last wash.

[0094] 6. Add secondary antibody: Dilute Goat Anti-Human IgG (H+L) / HRP secondary antibody to the working concentration, 100 μL / well, mix well, and incubate at room temperature for 1 h.

[0095] 7. Washing the plate: Use 300 μL of washing solution per well to wash the plate 3 times, and pat dry after the last wash.

[0096] 8. Color development: Mix solution A and solution B at a 1:1 ratio, add 200 μL to each well, and incubate at room temperature in the dark for 20 min.

[0097] 9. Termination: Add 50 μL of termination solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0098] The results showed that the affinity dissociation constant (Kd) of the anti-MPO monoclonal antibody H341 was 0.0088 nM.

[0099] II. EC50 Testing 1. Coating: Coat with 1 μg / mL antigen protein, 100 μL / well, overnight at 4℃.

[0100] 2. Sealing: Shake off the liquid inside the plate and pat dry. Add 2% BSA, 300 μL / well, seal, and incubate at room temperature for 1 h.

[0101] 3. Washing the plate: Use 300 μL of washing solution per well to wash the plate twice, and pat dry the plate after the last wash.

[0102] 4. Antibody dilution: The anti-MPO monoclonal antibody H341 prepared in Example 1 was diluted to 1 μg / mL, and then serially diluted three times to 10 wells. 100 μL of each well was added to the corresponding well plate, mixed well, and reacted at room temperature for 2 h.

[0103] 5. Washing the plate: Use 300 μL of washing solution per well to wash the plate 3 times, and pat dry after the last wash.

[0104] 6. Add secondary antibody: Dilute Goat Anti-Human IgG (H+L) / HRP secondary antibody to the working concentration, 100 μL / well, mix well, and incubate at room temperature for 1 h.

[0105] 7. Washing the plate: Use 300 μL of washing solution per well to wash the plate 3 times, and pat dry after the last wash.

[0106] 8. Color development: Mix solution A and solution B at a 1:1 ratio, add 200 μL to each well, and incubate at room temperature in the dark for 20 min.

[0107] 9. Termination: Add 50 μL of termination solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0108] The results are as follows Figure 2 As shown, the results indicate that the EC50 is 1.33 ng / mL.

[0109] The above results indicate that the anti-MPO monoclonal antibody H341 has good binding activity with MPO.

[0110] Example 3: Universality evaluation of anti-MPO monoclonal antibody H341 Anti-MPO monoclonal antibody H341 solution was diluted 10-fold and 100-fold with negative serum (serum in which anti-MPO antibodies were not detected in clinical tests), and then detected using anti-MPO antibody IgG assay kits from AESKU (3303), WOFEN (708700), and EUROIMMUN (EA1211-9601 G), respectively. For specific detection methods, please refer to the instructions for each kit.

[0111] The results are shown in Table 1. The results show that this anti-MPO monoclonal antibody H341 can react on all mainstream commercial kits.

[0112] Table 1. Reaction of anti-MPO monoclonal antibody H341 on different kits

[0113] Note: The CO value represents CutOff (a value lower than the CO value is negative).

[0114] Example 4. Stability evaluation of anti-MPO monoclonal antibody H341 The anti-MPO monoclonal antibody H341 solution was diluted 500-fold and 1000-fold with negative serum respectively, and the stability of anti-MPO monoclonal antibody H341 after 7 days of acceleration at 37°C (placed at 37°C for 7 days) and 10 freeze-thaw cycles at -20°C was evaluated respectively, while the corresponding antibody stored at 4°C to 8°C was used as a control. The treated antibodies and the control group antibodies were detected using the anti-MPO antibody IgG assay kit of the brand EUROIMMUN (EA 1211-9601 G).

[0115] Table 2. Stability test of anti-MPO monoclonal antibody

[0116] The results are shown in Table 2. The results indicate that the anti-MPO monoclonal antibody H341 has good stability. Specifically, the titer decrease after 10 freeze-thaw cycles at -20°C is less than 10%, and the titer decrease after 7 days of acceleration at 37°C is less than 20%, both showing good stability.

