Hybridoma cell, monoclonal antibody for resisting eosinophilic granulocyte peroxidase and application of monoclonal antibody

Abstract

The invention relates to the technical field of biology, in particular to a hybridoma cell, a monoclonal antibody for resisting eosinophilic granulocyte peroxidase and application of the monoclonal antibody. The invention provides a pair of hybridoma cell strains for generating monoclonal antibodies of eosinophilic granulocyte peroxidase antibodies, the pair of hybridoma cell strains are hybridoma cell strains 57 # and 58 #, the preservation number of the hybridoma cell strain 57 # is CCTCC (China Center For Type Culture Collection) NO: C2024297, and the preservation number of the hybridoma cell strain 58 # is CCTCC NO: C2024298. Hybridoma cell lines 57 # and 58 # for monoclonal antibodies are prepared for eosinophilic granulocyte peroxidase through a monoclonal antibody technology, the monoclonal antibodies have high specificity, and the prepared colloidal gold test strip and the magnetic particle light-emitting kit are high in sensitivity, good in specificity and high in sensitivity. Therefore, the eosinophilic granulocyte peroxidase can be rapidly detected.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to hybridoma cells, 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, including 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. Chronic cough is the most common complaint in respiratory medicine clinics. Eosinophilic bronchitis is one of the common causes of chronic cough in my country. Due to atypical symptoms and lack of effective examination methods, it is often misdiagnosed and mistreated, causing serious physical and mental pain to patients and their families, and also bringing 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 eosinophilic airway inflammation-related diseases.

[0003] Eosinophil-specific granules contain four basic proteins: major basic protein (MBP) is located in the crystalline nucleus of the granules, accounting for 50% of the total granule protein; eosinophil cationic 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. 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 test when used for human specimen detection; the ELISA method established by them has not undergone corresponding methodological evaluation experiments to evaluate its detection performance; the ELISA method has many steps and takes a long time to operate, requires certain equipment conditions, and requires batch processing of specimens, which cannot meet the needs of rapid clinical diagnosis. Summary of the invention

[0004] In view of this, the present invention provides hybridoma cells, monoclonal antibodies against eosinophil peroxidase and their applications. The present invention prepares hybridoma cell lines 57# and 58# for monoclonal antibodies against eosinophil peroxidase by monoclonal antibody technology, the monoclonal antibodies have high specificity, and the prepared colloidal gold test strips and magnetic particle luminescence kit have high sensitivity and good specificity, thereby being able to quickly detect eosinophil peroxidase.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a hybridoma cell line EPX57#, and the deposit number of the hybridoma cell is CCTCC NO: C2024297.

[0007] The present invention also provides a hybridoma cell line EPX58#, and the deposit number of the hybridoma cell is CCTCC NO: C2024298.

[0008] The present invention also provides the use of the hybridoma cell line EPX57# and / or the hybridoma cell line EPX58# in preparing monoclonal antibodies against eosinophil peroxidase.

[0009] The present invention also provides a monoclonal antibody against eosinophil peroxidase, wherein the monoclonal antibody is produced by the hybridoma cell line EPX57# and / or the hybridoma cell line EPX58#.

[0010] In some specific embodiments of the present invention, the subtype of the monoclonal antibody is IgG1.

[0011] The invention also provides application of the monoclonal antibody in preparing a product for detecting eosinophil peroxidase.

[0012] The present invention also provides the use of the monoclonal antibody in preparing products for eosinophilic airway inflammation-related diseases.

[0013] In some specific embodiments of the present invention, the eosinophilic airway inflammation-related disease includes one or more of bronchial asthma, cough variant asthma or eosinophilic bronchitis.

[0014] In some specific embodiments of the present invention, the product includes a reagent, a kit, a chip or a device.

[0015] The present invention also provides a reagent or a kit, comprising the monoclonal antibody.

[0016] In some specific embodiments of the present invention, the reagent and / or kit further comprises a solid phase carrier, and the solid phase carrier is selected from one of a multi-well plate, a micro-reaction plate concave hole, a magnetic bead, a cellulose membrane and a nylon membrane.

[0017] In some specific embodiments of the present invention, the monoclonal antibody comprises a label, and the label is selected from any one or more of a chromophore, colloidal gold, and an enzyme that generates a detectable signal. Preferably, the label is horseradish peroxidase.

[0018] In some specific embodiments of the present invention, the monoclonal antibody produced by the hybridoma cell line EPX57# is labeled with horseradish peroxidase, the monoclonal antibody produced by the hybridoma cell line EPX58# is coated with magnetic beads, and the eosinophil peroxidase is detected by a double antibody sandwich method.

