Recombinant monoclonal antibody of eosinophilic granulocyte peroxidase as well as preparation and application of recombinant monoclonal antibody

Abstract

The invention relates to the field of biotechnology and in vitro diagnosis, in particular to a recombinant monoclonal antibody of eosinophilic granulocyte peroxidase as well as preparation and application of the recombinant monoclonal antibody. The present invention provides an antibody that specifically binds to eosinophilic granulocyte peroxidase, which has a CDR1 region, a CDR2 region, and a CDR3 region. A pair of antibody pairs for specifically recognizing the eosinophilic granulocyte peroxidase is obtained through phage library screening, and the sensitivity of the pairs is higher, so that the eosinophilic granulocyte peroxidase is rapidly and accurately detected.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and in vitro diagnostics, and particularly to recombinant monoclonal antibodies against eosinophil peroxidase, their preparation, and applications. Background Technology

[0002] Eosinophilic airway inflammation-related diseases are a collective term for chronic inflammatory diseases characterized primarily by eosinophilic infiltration of the airways. They include common respiratory diseases such as bronchial asthma, cough-variant asthma, and eosinophilic bronchitis. Asthma is the most common eosinophilic airway inflammation disease in clinical practice and a serious public health problem worldwide. Currently, there are approximately 300 million asthma patients globally and about 30 million in my country. The prevalence of asthma varies from 1% to 30% in different countries and is showing an increasing trend year by year. The current incidence rate in China is 2%-3%, exceeding 4% in some areas, but the rate of increase is rapid, and the asthma mortality rate is one of the highest in the world, resulting in a significant disease burden. Chronic cough is the most common chief complaint in respiratory medicine outpatient clinics. Eosinophilic bronchitis, as one of the common causes of chronic cough in my country, is often misdiagnosed and mistreated due to atypical symptoms and a lack of effective diagnostic methods, causing severe physical and mental suffering for patients and their families, and imposing a heavy economic burden on society. Accurate and timely assessment of the type and severity of airway inflammation is crucial for the diagnosis and treatment of eosinophilic airway inflammation-related diseases. In addition, eosinophilic airway inflammation is closely related to various chronic respiratory diseases such as some chronic obstructive pulmonary diseases (COPD) and allergic bronchopulmonary aspergillosis.

[0003] Eosinophil-specific granules contain four basic proteins: major basic protein (MBP) (located in the lens nucleus of the granule, accounting for 50% of the total granule protein), eosinophil cationic protein (ECP), eosinophil peroxidase (EPX), and eosinophil-derived neurotoxin (EDN), which are located in the granule matrix. ECP and EDN have been used clinically as markers of activated eosinophils for a long time; however, because these two proteins have been shown not to be eosinophil-specific products and can be expressed in neutrophils and liver tissue, their specificity is low, making them unsuitable as eosinophil markers. While MBP is most abundant in eosinophil granules, it is also significantly expressed in mast cells and basophils, thus making it unsuitable as a marker. EPX, as an eosinophil-specific product, has not been reported to be expressed in other inflammatory cells and tissues. Recent studies have also shown that it exhibits no cross-reactivity with neutrophils, demonstrating high specificity and meeting the requirements for eosinophil-specific markers. Currently, some researchers abroad have established enzyme-linked immunosorbent assays (ELISA) for EPX, but this method has several shortcomings: the monoclonal antibody used is an anti-mouse EPX monoclonal antibody. Although some experiments have demonstrated the binding reaction between this antibody and human EPX antigen, allowing for the establishment of an ELISA method for detecting human EPX, the fact that this antibody is an anti-mouse EPX monoclonal antibody may affect the specificity and sensitivity of the detection when used in human samples; the established ELISA method has not undergone corresponding methodological evaluation experiments to assess its detection performance; the ELISA method involves multiple steps, is time-consuming, requires specific equipment, and necessitates batch processing of samples, failing to meet the needs of rapid clinical diagnosis. Therefore, the development of highly sensitive and specific antibodies that recognize EPX is urgently needed. Summary of the Invention

