Monoclonal antibody for resisting eosinophilic granulocyte peroxidase and application thereof

Abstract

The invention relates to the field of biotechnology and in vitro diagnosis, in particular to an anti-eosinophilic granulocyte peroxidase monoclonal antibody and application thereof. The antibody provided by the invention can directly and specifically capture eosinophilic granulocyte peroxidase with enzyme activity, and is used for biological research and clinical diagnosis of eosinophilic granulocytes. The monoclonal antibody for resisting eosinophilic granulocyte peroxidase is prepared by the following steps: taking recombinant eosinophilic granulocyte peroxidase as an immunogen to immunize a Balb / c mouse, then fusing the spleen with a myeloma cell NS1, and screening and purifying to obtain the monoclonal antibody for resisting eosinophilic granulocyte peroxidase. The prepared monoclonal antibody for resisting the eosinophilic granulocyte peroxidase specifically recognizes the epitope of the eosinophilic granulocyte peroxidase.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and in vitro diagnosis, and in particular to monoclonal antibodies against eosinophil peroxidase and applications thereof. Background Art

[0002] Eosinophilic airway inflammation-related diseases are a general term for chronic inflammatory diseases characterized by eosinophil infiltration of the airways. It includes 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 is a serious public health problem worldwide. There are currently about 300 million asthma patients in the world and about 30 million in my country. The prevalence of asthma in various countries ranges from 1% to 30%, and is increasing year by year. The incidence rate in China is currently 2%-3%, and in some areas it exceeds 4%, but the rate of increase is rapid, and the asthma mortality rate is one of the highest in the world. The resulting disease burden cannot be ignored. Chronic cough is the most common complaint in respiratory outpatient clinics. Eosinophilic bronchitis is one of the common causes of chronic cough in my country. It is often misdiagnosed and mistreated due to atypical symptoms and lack of effective examination methods. It causes serious physical and mental pain to patients and their families, and also brings a heavy economic burden to society. Accurate and timely evaluation of the type and severity of airway inflammation is the key to the diagnosis and treatment of diseases related to eosinophilic airway inflammation.

[0003] At present, the technologies for evaluating asthma airway inflammation can be divided into two categories: invasive and non-invasive. Although invasive detection technologies such as bronchoscopic mucosal biopsy, bronchoalveolar lavage, and pathological studies of surgical specimens are the gold standard, their invasiveness greatly restricts their operability as routine dynamic monitoring technologies. In recent years, a variety of non-invasive technologies have been increasingly used in the monitoring and evaluation of airway inflammation. Among them, the technologies that have been used clinically include induced sputum cell classification, exhaled nitric oxide analysis, and inflammatory mediator level detection (ECP, interleukin, etc.) of body fluid specimens (blood, urine). The above detection methods certainly have their own advantages, but they also have shortcomings: 1) Although the concentration of exhaled nitric oxide is positively correlated with the degree of eosinophilic airway inflammation, it lacks specificity and the detection cost is relatively expensive. 2) At present, the specimens for the detection of inflammatory mediator levels are mainly peripheral blood and urine. Such specimens have the advantages of convenient collection and mature detection technology, but the disadvantage is that peripheral body fluid indicators are difficult to truly and timely reflect airway inflammation. Moreover, the biological activity of peripheral blood inflammatory mediators is interfered by many factors, and their levels are not highly correlated with the levels in the lungs. 3) The proportion and number of sputum eosinophils can clarify the type and degree of airway inflammation, which is of great significance for the diagnosis and treatment of variant airway inflammatory diseases such as asthma, especially in the diagnosis of eosinophilic bronchitis, the proportion of sputum eosinophils greater than or equal to 2.5% is one of the diagnostic criteria. However, the shortcomings of induced sputum cell classification detection are that the detection procedure has many manual operation steps, requires skilled technicians and more instruments, is time-consuming, and reports cannot be issued in time, which is inefficient; and the proportion of eosinophils is not completely parallel to the severity of clinical symptoms. In our daily clinical work, we found that some asthma patients had complete symptom relief after effective treatment, but for a long period of time, their sputum eosinophil proportion did not drop to the normal range. However, the eosinophils of patients with severe asthma will show significant degranulation, and complete eosinophils (Eos) are rarely seen, so the test results will be lower than the actual number. In addition, some healthy people with allergic constitutions, although they do not have any symptoms of respiratory diseases, have a long-term increase in the proportion of eosinophils in their sputum that exceeds the normal range, suggesting that the sputum eosinophil proportion is insufficient in reflecting the degree of eosinophilic airway inflammation. In view of this, it is very important and urgent to find an Eos airway inflammation biomarker with excellent sensitivity and specificity and to establish a supporting rapid and simple detection technology based on this for the clinical diagnosis and treatment of asthma, and it has good economic and social benefits.