[0019] The present invention also provides a chip or a device, comprising the monoclonal antibody.

[0020] The present invention also provides a method for preparing the monoclonal antibody, comprising: injecting the hybridoma cell line EPX57# and / or the hybridoma cell line EPX58# into the peritoneal cavity of mice, collecting mouse ascites, purifying, and obtaining the monoclonal antibody.

[0021] The present invention provides the following beneficial effects:

[0022] The present invention provides a pair of hybridoma cell lines that produce monoclonal antibodies of eosinophil peroxidase antibodies: hybridoma cell lines 57# and 58#, wherein the preservation number of the hybridoma cell line 57# is CCTCC NO: C2024297, and the preservation number of the hybridoma cell line 58# is CCTCC NO: C2024298. The present invention prepares hybridoma cell lines 57# and 58# for monoclonal antibodies against eosinophil peroxidase by monoclonal antibody technology, wherein the monoclonal antibodies have high specificity, and the prepared colloidal gold test strips and magnetic particle luminescence kit have high sensitivity and good specificity, thereby being able to rapidly detect eosinophil peroxidase.

[0023] Biological Deposit Description

[0024] Biological materials: Hybridoma cell line EPX57#, deposited on September 3, 2024 in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024297.

[0025] Biological materials: Hybridoma cell line EPX58#, deposited on September 3, 2024 in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024298. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1Shows the electrophoresis purity test of purified ascites EPX 57# and EPX 58#. DETAILED DESCRIPTION

[0028] The present invention discloses hybridoma cells, 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 them. It is particularly important to point out that all similar substitutions 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.

[0029] The invention provides monoclonal antibodies against eosinophil peroxidase and applications thereof. The antibody pair of the invention can be used for double antibody sandwich method and for biological research and clinical diagnosis of eosinophils.

[0030] The present invention provides a hybridoma cell line that produces a monoclonal antibody against eosinophil peroxidase, wherein the hybridoma cell line is a hybridoma cell line EPX57#, the preservation number of the hybridoma cell line EPX57# is CCTCC NO: C2024297, the preservation address is China Center for Type Culture Collection, and the preservation time is September 3, 2024; or the hybridoma cell line is a hybridoma cell line EPX58#, the preservation number of the hybridoma cell line EPX58# is CCTCC NO: C2024298, the preservation address is China Center for Type Culture Collection, and the preservation time is September 3, 2024.

[0031] The present invention provides a monoclonal antibody against eosinophil peroxidase, wherein the monoclonal antibody is antibody EPX57#, which is an antibody produced by the hybridoma cell line EPX57#; or the monoclonal antibody is antibody EPX58#, which is an antibody produced by the hybridoma cell line EPX58#.

[0032] The present invention provides a pair of hybridoma cell lines that produce monoclonal antibodies against eosinophil peroxidase. The pair of hybridoma cell lines are hybridoma cell lines EPX57# and EPX58#. The preservation number of the hybridoma cell line EPX57# is CCTCC NO: C2024297, the preservation address is China Center for Type Culture Collection, and the preservation time is September 3, 2024; the preservation number of the hybridoma cell line EPX58# is CCTCC NO: C2024298, the preservation address is China Center for Type Culture Collection, and the preservation time is September 3, 2024.

[0033] The present invention provides a pair of monoclonal antibodies against eosinophil peroxidase, wherein the pair of monoclonal antibodies are antibodies EPX57# and EPX58#, wherein the antibody EPX57# is an antibody produced by the hybridoma cell line EPX57#; and the antibody EPX58# is an antibody produced by the hybridoma cell line EPX58#.

[0034] 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 linear epitope of eosinophil peroxidase.

[0035] Furthermore, the monoclonal antibody EPX57# is a coated or labeled antibody, and the monoclonal antibody EPX58# is a labeled or coated antibody.

[0036] The present invention provides use of the hybridoma cell line, the monoclonal antibody, the pair of hybridoma lines or the pair of monoclonal antibodies in preparing a kit for detecting eosinophil peroxidase.

[0037] Unless otherwise specified, the hybridoma cells, anti-eosinophil peroxidase monoclonal antibodies and raw materials and reagents used in their application provided by the present invention can all be purchased from the market.

