Monoclonal antibodies to eosinophil peroxidase and uses thereof

Abstract

The present application relates to the field of biotechnology and in vitro diagnosis, and relates to a monoclonal antibody of eosinophil peroxidase and application thereof. The antibody of the present application is mainly used for detecting the content of eosinophil peroxidase by western blot. The monoclonal antibody of eosinophil peroxidase of the present application is prepared by immunizing Balb / c mice with recombinant eosinophil peroxidase as an immunogen, then fusing the spleen with myeloma cells NS1, and screening and purifying. The prepared monoclonal antibody of eosinophil peroxidase specifically recognizes the linear epitope of eosinophil peroxidase.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and in vitro diagnostics, specifically to monoclonal antibodies against eosinophil peroxidase and their 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 clinically and is 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, 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.

[0003] Currently, techniques for evaluating airway inflammation in asthma can be divided into invasive and non-invasive categories. Invasive techniques, such as bronchoscopic mucosal biopsy, bronchoalveolar lavage, and pathological studies of surgical specimens, while considered the gold standard, are significantly limited by their invasiveness, hindering their practicality as routine dynamic monitoring techniques. In recent years, various non-invasive techniques have been increasingly widely used in the monitoring and assessment of airway inflammation. Those already in clinical use include induced sputum cell classification, exhaled nitric oxide analysis, and the detection of inflammatory mediator levels (ECP, interleukins, etc.) in body fluid specimens (blood, urine). While these methods have their own advantages, they also have drawbacks: 1) Although exhaled nitric oxide concentration is positively correlated with the degree of eosinophilic airway inflammation, its specificity is somewhat lacking, and the testing cost is relatively high. 2) Currently, the specimens used for detecting inflammatory mediator levels are mainly peripheral blood and urine. These specimens have advantages such as convenient collection and mature testing techniques, but the drawback is that peripheral body fluid indicators are difficult to accurately and timely reflect airway inflammation. Furthermore, the biological activity of peripheral blood inflammatory mediators is affected by various factors, and their levels are not highly correlated with those in the lungs. 3) The proportion and number of eosinophils in sputum can clearly indicate 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. In particular, in the diagnosis of eosinophilic bronchitis, a sputum eosinophil proportion 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 involves many manual steps, requires skilled technicians and a lot of equipment, is time-consuming, and cannot issue reports in a timely manner, resulting in low efficiency; moreover, the proportion of eosinophils is not completely parallel to the severity of clinical symptoms. In our daily clinical work, we have found that some asthma patients, after effective treatment, have completely relieved their symptoms, but their sputum eosinophil proportion does not decrease to the normal range for a long period of time. In severe asthma patients, eosinophils show significant degranulation, and few intact eosinophils (Eos) are seen, so their test results will be lower than the actual number. In addition, some healthy individuals with allergic constitutions, despite having no respiratory symptoms, exhibit a persistently elevated proportion of eosinophils in their sputum, suggesting that the sputum eosinophil percentage is insufficient in reflecting the degree of eosinophilic airway inflammation. Therefore, identifying Eosinophilic airway inflammation biomarkers with both high sensitivity and specificity, and establishing corresponding rapid and convenient detection techniques, is crucial and urgent for the clinical diagnosis and treatment of asthma, and has significant economic and social benefits.