[0004] In view of this, the present invention provides a recombinant monoclonal antibody against eosinophil peroxidase, its preparation and application.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a monoclonal antibody against eosinophil peroxidase:

[0007] (I) Its light chain CDR1, CDR2 and CDR3 have the amino acid sequences shown in SEQ ID No. 1, GAT and SEQ ID No. 2, respectively; or

[0008] Its light chain CDR1, CDR2, and CDR3 have the amino acid sequences shown in SEQ ID No. 18, RTS, and SEQ ID No. 19, respectively; and

[0009] (II) Its heavy chain CDR1, CDR2 and CDR3 have the amino acid sequences shown in SEQ ID No. 9, 10 and 11, respectively; or

[0010] Its heavy chain CDR1, CDR2, and CDR3 have the amino acid sequences shown in SEQ ID No. 26, 27, and 28, respectively; and

[0011] (III) A sequence based on the amino acid sequence shown in (I) or (II) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0012] (IV) A sequence that is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more homologous to any of the amino acid sequences shown in (I) to (III).

[0013] In some specific embodiments of the present invention, the monoclonal antibody:

[0014] (I) Its light chain CDR1, CDR2, and CDR3 have the amino acid sequences shown in SEQ ID No. 1, GAT, and SEQ ID No. 2, respectively; and

[0015] Its heavy chain CDR1, CDR2, and CDR3 have the amino acid sequences shown in SEQ ID No. 9, 10, and 11, respectively; or

[0016] (II) Its light chain CDR1, CDR2, and CDR3 have, respectively, the amino acid sequences shown in SEQ ID No. 18, RTS, and SEQ ID No. 19; and

[0017] Its heavy chain CDR1, CDR2, and CDR3 have the amino acid sequences shown in SEQ ID No. 26, 27, and 28, respectively; and

[0018] (III) A sequence based on the amino acid sequence shown in (I) or (II) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0019] (IV) A sequence that is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more homologous to any of the amino acid sequences shown in (I) to (III).

[0020] In some specific embodiments of the present invention, the monoclonal antibody:

[0021] (I) Its light chain FR1, FR2, FR3, and FR4 have the amino acid sequences shown in SEQ ID No. 3, 4, 5, and 6, respectively; or

[0022] Its light chain FR1, FR2, FR3, and FR4 have amino acid sequences as shown in SEQ ID No. 20, 21, 22, and 23, respectively;

[0023] and

[0024] (II) Its heavy chain FR1, FR2, FR3, and FR4 have the amino acid sequences shown in SEQ ID No. 12, 13, 14, and 15, respectively; or

[0025] Its heavy chain FR1, FR2, FR3, and FR4 have the amino acid sequences shown in SEQ ID No. 29, 30, 31, and 32, respectively;

[0026] (III) A sequence based on the amino acid sequence shown in (I) or (II) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0027] (IV) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with any of the amino acid sequences shown in (I) to (III).

[0028] In some specific embodiments of the present invention, the monoclonal antibody:

[0029] (I) Its light chain FR1, FR2, FR3, and FR4 have the amino acid sequences shown in SEQ ID No. 3, 4, 5, and 6, respectively; and

[0030] Its heavy chain FR1, FR2, FR3, and FR4 have the amino acid sequences shown in SEQ ID No. 12, 13, 14, and 15, respectively; or

[0031] (II) Its light chain FR1, FR2, FR3, and FR4 have the amino acid sequences shown in SEQ ID No. 20, 21, 22, and 23, respectively; and

[0032] Its heavy chain FR1, FR2, FR3, and FR4 have the amino acid sequences shown in SEQ ID No. 29, 30, 31, and 32, respectively;

[0033] (III) A sequence based on the amino acid sequence shown in (I) or (II) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0034] (IV) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with any of the amino acid sequences shown in (I) to (III).

[0035] In some specific embodiments of the present invention, the monoclonal antibody:

[0036] (I) Its light chain variable region has an amino acid sequence as shown in SEQ ID No. 7 or SEQ ID No. 24; and

[0037] (II) Its heavy chain variable region has an amino acid sequence as shown in SEQ ID No. 16 or SEQ ID No. 33; or

[0038] (III) A sequence based on the amino acid sequence shown in (I) or (II) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0039] (IV) A sequence that is more than 80% homologous to the amino acid sequence shown in any of (I) to (III).