[0117] The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented within a wide range with equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experiments. Although the present invention has been presented with specific examples, it should be understood that further improvements can be made to the present invention. In summary, according to the principle of the present invention, the present application is intended to cover any variations, uses or improvements of the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in the present application. Some applications of the essential features can be carried out within the scope of the appended claims below.

Claims

1. An anti-MPO monoclonal antibody, characterized in that: The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the anti-MPO monoclonal antibody are shown in positions 26-33, 51-58, and 97-111 of SEQ ID No. 1, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the anti-MPO monoclonal antibody are shown in positions 26-34, 52-54, and 91-102 of SEQ ID No. 2, respectively.

2. The anti-MPO monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is any one of the following A1)-A3): A1) The amino acid sequence is that of the protein shown in SEQ ID No. 1; A2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 1; A3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1; The amino acid sequence of the light chain variable region is any one of the following B1)-B3): B1) The amino acid sequence is that of the protein shown in SEQ ID No. 2; B2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 2; B3) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.

2.

3. The anti-MPO monoclonal antibody according to claim 1 or 2, characterized in that: The heavy chain constant region of the anti-MPO monoclonal antibody is the heavy chain constant region of human IgG1; The light chain constant region of the anti-MPO monoclonal antibody is the human Lambda-type light chain constant region.

4. Any of the following biological materials: C1) The active fragment of the antibody is derived from any of the following from the anti-MPO monoclonal antibody of any of claims 1-3: Fab fragment, Fab′ fragment, F(ab′)2 fragment, or single-chain antibody; C2) A nucleic acid molecule, wherein the nucleic acid molecule encodes the anti-MPO monoclonal antibody as described in any one of claims 1-3 or the active fragment as described in C1); C3) Expression cassettes, recombinant vectors, recombinant cells or recombinant microorganisms containing the nucleic acid molecules described in C2).

5. The biomaterial according to claim 4, characterized in that: In the nucleic acid molecule described in C2), the nucleotide sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region encoding the anti-MPO monoclonal antibody are shown in positions 76-99, 151-174 and 289-333 of SEQ ID No. 5, respectively. In the nucleic acid molecule described in C2), the nucleotide sequences of CDR1, CDR2 and CDR3 in the light chain variable region encoding the anti-MPO monoclonal antibody are shown in positions 76-102, 154-162 and 271-306 of SEQ ID No. 6, respectively.

6. The biomaterial according to claim 4, characterized in that: In the nucleic acid molecule described in C2), the nucleotide sequence encoding the heavy chain variable region of the anti-MPO monoclonal antibody is a sequence having at least 75% identity with SEQ ID No. 5; In the nucleic acid molecule described in C2), the nucleotide sequence encoding the light chain variable region of the anti-MPO monoclonal antibody is a sequence having at least 75% identity with SEQ ID No.

6.

7. The use of the anti-MPO monoclonal antibody according to any one of claims 1-3 in any one of the following X1)-X2): X1) Quality control materials, calibrators, or positive sample analogs for the preparation of anti-MPO antibody detection reagents; X2) Prepare products for detecting MPO protein.

8. The use of the biomaterial according to any one of claims 4-6 in any one of the following Y1)-Y3): Y1) Prepare the anti-MPO monoclonal antibody according to any one of claims 1-3; Y2) Preparation of quality control materials, calibrators, or positive sample analogs for anti-MPO antibody detection reagents; Y3) Prepare products for detecting MPO protein.

9. A product for detecting anti-MPO antibodies, comprising the anti-MPO monoclonal antibody as described in any one of claims 1-3.

10. A method for preparing the anti-MPO monoclonal antibody according to any one of claims 1-3, comprising the following steps: expressing the encoding gene of the anti-MPO monoclonal antibody according to any one of claims 1-3 in a host cell to obtain the anti-MPO monoclonal antibody.

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