[0004] Studies have found that Eosinophils (EOs) play a crucial role in the pathogenesis of diseases such as asthma through degranulation in airway tissues. This is because degranulation releases various toxic proteins, cytokines, and lipid mediators, which directly or indirectly damage the local airway structure, causing airway inflammation and increased airway reactivity, leading to recurrent symptoms such as wheezing, shortness of breath, chest tightness, and / or cough. Studies have found that a large number of degranulated eosinophils (Eosinophils) are observed in the upper airway mucosa of patients with allergic rhinitis 24 hours after allergen exposure, surrounded by a wide distribution of free particles. Normally, Eos, which are in a resting state in the blood, are activated upon exposure to relevant active mediators during their migration to the site of inflammation. However, these activated Eos remain relatively stable and do not degranulate until further stimulation by inflammatory mediators or activation of certain cell membrane receptors. Furthermore, the degranulation process is not a one-time event; the amount of particles released depends on the type and intensity of the stimulation to the Eos and the matrix proteins to which the Eos adhere. Compared to the number or proportion of eosinophils, the concentration of eosinophil degranulation products is a more accurate and timely indicator of the degree of eosinophilic airway inflammation and has a higher correlation with the clinical symptoms of asthma patients. Among the degranulation products of eosinophils, cytokines (interleukins), chemokines, growth factors (TGF), and lipid mediators (LTs, PGs) are mediators co-secreted by various inflammatory cells and structural cells. They lack cell specificity and often function in a network manner, making them susceptible to numerous influencing 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 lens nucleus of the granule, accounting for 50% of the total granule protein), eosinophilic protein (ECP), eosinophil peroxidase (EPX), and eosinophil-derived neurotoxin (EDN) 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 has the highest content 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 has no cross-reactivity with neutrophils, exhibits high specificity, and meets the requirements for eosinophil-specific markers. Regarding the selection of specimen sources, peripheral blood testing for inflammatory cells and mediators is a traditional method, convenient for specimen collection and technically mature. However, its drawback lies in the difficulty of accurately and timely reflecting airway inflammation in peripheral blood indicators. The biological activity of peripheral blood cytokines is affected by various factors, and their levels are not highly correlated with intrapulmonary levels. Therefore, selecting airway secretions (induced sputum specimens) for testing provides a more objective, timely, and accurate reflection of the patient's airway inflammation status. Moreover, due to its non-invasive nature, it is more suitable for continuous dynamic monitoring over a short period.

[0006] Currently, some researchers abroad have established enzyme-linked immunosorbent assay (ELISA) methods for detecting EPX. However, this method has several shortcomings: the monoclonal antibody used in this method is an anti-mouse EPX monoclonal antibody. Although some experiments have been conducted to demonstrate that this antibody binds to human EPX antigen, thus enabling 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 many steps, is time-consuming, requires certain equipment conditions, and requires batch processing of samples, which cannot meet the needs of rapid clinical diagnosis. Therefore, the preparation of antibodies that specifically recognize EPX is urgently needed. Summary of the Invention

[0007] In view of this, the present invention provides a monoclonal antibody against eosinophil peroxidase and its application.

[0008] This invention provides a monoclonal antibody against eosinophil peroxidase and its applications. The antibody of this invention is primarily used for Western blot detection of eosinophil peroxidase levels.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

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

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

[0012] (2) Its light chain CDR1, CDR2 and CDR3 have amino acid sequences as shown in SEQ ID No. 4, 5 and 6 respectively; or

[0013] (3) A sequence based on the amino acid sequence shown in (1) or (2) by substitution, deletion, addition and / or replacement of one or more amino acids; or

[0014] (4) A sequence that is more than 80% homologous 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 an amino acid sequence as shown in SEQ ID No. 8; and

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

[0018] (7) A sequence based on the amino acid sequence shown in (5) or (6) by substitution, deletion, addition and / or replacement of one or more amino acids; or

[0019] (8) A sequence that is more than 80% homologous to the amino acid sequence shown in any of (5) to (7).

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

[0021] (9) Its heavy chain variable region has a nucleotide sequence as shown in SEQ ID No. 7; and

[0022] (10) Its light chain variable region has a nucleotide sequence as 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 substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (9) or (10), and which has the same or similar function to the nucleotide sequence shown in (9) or (10); or

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

[0026] The present invention also provides the use of the monoclonal antibody in the preparation of reagents and / or kits for detecting eosinophil peroxidase.

[0027] In some specific embodiments of the present invention, the eosinophil peroxidase is an eosinophil peroxidase with a linear epitope.

[0028] The present invention also provides the use of the monoclonal antibody in the preparation of reagents and / or kits for diagnosing eosinophil counts.

[0029] The present invention also provides the use of the monoclonal antibody in the preparation of reagents and / or kits for detecting eosinophilic airway inflammation-related diseases;

[0030] The diseases associated with eosinophilic airway inflammation include bronchial asthma, cough variant asthma, and / or eosinophilic bronchitis.

[0031] Based on the above research, the present invention also provides reagents, including the monoclonal antibody.

[0032] The present invention also provides a kit comprising the monoclonal antibody or the reagent.

[0033] The present invention also provides an apparatus comprising any of the following and acceptable components:

[0034] (1) the monoclonal antibody; and / or

[0035] (2) The reagents.