[0040] In some specific embodiments of the present invention, the monoclonal antibody:

[0041] (I) Its light chain variable region has an amino acid sequence as shown in SEQ ID No. 7; and

[0042] Its heavy chain variable region has an amino acid sequence as shown in SEQ ID No. 16; or

[0043] (II) Its light chain variable region has an amino acid sequence as shown in SEQ ID No. 24; and

[0044] Its heavy chain variable region has an amino acid sequence as shown in SEQ ID No. 33; or

[0045] (III) A sequence based on the amino acid sequence shown in (I) or (II) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0046] (IV) A sequence that is more than 80% homologous to the amino acid sequence shown in any of (I) to (III).

[0047] In some specific embodiments of the present invention, the monoclonal antibody:

[0048] (I) Its light chain variable region has a nucleotide sequence as shown in SEQ ID No. 8 or SEQ ID No. 25; and

[0049] Its heavy chain variable region has a nucleotide sequence as shown in SEQ ID No. 17 or SEQ ID No. 34; or

[0050] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0051] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0052] (IV) Nucleotide sequences that have at least 80% sequence homology with the nucleotide sequences described in (I) to (III).

[0053] In some specific embodiments of the present invention, the monoclonal antibody:

[0054] (I) Its light chain variable region has a nucleotide sequence as shown in SEQ ID No. 8; and

[0055] Its heavy chain variable region has a nucleotide sequence as shown in SEQ ID No. 17;

[0056] or

[0057] Its light chain variable region has a nucleotide sequence as shown in SEQ ID No. 25; and

[0058] Its heavy chain variable region has a nucleotide sequence as shown in SEQ ID No. 34;

[0059] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0060] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0061] (IV) Nucleotide sequences that have at least 80% sequence homology with the nucleotide sequences described in (I) to (III).

[0062] In some specific embodiments of the present invention, the monoclonal antibody is of the IgG subtype and the light chain is the κ chain.

[0063] In a second aspect, the present invention also provides a biological material comprising at least one of the following: a) to e)

[0064] a) A nucleic acid molecule encoding the monoclonal antibody;

[0065] b) The vector backbone and the recombinant vector encoding the nucleic acid of the monoclonal antibody;

[0066] c) The host that secretes the monoclonal antibody;

[0067] d) The monoclonal antibody labeled with chemicals or biolabels;

[0068] e) The monoclonal antibody conjugated to the vector.

[0069] Thirdly, the present invention also provides the application of the monoclonal antibody or the biological material in the preparation of detection reagents and / or detection kits for detecting eosinophil peroxidase;

[0070] Preferably, the monoclonal antibody specifically recognizes eosinophil peroxidase.

[0071] Fourthly, the present invention also provides the use of the monoclonal antibody or the biological material in the preparation of detection reagents and / or detection kits for detecting eosinophilic airway inflammation-related diseases;

[0072] Preferably, the eosinophilic airway inflammation-related diseases detected include bronchial asthma, cough variant asthma, and / or eosinophilic bronchitis.

[0073] Fifthly, the present invention also provides detection reagents, including the monoclonal antibody or the biological material.

[0074] In a sixth aspect, the present invention also provides a detection kit, comprising the monoclonal antibody, the biological material, or the detection reagent.

[0075] This invention provides monoclonal antibodies against eosinophil peroxidase, comprising coating antibodies and / or enzyme-labeled antibodies. Furthermore, this invention provides the use of the above-described monoclonal antibody or a pair of monoclonal antibodies in the preparation of detection reagents or kits for detecting eosinophil peroxidase. The monoclonal antibody pair of this invention can be used in a double-antibody sandwich assay for biological research and clinical diagnosis of eosinophilia.

[0076] Biological Preservation Instructions

[0077] Biological material: Hybridoma cell line EPX57#, deposited on September 3, 2024 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO: C2024297.