[0038] The present invention will be further described below in conjunction with embodiments:

[0039] Example 1 Preparation of anti-eosinophil peroxidase monoclonal antibody

[0040] 1.1 Immunization of mice

[0041] The 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, with an initial 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 indirectly by a 96-well luminescent plate coated with eosinophil peroxidase. The serum titer results are shown in Table 1, which show that the mouse serum titer is greater than 8 K, which can be fused and screened for monoclonal antibodies.

[0042] Table 1 Mouse serum titer

[0043]

[0044] 1.2 Hybridoma cell preparation

[0045] The mice were given an 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).

[0046] About 6 to 7 days after fusion, the specific antibody content in the cell culture supernatant was detected by indirect method using a 96-well luminescent plate coated with natural eosinophil peroxidase, and positive wells with luminescence values ​​not less than 10,000 were selected for three rounds of subcloning using the limiting dilution method, and finally hybridoma cell lines EPX57#~EPX61# that can stably secrete anti-eosinophil peroxidase were obtained. The antibody subtype was detected by indirect method using a 96-well luminescent plate coated with eosinophil peroxidase. The results are shown in Table 2. The results show that all 7 antibodies are IgG subtypes, indicating that the antibodies can be purified using the SPA process.

[0047] Table 2 Identification of antibody subtypes

[0048]

[0049] 1.3 Purification of monoclonal antibodies against eosinophil peroxidase

[0050] The obtained mouse hybridoma cells EPX57#~EPX61# which can stably secrete anti-eosinophil peroxidase were injected into 5 ~10 7 cells into the peritoneal cavity of mice, and the ascites was collected 7 to 14 days later to obtain antibodies EPX57#~EPX61#.

[0051] IgG subtype antibodies were purified by SPA to obtain monoclonal antibodies against eosinophil peroxidase ( Figure 1 ), Figure 1 The purity of the preferred antibodies 57# and 58# was above 95%.

[0052] Example 2 Evaluation of anti-eosinophil peroxidase monoclonal antibodies

[0053] The anti-eosinophil peroxidase monoclonal antibodies (EPX-57#~EPX-61#) used in this example were prepared in Example 1.

[0054] The coated antibody was coated on magnetic beads, the labeled antibody was diluted 500 times, and the double antibody sandwich method was used to detect the sample treatment solution, 1 μg / ml natural eosinophil peroxidase (creative-enzymes, NATE-0228), 5 negative samples and 5 positive samples (sample EOS value>2.5 is positive clinical, <2.5 is negative clinical). The results are shown in Tables 3 and 4. The greater the P / N positive and negative contrast, the higher the sensitivity of the paired antibodies. The sensitivity of EPX57# and EPX58# pairing is the highest among the existing pairings, and EPX57# and EPX58# pairing specifically recognize EPX. Therefore, EPX57# and EPX58# antibodies are preferred for preservation research.

[0055] Table 3 Double antibody sandwich assay for eosinophil peroxidase

[0056]

[0057] Table 4 Specific recognition of eosinophil peroxidase by 57# and 58# pairing

[0058]

[0059] 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. Hybridoma cell line EPX57#, characterized in that: The deposit number of the hybridoma cell is CCTCC NO: C2024297.

2. Hybridoma cell line EPX58#, characterized in that: The deposit number of the hybridoma cell is CCTCC NO: C2024298.

3. Use of the hybridoma cell line EPX57# according to claim 1 and / or the hybridoma cell line EPX58# according to claim 2 in the preparation of monoclonal antibodies against eosinophil peroxidase.

4. A monoclonal antibody against eosinophil peroxidase, characterized in that The monoclonal antibody is produced by the hybridoma cell line EPX57# according to claim 1 and / or the hybridoma cell line EPX58# according to claim 2.

5. The monoclonal antibody according to claim 4, characterized in that The subtype of the monoclonal antibody is IgG1.

6. Use of the monoclonal antibody according to claim 4 or 5 in the preparation of a product for detecting eosinophil peroxidase.

7. Use of the monoclonal antibody according to claim 4 or 5 in the preparation of products for eosinophilic airway inflammation-related diseases.

8. A reagent or a kit, characterized in that Comprising the monoclonal antibody as claimed in claim 4 or 5.

9. A chip or device, characterized in that Comprising the monoclonal antibody as claimed in claim 4 or 5.

10. The method for preparing a monoclonal antibody according to claim 4 or 5, characterized in that: include: The hybridoma cell line EPX57# described in claim 1 and / or the hybridoma cell line EPX58# described in claim 2 are injected into the mouse peritoneal cavity, and the mouse ascites is collected and purified to obtain the monoclonal antibody.

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