[0004] Studies have found that Eos degranulation in airway tissue plays a key role in the pathogenesis of diseases such as asthma, because it can release a variety of toxic proteins, cytokines, and lipid mediators through degranulation. These substances have direct or indirect damaging effects on the local tissue structure of the airway, causing airway inflammation and increased airway responsiveness, leading to recurrent symptoms such as wheezing, shortness of breath, chest tightness, and (or) coughing. et al. found that a large number of degranulated Eos can be seen in the upper airway mucosal tissue of allergic rhinitis patients 24 hours after allergen stimulation, and there are extensive free particles around them. Usually, Eos in a resting state in the blood is activated when it migrates to the inflammatory site after being exposed to relevant active mediators. However, these activated Eos are in a relatively stable state before being further stimulated by inflammatory mediators or certain cell membrane receptors are excited, and they will not degranulate. Moreover, the degranulation process is not completed in one go. The amount of particles released depends on the type and intensity of the stimulation to Eos and the matrix protein to which Eos adheres. Compared with the number or proportion of eosinophils, the concentration of eosinophil degranulation products can more accurately and timely reflect the degree of eosinophilic airway inflammation, and is more closely related to the clinical symptoms of asthma patients. Among the degranulation products of eosinophils, cytokines (interleukins), chemokines, growth factors (TGF), and ester mediators (LTs, PGs) are mediators secreted by a variety of inflammatory cells and structural cells. They are non-cell specific and often work in a network form, affected by many factors. In contrast, Eos-specific granules are more suitable as biological markers for Eos degranulation.

[0005] Eosinophil-specific granules contain four basic proteins: major basic protein (MBP) (located in the crystalline nucleus of the granules, accounting for 50% of the total granule protein), eosinophilcationic protein (ECP), Eos peroxidase (EPX) and Eos-derived neurotoxin (EDN) are located in the matrix of the granules. ECP and EDN have been used as markers of activated eosinophils in clinical testing for a long time, but since these two proteins have been proven not to be specific products of eosinophils and can be expressed in neutrophils and liver tissue, their specificity is low and they are not suitable as markers of eosinophils. Although MBP has the highest content in eosinophil granules, it is also not suitable because it is also significantly expressed in mast cells and basophils. As a specific product of eosinophils, EPX has not been reported to be expressed in other inflammatory cells and tissue cells. The latest research also shows that it has no cross-reaction with neutrophils and has high specificity, meeting the requirements of eosinophil-specific markers. In terms of the selection of the source of test specimens, the detection of inflammatory cells and mediators through peripheral blood is a traditional method. The specimen collection is convenient and the detection technology is mature. Its disadvantage is that it is difficult for peripheral blood indicators to truly and timely reflect airway inflammation. The biological activity of peripheral blood cytokines is interfered by many factors, and its level is not highly correlated with the level in the lungs. Therefore, the selection of airway secretions (induced sputum specimens) for testing can more objectively, promptly and accurately reflect the patient's airway inflammation, and because of its non-invasiveness, it is more suitable for continuous dynamic monitoring in a short period of time.

[0006] At present, foreign scholars have established an enzyme linked immunosorbent assay (ELISA) for EPX, but this method has several shortcomings: the monoclonal antibody used in this method is an anti-mouse EPX monoclonal antibody. Although scholars have conducted some experiments to prove that the antibody has a binding reaction with the human EPX antigen, and thus the antibody can be used to establish an ELISA method for detecting human EPX, the antibody is an anti-mouse EPX monoclonal antibody, which may affect the specificity and sensitivity of the detection when used for human specimen detection; the ELISA method established by them has not conducted corresponding methodological evaluation experiments to evaluate its detection performance; the ELISA method has many steps and takes a long time, requires certain equipment conditions, and requires batch processing of specimens, which cannot meet the needs of clinical rapid diagnosis. Therefore, it is urgent to prepare antibodies that specifically recognize EPX. Summary of the invention

[0007] In view of this, the present invention provides monoclonal antibodies against eosinophil peroxidase and applications thereof.