[0078] Biological material: Hybridoma cell line EPX58#, deposited on September 3, 2024 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO: C2024298. Detailed Implementation

[0079] This invention discloses a recombinant monoclonal antibody against eosinophil peroxidase, its preparation, and its application. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0080] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0081] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0082] Example 1: Preparation of monoclonal antibody against eosinophil peroxidase

[0083] 1.1 Mouse Immunization

[0084] Purchased natural eosinophil peroxidase (creative-enzymes, NATE-0228) was fully emulsified with Freund's complete adjuvant and intraperitoneally immunized 5-week-old female Balb / c mice at an initial dose of 100 μg / mouse. Second and third immunizations were administered at 21 and 42 days after the first immunization, respectively, with a dose of 50 μg / mouse for each immunization. Approximately 10 days after the third immunization, blood was collected from the tail, and serum titers were determined using an indirect method with 96-well luminescent plates coated with eosinophil peroxidase. The serum titer results are shown in Table 1. The results indicated that the mouse serum titers were greater than 8K, qualifying for the fusion screening of monoclonal antibodies.

[0085] Table 1: Mouse serum titers

[0086]

[0087] 1.2 Phage Library Construction

[0088] Mice were given a booster immunization via intrasplenic administration at a dose of 100 μg per mouse. Three days post-boost immunization, the spleens were harvested for library construction. The complementarity-determining region (CDR) of the anti-eosinophil peroxidase antibody was determined by antibody cDNA sequencing and antibody analysis based on the IMGT database. Subsequent PCR amplification was performed to obtain the mutated and non-mutated regions. The scFv fragment was obtained by overlap extension PCR. The PCR product was cloned into the Pcomb3xss vector according to the manufacturer's instructions. Electrophoresis on a 2.5% agarose gel was performed to verify successful transformation. Subsequently, the vector was transformed into recipient *E. coli* TG1 cells prepared using the kit via electroporation to construct the antibody library.

[0089] 1.3 Phage Display Library Screening

[0090] Eosinophil peroxidase was immobilized on ELISA plates / magnetic beads (1–10 μg / mL, incubated overnight at 4°C) and blocked with casein. A phage library (10 μg / mL) was then added. 11 ~10 13 PFU (Polydioxanone), incubated at room temperature for 1-2 hours. Negative controls consisted of uncoated magnetic beads / blank beads. Washing was performed with PBS + 0.1% Tween-20, followed by elution with acidic glycine buffer (pH 2.2). The eluent was neutralized with Tris-HCl (pH 8.0) and used to infect logarithmic-phase E. coli. Helper phages were added to rescue the phage particles, and the mixture was incubated overnight at 37°C. The phages were purified by NaCl precipitation for the next round of screening. This process was repeated three times before testing.

[0091] 1.4 Screening of positive clones

[0092] First, the phages obtained in step 1.3 are subjected to ELISA to check for enrichment. If enrichment is found, single phage clones are selected and tested with ELISA. Those that test positive are the desired single clones and sent for sequencing.

[0093] ELISA: Coat eosinophil peroxidase protein at a concentration of 1 μg / mg into each well (50 μL), incubate overnight at 4°C, wash 3 times, and blot dry. Add casein (150 μL) to each well, block at 37°C for 2 h, wash 3 times, and blot dry. Add phage (50 μL) to each well, incubate at 37°C for 0.5 h, and wash 5 times. Add enzyme-labeled phage antibody (50 μL) to each well, incubate at 37°C for 0.5 h, wash 5 times, and blot dry. Add chemiluminescence buffer (100 μL) to each well, and detect the luminescence value using a chemiluminescence analyzer.

[0094] 1.5 Sequence recombination expression, purification and labeling

[0095] The sequenced data was inserted into the pcmv3 vector to construct a plasmid. The plasmid was then transfected into 293 cells, and the supernatant was collected after 2 days. The supernatant was run on an SDS-PAGE gel to determine antibody expression, and the expression level was also detected by ELISA, as described in section 1.4. After confirming antibody expression, a stable cell line was constructed, cultured, purified, and HRP-labeled.