[0036] The present invention also provides a method for preparing the monoclonal antibody, comprising the following steps:

[0037] Step a: Immunize mice with recombinant EPX antigen gene protein as an immunogen;

[0038] Step b: Fuse the spleen cells of the mouse described in step a with myeloma cells to obtain hybridoma cells;

[0039] Step c: Screen and purify the hybridoma cells described in step b;

[0040] Step d: Obtain the monoclonal antibody from the hybridoma cell culture described in step c;

[0041] The mice include Balb / c mice; and / or

[0042] The myeloma cells include myeloma cells NS1.

[0043] In some specific embodiments of the present invention, the preparation method of the recombinant EPX antigen gene protein is as follows: the eosinophil peroxidase gene EPX is constructed into pET32a, transformed into a host for expression, positive expression strains are screened and cultured, centrifuged, resuspended, and sonicated. The supernatant obtained by centrifugation is mixed with loading buffer to obtain a precipitate, which is dissolved in urea. The supernatant is then centrifuged to obtain the recombinant EPX antigen gene protein.

[0044] In some specific embodiments of the present invention, the resuspension is performed using a 20 mmol / L Tris-HCl buffer solution with a pH of 8.0;

[0045] The conditions for ultrasonic fragmentation include: 300W ultrasound, 3 seconds of ultrasound with 3 seconds intervals, for a total of 18 minutes.

[0046] The concentration of the urea is 8M.

[0047] This invention provides a monoclonal antibody against eosinophil peroxidase and its applications. The monoclonal antibody against eosinophil peroxidase of this invention is prepared by immunizing Balb / c mice with recombinant eosinophil peroxidase as an immunogen, then fusing the spleen with myeloma cells NS1, followed by screening and purification. The prepared monoclonal antibody against eosinophil peroxidase specifically recognizes the linear epitope of eosinophil peroxidase. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0049] Figure 1 This illustrates the prokaryotic recombinant expression of eosinophil peroxidase in Example 1;

[0050] Figure 2In Example 3, the WB platform eosinophil peroxidase antibody recognizes eosinophil peroxidase in induced sputum; among them, 5054 is a positive clinical sample containing 87% eosinophils; 5056B is a negative clinical sample containing 0% eosinophils.

[0051] Figure 3 In Example 3, the WB platform eosinophil peroxidase antibody does not recognize or weakly recognizes natural myeloperoxidase; where A is an SDS-PAGE image of MPO protein; B and C are WB images of EPX antibody recognizing natural myeloperoxidase.

[0052] Figure 4 The WB platform in Example 3 shows the recognition of prokaryotic recombinant eosinophil peroxidase by eosinophil peroxidase; where A is an SDS-PAGE image of EPX recombinant protein; B and C are WB images of EPX antibody recognizing EPX recombinant protein;

[0053] Figure 5 In Example 3, the WB platform EPX recognizes prokaryotic recombinant expression of eosinophil peroxidase. Detailed Implementation

[0054] This invention discloses a monoclonal antibody against eosinophil peroxidase and its applications. 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.

[0055] This invention provides an antibody that recognizes a linear epitope and inhibits eosinophil peroxidase activity.

[0056] Specifically, firstly, the present invention provides a monoclonal antibody or fragment thereof against eosinophil peroxidase, wherein the monoclonal antibody or fragment thereof is capable of specifically binding to eosinophil peroxidase, and wherein the monoclonal antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the CDR region sequence of which is as follows:

[0057] CDR of heavy chain variable region:

[0058] CDR1: GFTFSSFG (SEQ ID No. 1)

[0059] CDR2: ITGGSRSI (SEQ ID No. 2)

[0060] CDR3:ARLGYDRGFAY(SEQ ID No.3)

[0061] CDR of the variable region of light chain:

[0062] CDR1: QDINSY (SEQ ID No. 4)

[0063] CDR2: RAN (SEQ ID No. 5)

[0064] CDR3: LQYDEFPFT (SEQ ID No. 6)

[0065] The base sequence of the heavy chain variable region: (SEQ ID No. 7)

[0066]

[0067] Amino acid sequence: (SEQ ID No. 8)

[0068]

[0069] Base sequence of the light chain variable region: (SEQ ID No. 9)

[0070]

[0071] Amino acid sequence: (SEQ ID No. 10)

[0072] DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLIDGVPSRFSGSGSGQDFSLPISTLEYEDMGIYYCLQYDEFPFTFGGSGTKLEIK

[0073] The monoclonal antibody against eosinophil peroxidase provided by this invention and the raw materials and reagents used in its application are all commercially available.