[0008] The present invention provides monoclonal antibodies against eosinophil peroxidase and applications thereof. The antibodies of the present invention can directly and specifically capture eosinophil peroxidase with enzyme activity and are used in biological research and clinical diagnosis of eosinophils.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a monoclonal antibody against eosinophil peroxidase.

[0011] (1), the CDR1, CDR2 and CDR3 of its heavy chain have the amino acid sequences shown in SEQ ID No. 1, 2 and 3, respectively; and

[0012] (2) CDR1, CDR2 and CDR3 of its light chain have the amino acid sequences shown in SEQ ID Nos. 4, 5 and 6, respectively; or

[0013] (3) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1) or (2); or

[0014] (4) A sequence having a homology of 80% or more to the amino acid sequence shown in any one of (1) to (3).

[0015] In some specific embodiments of the present invention, the monoclonal antibody,

[0016] (5) Its heavy chain variable region has the amino acid sequence shown in SEQ ID No. 8; and

[0017] (6) Its light chain variable region has the amino acid sequence shown in SEQ ID No. 10; or

[0018] (7) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (5) or (6); or

[0019] (8) A sequence having a homology of 80% or more to the amino acid sequence shown in any one of (5) to (7).

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

[0021] (9), whose heavy chain variable region has the nucleotide sequence shown in SEQ ID No.7; and

[0022] (10), whose light chain variable region has the nucleotide sequence shown in SEQ ID No.9; or

[0023] (11) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (9) but is different from the nucleotide sequence shown in (9) due to the degeneracy of the genetic code; or

[0024] (12) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (9) or (10), and having the same or similar function as the nucleotide sequence shown in (9) or (10); or

[0025] (13) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in (9) to (12).

[0026] The present invention also provides the use of the monoclonal antibody in preparing a reagent and / or a kit for detecting eosinophil peroxidase.

[0027] In some specific embodiments of the present invention, the monoclonal antibody specifically recognizes eosinophil peroxidase.

[0028] Based on the above research, the present invention also provides the use of the monoclonal antibody in the preparation of a reagent and / or a kit for detecting eosinophilic airway inflammation-related diseases;

[0029] The diseases detected to be associated with eosinophilic airway inflammation include bronchial asthma, cough variant asthma and / or eosinophilic bronchitis.

[0030] The present invention also provides a reagent comprising the monoclonal antibody.

[0031] The present invention also provides a kit, comprising the monoclonal antibody or the reagent.

[0032] The present invention also provides a device coated with any of the following items and acceptable components:

[0033] (1), the monoclonal antibody; and / or

[0034] (2) the reagent.

[0035] The present invention also provides a method for preparing the monoclonal antibody, comprising the following steps:

[0036] Step a, immunizing mice with EPX antigen gene recombinant protein as an immunogen;

[0037] Step b, fusing the spleen cells of the mouse in step a with myeloma cells to obtain hybridoma cells;

[0038] Step c, screening and purifying the hybridoma cells described in step b;

[0039] Step d, obtaining the monoclonal antibody from the culture of the hybridoma cells in step c;

[0040] The mice include Balb / c mice; and / or

[0041] The myeloma cells include myeloma cells NS1.

[0042] In some specific embodiments of the present invention, the preparation method of the EPX antigen gene recombinant protein is as follows: the eosinophil peroxidase gene EPX is constructed into pET32a, transformed into a host for expression, positive expression strains are screened for culture, centrifuged, resuspended, and ultrasonically disrupted, the supernatant obtained by centrifugation is mixed with loading buffer to obtain a precipitate, the precipitate is dissolved with urea, and the supernatant is obtained by centrifugation to obtain the EPX antigen gene recombinant protein.

[0043] In some specific embodiments of the present invention, the resuspension is carried out using 20 mmol / L Tris-HCl buffer with a pH of 8.0;

[0044] The ultrasonic disruption conditions include: ultrasonic 300W, ultrasonic 3s with intervals of 3s for a total of 18 minutes;

[0045] The concentration of the urea is 8M.