[0096] (1) EPX 182 kh65 (coated antibody):

[0097] The amino acid sequences of the three CDR regions of its light chain are as follows:

[0098] EDIYNR (as shown in SEQ ID No. 1);

[0099] GAT;

[0100] QQYWSTPYT (as shown in SEQ ID No. 2);

[0101] The amino acid sequences of the four FR regions of its light chain are as follows:

[0102] DIQMTQSSSSFSVSLGDRVTITCKAS (as shown in SEQ ID No. 3);

[0103] LAWYQQKPGNAPRLLIS (as shown in SEQ ID No. 4);

[0104] SLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYC (as shown in SEQ ID No. 5);

[0105] FGGGTKLEIKRAD (as shown in SEQ ID No. 6);

[0106] The amino acid sequence of its light chain variable region is shown in SEQ ID No. 7: DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPYTFGGGTKLEIKRAD;

[0107] The nucleotide sequence of its light chain variable region is as SEQ ID Shown in No.8: GACATCCAGATGACACAATCTTCATCCTCCTTTTCTGTATCTCTAGGAGACAGAGTCACCATTACTTGCAAGGCAAGTGAGGACATATATAATCGGTTAGCCTGGTATCAGCAGAAACCAGGAAATGCTCCTAGGCTCTTAATATCTGGTGCAACCAGTTTG GAAACTGGGGTTCCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCATTACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAGTATTGGAGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGAT;

[0108] Its light chain constant region is mouse κ.

[0109] The amino acid sequences of the three CDR regions of its heavy chain are as follows:

[0110] GYTLSSYW (as shown in SEQ ID No. 9);

[0111] ILPGVGTT (as shown in SEQ ID No. 10);

[0112] AEGSSLYYFDN (as shown in SEQ ID No. 11);

[0113] The amino acid sequences of the four FR regions of its heavy chain are as follows:

[0114] QAYLQQSGAALMKPGASVKISCKTT (as shown in SEQ ID No. 12);

[0115] IEWIKQRPGHGLEWIGE (as shown in SEQ ID No. 13);

[0116] NYNEKFKDKATFTTETSSNTVYMQLNSLTSEDSAVYYC (as shown in SEQ ID No. 14);

[0117] WGQGTTLTVSA (as shown in SEQ ID No. 15);

[0118] The amino acid sequence of its heavy chain variable region is shown in SEQ ID No. 16: QAYLQQSGAALMKPGASVKISCKTTGYTLSSYWIEWIKQRPGHGLEWIGEILPGVGTTNYNEKFKDKATFTTETSSNTVYMQLNSLTSEDSAVYYCAEGSSLYYFDNWGQGTTLTVSA

[0119] The nucleotide sequence of its heavy chain variable region is as SEQ ID No.17 shows: CAGGCTTATCTACAGCAGTCTGGAGCTGCGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGTAAGACTACTGGCTACACACTCAGTAGTTACTGGATAGAGTGGATAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAGTTGGTACTAC TAATTATAATGAGAAGTTCAAGGACAAGGCCACATTCACTACAGAAACATCCTCCAACACAGTCTACATGCAACTCAACAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAGAGGGTAGTAGCCTGTACTACTTTGACAACTGGGGCCAAGGCACCACTCTCACTGTCTCTGCA

[0120] Its heavy chain constant region is mouse IGG1.

[0121] (2) HRP-EPX150-kh26 (enzyme-labeled antibody):

[0122] The amino acid sequences of the three CDR regions of the light chain are as follows:

[0123] SSISSNY (as shown in SEQ ID No. 18);

[0124] RTS;

[0125] QQGSSIPLT (as shown in SEQ ID No. 19);

[0126] The amino acid sequences of the four FR regions of its light chain are as follows:

[0127] ENVLTQSPTTMAASPGEKITITCSAS (as shown in SEQ ID No. 20);

[0128] LHWYQQKPGFSPKLLIY (as shown in SEQ ID No. 21);

[0129] NLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYC (as shown in SEQ ID No. 22);

[0130] FGAGTKLELK (as shown in SEQ ID No. 23);

[0131] The amino acid sequence of its light chain variable region is shown in SEQ ID No. 24: ENVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIPLTFGAGTKLELK

[0132] The nucleotide sequence of the light chain variable region is as SEQ ID No.25 shows: GAAAATGTGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACATC CAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTAGTAGTATACCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA;

[0133] Its light chain constant region is mouse κ.