[0074] The present invention will be further illustrated below with reference to the embodiments:

[0075] Example 1: Recombinant expression of eosinophil peroxidase in Escherichia coli

[0076] 1.1 Conversion

[0077] The eosinophil peroxidase gene EPX (NCBI 8288) was synthesized and constructed into the expression vector pET32a. pET32a-EPX was transformed into E coli BL21 expression host bacteria, and positive expression bacteria were screened.

[0078] 1.2 Induced Expression

[0079] The selected positive expression strains containing the recombinant expression vector pET32a-EPX were inoculated into LB medium and cultured overnight at 37°C. The next day, the culture was scaled up at a 1:100 ratio until the bacterial culture reached OD. 600 When the concentration of the bacterial culture was 0.6, IPTG was added to the bacterial culture until the final concentration was 0.4 mmol / L, and the culture was induced at 37°C for 12 h.

[0080] 1.3 Purification

[0081] The induced bacterial culture was centrifuged at 12000 rpm for 10 min to enrich the bacterial cells. The cells were resuspended in 20 mmol / L Tris-HCl buffer (pH 8.0) at a volume ratio of 1:10. The cells were then sonicated on ice (300 W, 3 s intervals, 3 s for a total of 18 min) until the suspension was clear. The suspension was then centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected, and 80 μl of the supernatant was added to 20 μl of 5×SDS loading buffer. The precipitate was collected and dissolved in 1 mL of 8M urea. The suspension was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain the recombinant EPX antigen gene protein. Its SDS-PAGE image is shown below. Figure 1 .

[0082] Example 2: Preparation of anti-eosinophil peroxidase monoclonal antibody

[0083] 2.1 Mouse Immunization

[0084] The recombinant EPX antigen gene protein obtained in Example 1 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. A second and third immunization were administered 21 and 42 days after the first immunization, respectively, at 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 plates coated with eosinophil peroxidase.

[0085] 2.2 Hybridoma cell preparation

[0086] Indirect methods were selected to detect serum titers greater than 10. 4 Mice were given an intrasplenic booster immunization at a dose of 100 μg per mouse. Three days after the booster immunization, the spleen of the mice was harvested and fused with mouse myeloma cells NS1 at a ratio of 10:1. The fused cells were then cultured on DMEM medium (Gibco) containing HAT.

[0087] Approximately 6-7 days after fusion, the content of specific antibodies in the cell culture supernatant was detected indirectly using a 96-well plate coated with eosinophil peroxidase. Positive wells with an OD value of not less than 0.5 were selected for three rounds of subcloning using the limiting dilution method, ultimately yielding hybridoma cell lines EPX-1 to EPX-29 that can stably secrete anti-eosinophil peroxidase.

[0088] 2.3 Obtaining monoclonal antibodies against eosinophil peroxidase

[0089] The mouse hybridoma cells EPX-1 to EPX-29, which can stably secrete anti-eosinophil peroxidase, were injected into the peritoneal cavity of mice, and the ascites fluid was collected to obtain antibodies EPX-1 to EPX-29.

[0090] 2.4 Identification of anti-eosinophil peroxidase monoclonal antibody subtypes

[0091] The recombinant EPX antigen gene protein obtained in Example 1 was diluted to 1 μg / mL with PBS, coated onto 96-well ELISA plates overnight, and blocked with 1% Casein. The anti-eosinophil peroxidase monoclonal antibodies EPX-1 to EPX-29 (concentration 5 mg / mL) prepared in 2.3 were diluted 1:1000 with 0.05 mmol / L, pH 9.6 CB buffer. The isotypes of the anti-eosinophil peroxidase antibodies EPX-1 to EPX-29 prepared in 2.3 were indirectly identified using the mouse monoclonal antibody isotype (Sigma, ISO2-1KT) identification reagent. The results are shown in Table 1.