[0046] The present invention provides an anti-eosinophil peroxidase monoclonal antibody and its application. The anti-eosinophil peroxidase monoclonal antibody of the present invention is obtained by immunizing Balb / c mice with recombinant eosinophil peroxidase as an immunogen, then taking the spleen and fusing it with myeloma cells NS1, and then screening and purifying it. The prepared anti-eosinophil peroxidase monoclonal antibody specifically recognizes the epitope of eosinophil peroxidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0048] Figure 1 The figure shows the prokaryotic recombinant expression of eosinophil peroxidase in Example 1. DETAILED DESCRIPTION

[0049] The present invention discloses monoclonal antibodies against eosinophil peroxidase and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0050] The antibody of the present invention can directly and specifically capture eosinophil peroxidase with enzyme activity, and is used in biological research and clinical diagnosis of eosinophils.

[0051] The invention provides a monoclonal antibody which specifically recognizes the eosinophil peroxidase activity.

[0052] Specifically, first, the present invention provides an anti-eosinophil peroxidase monoclonal antibody or a fragment thereof, wherein the monoclonal antibody or the fragment thereof can specifically bind to eosinophil peroxidase, and the monoclonal antibody or the fragment thereof comprises a heavy chain variable region and a light chain variable region, and the CDR region sequence of the heavy chain variable region is as follows:

[0053] Heavy chain variable region CDRs:

[0054] CDR1: GYTFSSYW (SEQ ID No. 1)

[0055] CDR2: ILPGSGST (SEQ ID No. 2)

[0056] CDR3:ARRGGNAWFAY(SEQ ID No.3)

[0057] CDRs of light chain variable region:

[0058] CDR1:KSVSTSGYSY(SEQ ID No.4)

[0059] CDR2: LVS (SEQ ID No. 5)

[0060] CDR3: QHIRRAYT (SEQ ID No. 6)

[0061] The base sequence of the heavy chain variable region:

[0062] CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGACGGGGGGGTAACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID No.7)

[0063] Amino acid sequence:

[0064] QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLE

[0065] WIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARRGGNAWFAYWGQGTLVTVSA(SEQ ID No.8)

[0066] Base sequence of the light chain variable region:

[0067] GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGAGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG(SEQ ID No.9)

[0068] Amino Acid Sequence:

[0069] DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPP RLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRRAYT FGGGTKLEIK(SEQ ID No.10)

[0070] The anti-eosinophil peroxidase monoclonal antibody provided by the invention and the raw materials and reagents used in its application can all be purchased from the market.

[0071] The present invention will be further described below in conjunction with embodiments:

[0072] Example 1 Preparation of eosinophil peroxidase prokaryotic immunogen

[0073] 1.1 Conversion

[0074] The eosinophil peroxidase gene EPX (NCBI 8288) was synthesized and constructed into the expression vector pET32a. pET32a-EPX was transformed into the E coli .BL21 expression host bacteria, and positive expression bacteria were screened.

[0075] 1.2 Inducible expression

[0076] The positive expression strain containing the recombinant expression vector pET32a-EPX was inoculated into LB medium and cultured at 37°C overnight. The next day, the culture was expanded at 1:100 until the bacterial solution OD 600 =0.6, add IPTG to the bacterial solution to a final concentration of 0.4 mmol / L and induce at 37°C for 12 h.

[0077] 1.3 Purification

[0078] The induced bacterial solution was centrifuged at 12000r / min for 10min to enrich the bacteria, and the bacteria were resuspended in 20mmol / L Tris-HCl buffer with a pH of 8.0 at a volume ratio of 1:10, and the suspension was broken by ice bath ultrasound (ultrasound 300W, ultrasound 3s interval 3s total 18min) until the bacterial suspension was clear, and centrifuged at 4℃, 12000r / min for 10min, and the precipitate was taken. The precipitate was dissolved with 1mL 8M urea, centrifuged at 8000r / min for 10min, and the supernatant was collected to obtain the EPX antigen gene recombinant protein. Its SDS-PAGE picture is as follows Figure 1 .