[0134] The amino acid sequences of the three CDR regions of its heavy chain are as follows:

[0135] RFTFTDYN (as shown in SEQ ID No. 26);

[0136] IYPYNGGT (as shown in SEQ ID No. 27);

[0137] ARWGPYYRYDVRHFDV (as shown in SEQ ID No. 28);

[0138] The amino acid sequences of the four FR regions of its heavy chain are as follows:

[0139] DVQLQQSGPELVKPGASVKISCKAS (as shown in SEQ ID No. 29);

[0140] IHWVKQSHGKSLEWIGY (as shown in SEQ ID No. 30);

[0141] GYNQKFKSKATLTVDTSSSTAYMELRSLTSEDSAVYYC (as shown in SEQ ID No. 31);

[0142] WGAGTTVTVSS (as shown in SEQ ID No. 32);

[0143] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.33: DVQLQQSGPELVKPGASVKISCKASRFTFTDYNIHWVKQSHGKSLEWIGYIYPYNGGTGYNQKFKSKATLTVDTSSSTAYMELRSLTSEDSAVYYCARWGPYYRYDVRHFDVWGAGTTVTVSS

[0144] The nucleotide sequence of the heavy chain variable region is as SEQ ID Shown in NO.34: GATGTACAGCTTCAGCAGTCAGGACCTGAGCTGGAAACCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTAGATTCACATTCACTGACTACAACATACACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTTATCCTTACAATGGTGGTACTGGCTAC AACCAGAAGTTCAAGAGCAAGGCCACATTGACTGTAGACACTTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAAGACTCTGCAGTCTATTACTGTGCAAGATGGGGTCCCTACTATAGGTACGACGTAAGGCACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

[0145] Its heavy chain constant region is mouse IGG1.

[0146] EPX 270 kh76:

[0147] Light chain variable region (as shown in SEQ ID No. 35): EIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPDRFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIPTFGSGTKLEIKRAD

[0148] Heavy chain variable region (as shown in SEQ ID No. 36):

[0149] EVQLQQSGPELVKPGASVKISCKASRFTFTDYNIHWVKQSHGKSLEWIGYIYPYNGGTGYNQKFKSKATLTVDTSSSTAYMELRSLTSEDSAVYYCARWGPYYRYDVRHFDVWGAGTTVTVSS

[0150] HRP-EPX30-kh79:

[0151] Light chain variable region (as shown in SEQ ID No. 37):

[0152] DIQMTQTTSSLSASLGDRVTISCRASQDISNDLNWFQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGKTLPLTFGAGTKLELK

[0153] Heavy chain variable region (as shown in SEQ ID No. 38):

[0154] EVQLQQSGPELVKPGASVKISCKASRFTFTDYNIHWVKQSHGKSLEWIGYIYPYNGGTGYNQKFKSKATLTVDTSSSTAYMELRSLTSEDSAVYYCARWGPYYRYDVRHFDVWGAGTTVTVSS

[0155] Example 2 Evaluation of anti-eosinophil peroxidase monoclonal antibody

[0156] Experimental group: The anti-eosinophil peroxidase monoclonal antibodies (EPX 182 kh65, HRP-EPX150-kh26, EPX 270 kh76 and HRP-EPX30-kh79) used in this example were prepared in Example 1.

[0157] Control group:

[0158] EPX-58#: Accession number CCTCC NO: C2024298;

[0159] EPX-57#: Accession number is CCTCC NO: C2024297.