[0092] Table 1. Results of Anti-eosinophil Peroxidase Antibody Subtype Identification

[0093]

[0094]

[0095] As shown in Table 1, except for EPX6 and EPX23, which are IgM subtypes, the remaining anti-eosinophil peroxidase monoclonal antibodies are all IgG1 subtypes.

[0096] 2.5 Purification of monoclonal antibodies against eosinophil peroxidase

[0097] The IgG1 subtype antibody was purified by SPA, and the IgM subtype antibody was purified by water dialysis, thus obtaining a monoclonal antibody against eosinophil peroxidase with a purity of over 90%.

[0098] Example 3 Evaluation of anti-eosinophil peroxidase monoclonal antibody

[0099] Clinical samples of induced sputum from the hospital, natural eosinophil peroxidase (creative-enzymes, NATE-0228), natural myeloperoxidase (AROTEC Diagnostics, ATM01-10), and recombinant protein of the EPX antigen gene prepared in Example 1 were diluted with 2× loading buffer (0.2M Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 4% β-mercaptoethanol, 0.4% bromophenol blue). The samples were boiled for 5 minutes and separated on 10% SDS-PAGE with 15 μL of protein extract. The total protein separated by SDS-PAGE was directly transferred to a PVDF membrane for immunoblotting without staining. Western blotting was performed using the primary antibody anti-His and the purified anti-eosinophil peroxidase monoclonal antibody (EPX-1~EPX-29) prepared in Example 2, and the results were visualized using an ECL detection system. The visualization effect is as shown in the figure. Figure 2 , Figure 3 , Figure 4 , Figure 5 . Figure 2 In the study, sample 5056B was negative and sample 5054 was positive. The results showed that, except for EPX24, EPX25, and EPX28, all antibodies could specifically recognize eosinophil peroxidase induced in sputum. Figure 3 The results showed that EPX4, EPX6, EPX17, EPX18, EPX19, and EPX20 had no crossover with natural myeloperoxidase, EPX21, EPX25, and EPX28 had weak crossover with natural myeloperoxidase, and EPX1 had strong crossover with natural myeloperoxidase. Figure 4 The results showed that EPX4, EPX6, EPX17, EPX18, EPX19, EPX20, EPX21, EPX25, and EPX28 recognized native eosinophil peroxidase, and EPX4 had a stronger recognition ability than the other antibodies. Figure 5 EPX4 demonstrates its recognition of recombinant eosinophil peroxidase. In summary, EPX4 exhibits strong recognition of both recombinant and native eosinophil peroxidase, surpassing the recognition ability of other antibodies, and does not recognize native myeloperoxidase. Therefore, the EPX4 antibody can be used for the specific identification of eosinophil peroxidase and for the clinical diagnosis of eosinophils in induced sputum.

[0100] 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, (1) The CDR1, CDR2, and CDR3 of its heavy chain are as shown in SEQ ID No. 1, 2, and 3, respectively, and (2) The CDR1, CDR2 and CDR3 of its light chain are as shown in the amino acid sequences of SEQ ID No.4, RAN and SEQ ID No.6 respectively.

2. The monoclonal antibody as described in claim 1, characterized in that, (5) Its heavy chain variable region has an amino acid sequence as shown in SEQ ID No. 8; and (6) Its light chain variable region has an amino acid sequence as shown in SEQ ID No.

10.

3. A nucleic acid encoding the monoclonal antibody as described in claim 1 or 2, characterized in that, (9) Its heavy chain variable region has a nucleotide sequence as shown in SEQ ID No. 7; and (10) Its light chain variable region has a nucleotide sequence as shown in SEQ ID No.

9.

4. The use of the monoclonal antibody as described in any one of claims 1 to 3 in the preparation of reagents and / or kits for detecting eosinophil peroxidase.

5. Use of the monoclonal antibody as described in any one of claims 1 to 3 in the preparation of reagents and / or kits for diagnosing eosinophil count.

6. The use of the monoclonal antibody as described in any one of claims 1 to 3 in the preparation of reagents and / or kits for detecting eosinophilic airway inflammation-related diseases; The diseases associated with eosinophilic airway inflammation include bronchial asthma, cough variant asthma, and / or eosinophilic bronchitis.

7. A reagent, characterized in that, Including monoclonal antibodies as described in any one of claims 1 to 3.

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

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

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