[0079] Example 2 Preparation of anti-eosinophil peroxidase monoclonal antibody

[0080] 2.1 Immunization of mice

[0081] The EPX antigen gene recombinant protein prepared in Example 1 was fully emulsified with Freund's complete adjuvant and intraperitoneally immunized 5-week-old female Balb / c mice, with an initial immunization dose of 100 μg / mouse; the second and third immunizations were performed 21 days and 42 days after the first immunization, respectively, with an immunization dose of 50 μg / mouse. Blood was collected from the tail about 10 days after the third immunization, and the serum titer was detected by an indirect method using a 96-well plate coated with eosinophil peroxidase.

[0082] 2.2 Hybridoma cell preparation

[0083] Select indirect method to detect serum titer greater than 10 4 The mice were given intrasplenic booster immunization, with an immunization dose of 100 μg / mouse. Three days after the booster immunization, the spleen of the mice was taken and fused with mouse myeloma cells NS1 at a ratio of 10:1, and the fused cells were cultured in DMEM medium containing HAT (Gibco).

[0084] About 6-7 days after fusion, the specific antibody content in the cell culture supernatant was detected indirectly using a 96-well plate coated with eosinophil peroxidase, and positive wells with OD values ​​not less than 0.5 were selected for three rounds of subcloning using the limiting dilution method, ultimately obtaining hybridoma cell lines EPX-1# to EPX-29# that could stably secrete anti-eosinophil peroxidase.

[0085] 2.3 Purification of monoclonal antibodies against eosinophil peroxidase

[0086] The obtained mouse hybridoma cells EPX-1#~EPX-29# capable of stably secreting anti-eosinophil peroxidase were injected into the peritoneal cavity of mice, and the ascites was collected to obtain antibodies EPX-1#~EPX-29#.

[0087] 2.4 Identification of anti-eosinophil peroxidase monoclonal antibody subtypes

[0088] The EPX antigen gene recombinant protein prepared in Example 1 was diluted to 1 μg / mL with PBS, coated on a 96-well ELISA plate overnight, and blocked with 1% Casein. The anti-eosinophil peroxidase monoclonal antibodies EPX-1#~EPX-29# (concentration of 5 mg / mL) prepared in 2.3 were diluted with 0.05 mmol / L, pH=9.6 CB buffer at a ratio of 1:1000. The subtypes of the anti-eosinophil peroxidase antibodies EPX-1#~EPX-29# prepared in 2.3 were identified indirectly using a mouse monoclonal antibody subtype identification reagent (sigma, ISO2-1KT). The results are shown in Table 1:

[0089] Table 1 Results of identification of anti-eosinophil peroxidase antibody subtypes

[0090]

[0091]

[0092] As shown in Table 1, except for EPX6# and EPX23# which are IgM subtypes, the subtypes of the anti-eosinophil peroxidase monoclonal antibodies are all IgG1 subtypes.

[0093] 2.5 Purification of monoclonal antibodies against eosinophil peroxidase

[0094] IgG1 subtype antibodies were purified by SPA, and IgM subtype antibodies were purified by water dialysis, thereby obtaining anti-eosinophil peroxidase monoclonal antibodies with a purity of more than 90%.

[0095] Example 3 Evaluation of anti-eosinophil peroxidase monoclonal antibodies

[0096] The anti-eosinophil peroxidase monoclonal antibodies (EPX-1# to EPX-29#) used in this example were prepared in Example 2.

[0097] 3.1 Characterization of the peroxidase activity of native eosinophil peroxidase and myeloperoxidase.

[0098] Eosinophil peroxidase and myeloperoxidase have peroxidase activity and can catalyze TMB color development. Now 500ng and 100ng of natural eosinophil peroxidase (creative-enzymes, NATE-0228) and 12.5ng of myeloperoxidase (AROTEC Diagnostics, ATM01-10) are mixed with TMB color development solution respectively. After termination, the OD value is read. The results are shown in Table 2. See the table below:

[0099] Table 2 Identification of eosinophil peroxidase and myeloperoxidase enzyme activities

[0100] antigen OD value 500ng EPX 3.275 100ng EPX 1.259 12.5ng MPO 2.424

[0101] The results in Table 2 show that both eosinophil peroxidase and myeloperoxidase have peroxidase activity, and the enzymatic activity of myeloperoxidase is stronger than that of eosinophil peroxidase.