[0160] Preparation method of test reagents:

[0161] Preparation of magnetic microparticle suspension: The preparation process includes washing and activating the magnetic microparticles, coating with antibodies, washing after coating, and blocking the magnetic microparticles. A certain volume of magnetic microparticles with carboxyl groups on their surface is placed in a glass bottle. The microparticles are adsorbed to the bottom of the bottle using a magnet, and the supernatant is removed. 5-10 volumes of 0.02M PBS (pH 7.5) are added, and the above operation is repeated 3 times. EDC and NHS are dissolved separately in 0.1M MES buffer (pH 5.0), and 1.5 ml of each is added to the magnetic microparticles. The mixture is gently shaken at room temperature for 30 minutes. The magnetic microparticles are adsorbed to the bottom using a magnet, and the supernatant is removed. 3 ml of 0.1M MES buffer (pH 5.0) is added to resuspend the magnetic microparticles. The above operation is repeated twice. Take 0.1-0.5 mg of EPX-specific antibody EPX 182 kH65 and add it to the activated magnetic microparticles; then add 0.1 M acetate buffer (pH 5.0); gently shake the mixture at room temperature for 15-60 minutes; use a magnet to adsorb the magnetic microparticles to the bottom, remove the supernatant, and wash three times with 5-10 volumes of Tris buffer (pH 8.0) containing 1% BSA and 0.1% preservative P300, 20 minutes each time; add 30 ml of Tris buffer (pH 8.0) containing 1% BSA and 0.1% preservative P300, and store at 2-8°C.

[0162] The sample diluent is prepared by using a buffer solution, which can be Tris, MOPS, HEPES, PBS, or Bis-Tris propane. The buffer concentration is 0.02M and the buffer pH is 7.2-7.4.

[0163] Preparation of enzyme conjugates: Horseradish peroxidase (HRP) was activated using a conventional modified sodium periodate method. An EPX-specific antibody (e.g., HRP-EPX150-kh26) was added, and the reaction was incubated overnight at 2–8°C. Sodium borohydride was added to reduct the enzyme conjugate. Unreacted reagents were removed by dialyzing. 50% glycerol was added, and the mixture was stored at -20°C. Before use, the mixture was diluted 1:500 to 1:5w in Tris buffer containing 1% BSA and 0.1% P300 preservative, and stored at 2–8°C.

[0164] On-machine testing and result analysis:

[0165] The fully automated immunoassay procedure using Antu Bio's fully automated immunoassay analyzer is as follows:

[0166] Place the sample container in the instrument's sample holder; load the sample holder and enter the sample information in the instrument's software interface; select "Run" to start the test, and the instrument will perform the following operations:

[0167] 1) Transfer the sample rack to the sample aspiration position and load the reaction vessel into the sample loading position.

[0168] 2) Complete the dispensing of 50µL sample or 50µL positive and negative controls, 20µL magnetic microparticle suspension, 50µL sample diluent and 50µL enzyme conjugate.

[0169] 3) Mix the reaction solution thoroughly and incubate at 37°C for 34 minutes.

[0170] 4) After the incubation is complete, use a cleaning solution to clean and separate the reaction solution.

[0171] 5) Complete the dispensing of 50µL substrate A solution and 50µL substrate B solution (substrate solution for fully automated immunoassay system, Zhengzhou Antu Bioengineering Co., Ltd., Henan Zhengzhou Medical Device Registration No. 20140023).

[0172] 6) Mix the reaction solution thoroughly and measure the luminescence intensity.

[0173] 7) The instrument automatically reports the test results.

[0174] The above-described double-antibody sandwich method was used to detect serially diluted 0.5 ug / ml natural eosinophil peroxidase (creative-enzymes, NATE-0228), which is the natural antigen in Table 2. A luminescence value above 200,000 indicates effective detection. The lower the limit of detection (LOD), the better. As shown in Table 2, the LOD for EPX-58# and PEX-57# is 62.5 ng / ml, the LOD for the paired antibodies EPX182-kh65 and EPX150-kh26 is 1 ng / ml, and the LOD for EPX270-kh76 and EPX30-kh79 is 4 ng / ml. The gradient of the antigen at each LOD meets the requirements; therefore, the preferred antibodies are EPX 182 kh65 and the HRP-EPX150-kh26 pair.

[0175] Table 2: Double-antibody sandwich assay for eosinophil peroxidase

[0176]

[0177]

[0178] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against eosinophil peroxidase, characterized in that, (I) Its light chain CDR1, CDR2 and CDR3 have the amino acid sequences shown in SEQ ID No. 1, GAT and SEQ ID No. 2, respectively; or Its light chain CDR1, CDR2, and CDR3 have the amino acid sequences shown in SEQ ID No. 18, RTS, and SEQ ID No. 19, respectively; and (II) Its heavy chain CDR1, CDR2 and CDR3 have the amino acid sequences shown in SEQ ID No. 9, 10 and 11, respectively; or Its heavy chain CDR1, CDR2, and CDR3 have the amino acid sequences shown in SEQ ID No. 26, 27, and 28, respectively; and (III) A sequence based on the amino acid sequence shown in (I) or (II) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (IV) A sequence that is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more homologous to any of the amino acid sequences shown in (I) to (III).

2. The monoclonal antibody as described in claim 1, characterized in that, (I) Its light chain FR1, FR2, FR3, and FR4 have the amino acid sequences shown in SEQ ID No. 3, 4, 5, and 6, respectively; or Its light chain FR1, FR2, FR3, and FR4 have amino acid sequences as shown in SEQ ID No. 20, 21, 22, and 23, respectively; and (II) Its heavy chain FR1, FR2, FR3, and FR4 have the amino acid sequences shown in SEQ ID No. 12, 13, 14, and 15, respectively; or Its heavy chain FR1, FR2, FR3, and FR4 have the amino acid sequences shown in SEQ ID No. 29, 30, 31, and 32, respectively; (III) A sequence based on the amino acid sequence shown in (I) or (II) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (IV) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with any of the amino acid sequences shown in (I) to (III).

3. The monoclonal antibody as described in claim 1 or 2, characterized in that, (I) Its light chain variable region has an amino acid sequence as shown in SEQ ID No. 7 or SEQ ID No. 24; and (II) Its heavy chain variable region has an amino acid sequence as shown in SEQ ID No. 16 or SEQ ID No. 33; or (III) A sequence based on the amino acid sequence shown in (I) or (II) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (IV) A sequence that is more than 80% homologous to the amino acid sequence shown in any of (I) to (III).

4. The monoclonal antibody according to any one of claims 1 to 3, characterized in that, (I) Its light chain variable region has a nucleotide sequence as shown in SEQ ID No. 8 or SEQ ID No. 25; and Its heavy chain variable region has a nucleotide sequence as shown in SEQ ID No. 17 or SEQ ID No. 34; or (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or (IV) Nucleotide sequences that have at least 80% sequence homology with the nucleotide sequences described in (I) to (III).

5. The monoclonal antibody according to any one of claims 1 to 4, characterized in that, Its subtype is IgG, and its light chain is κ chain.

6. A biomaterial, characterized in that, It includes at least one of the following: a) to e) a) A nucleic acid molecule encoding the monoclonal antibody as described in any one of claims 1 to 5; b) A vector backbone and a recombinant vector encoding the nucleic acid of any one of the monoclonal antibodies as described in claims 1 to 5; c) A host that secretes the monoclonal antibody as described in any one of claims 1 to 5; d) Chemically or biologically labeled monoclonal antibodies as described in any one of claims 1 to 5; e) A monoclonal antibody conjugated to a vector as described in any one of claims 1 to 5.

7. The use of the monoclonal antibody as described in any one of claims 1 to 5 or the biomaterial as described in claim 6 in the preparation of detection reagents and / or detection kits for detecting eosinophil peroxidase; Preferably, the monoclonal antibody specifically recognizes eosinophil peroxidase.

8. The use of the monoclonal antibody as described in any one of claims 1 to 5 or the biomaterial as described in claim 6 in the preparation of diagnostic reagents and / or kits for detecting eosinophilic airway inflammation-related diseases; Preferably, the eosinophilic airway inflammation-related diseases detected include bronchial asthma, cough variant asthma, and / or eosinophilic bronchitis.

9. A detection reagent, characterized in that, Includes the monoclonal antibody as described in any one of claims 1 to 5 or the biological material as described in claim 6.

10. A test kit, characterized in that, It includes the monoclonal antibody as described in any one of claims 1 to 5, the biomaterial as described in claim 6, or the detection reagent as described in claim 9.

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