[0102] 3.2 Antibody capture of eosinophil peroxidase

[0103] In view of the peroxidase activity of eosinophil peroxidase, anti-eosinophil peroxidase monoclonal antibodies (EPX-1#~EPX-29#) were coated on the ELISA plate and then natural eosinophil peroxidase was directly added for incubation. Eosinophil peroxidase was diluted to 1 μg / mL with purified water, and 50 μL (50 ng) was added to each well. After color development, it can be determined whether the antibody and eosinophil peroxidase are bound. The results are shown in Table 3 and the table below:

[0104] Table 3 EPX-6# can capture native eosinophil peroxidase

[0105]

[0106]

[0107] The results in Table 3 show that the signal value generated by EPX 6# antibody capturing eosinophil peroxidase is significantly higher than that of other antibodies, and EPX6# captures eosinophil peroxidase.

[0108] 3.3EPX6# antibody specifically recognizes eosinophil peroxidase

[0109] Since the homology between eosinophil peroxidase and myeloperoxidase is high, at 67%, the capture method is now used to identify the specificity of EPX6#. The coating concentration of EPX6# antibody is 5μg / mL, and the gradient of natural eosinophil peroxidase and myeloperoxidase are captured. The results are shown in Table 4, as shown in the table below:

[0110] Table 4 EPX6# specific recognition of EPX

[0111]

[0112] Since the OD value is positively correlated with positivity, the results in Table 4 indicate that EPX6# can specifically recognize natural eosinophil peroxidase.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A monoclonal antibody against eosinophil peroxidase, characterized in that (1), the CDR1, CDR2 and CDR3 of its heavy chain have the amino acid sequences shown in SEQ ID No. 1, 2 and 3, respectively; and (2) CDR1, CDR2 and CDR3 of its light chain have the amino acid sequences shown in SEQ ID Nos. 4, 5 and 6, respectively; or (3) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1) or (2); or (4) A sequence having a homology of 80% or more to the amino acid sequence shown in any one of (1) to (3).

2. The monoclonal antibody according to claim 1, characterized in that (5) Its heavy chain variable region has the amino acid sequence shown in SEQ ID No. 8; and (6) Its light chain variable region has the amino acid sequence shown in SEQ ID No. 10; or (7) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (5) or (6); or (8) A sequence having a homology of 80% or more to the amino acid sequence shown in any one of (5) to (7).

3. The monoclonal antibody according to claim 1 or 2, characterized in that (9), whose heavy chain variable region has the nucleotide sequence shown in SEQ ID No.7; and (10), whose light chain variable region has the nucleotide sequence shown in SEQ ID No.9; or (11) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (9) but is different from the nucleotide sequence shown in (9) due to the degeneracy of the genetic code; or (12) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (9) or (10), and having the same or similar function as the nucleotide sequence shown in (9) or (10); or (13) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in (9) to (12).

4. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a reagent and / or a kit for detecting eosinophil peroxidase.

5. The use according to claim 4, characterized in that The monoclonal antibody specifically recognizes eosinophil peroxidase.

6. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a reagent and / or a kit for detecting diseases associated with eosinophilic airway inflammation; The diseases detected to be associated with eosinophilic airway inflammation include bronchial asthma, cough variant asthma and / or eosinophilic bronchitis.

7. A reagent, characterized in that The invention comprises the monoclonal antibody according to any one of claims 1 to 3.

8. A kit, characterized in that Comprising the monoclonal antibody according to any one of claims 1 to 3 or the reagent according to claim 7.

9. The device, characterized in that The package contains any of the following and acceptable components: (1) The monoclonal antibody according to any one of claims 1 to 3; and / or (2) The reagent as described in claim 7.

10. The method for preparing a monoclonal antibody according to any one of claims 1 to 3, characterized in that: The steps include: Step a, immunizing mice with EPX antigen gene recombinant protein as an immunogen; Step b, fusing the spleen cells of the mouse in step a with myeloma cells to obtain hybridoma cells; Step c, screening and purifying the hybridoma cells described in step b; Step d, obtaining the monoclonal antibody from the culture of the hybridoma cells in step c; The mice include Balb / c mice; and / or The myeloma cells include myeloma cells NS1.

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