CN114573693A - Preparation method of immunoregulatory molecule MIF antibody in auditory development - Google Patents
Preparation method of immunoregulatory molecule MIF antibody in auditory development Download PDFInfo
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- CN114573693A CN114573693A CN202210439715.1A CN202210439715A CN114573693A CN 114573693 A CN114573693 A CN 114573693A CN 202210439715 A CN202210439715 A CN 202210439715A CN 114573693 A CN114573693 A CN 114573693A
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- 102000034655 MIF Human genes 0.000 title claims abstract description 30
- 238000011161 development Methods 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 230000004957 immunoregulator effect Effects 0.000 title claims description 7
- 238000000034 method Methods 0.000 claims abstract description 44
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 33
- 210000004027 cell Anatomy 0.000 claims abstract description 28
- 239000000427 antigen Substances 0.000 claims abstract description 18
- 102000036639 antigens Human genes 0.000 claims abstract description 18
- 108091007433 antigens Proteins 0.000 claims abstract description 18
- 230000003053 immunization Effects 0.000 claims abstract description 18
- 206010035226 Plasma cell myeloma Diseases 0.000 claims abstract description 12
- 238000002649 immunization Methods 0.000 claims abstract description 12
- 201000000050 myeloid neoplasm Diseases 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 12
- 210000004989 spleen cell Anatomy 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 11
- 238000010367 cloning Methods 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 230000001105 regulatory effect Effects 0.000 claims abstract description 7
- 238000002965 ELISA Methods 0.000 claims abstract description 6
- 241001465754 Metazoa Species 0.000 claims abstract description 6
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- 210000002966 serum Anatomy 0.000 claims abstract description 6
- 238000000746 purification Methods 0.000 claims description 23
- 206010003445 Ascites Diseases 0.000 claims description 10
- 238000011091 antibody purification Methods 0.000 claims description 9
- 230000008014 freezing Effects 0.000 claims description 9
- 238000007710 freezing Methods 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 238000010370 cell cloning Methods 0.000 claims description 5
- 230000007910 cell fusion Effects 0.000 claims description 5
- 238000003113 dilution method Methods 0.000 claims description 5
- 238000005342 ion exchange Methods 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 238000010257 thawing Methods 0.000 claims description 5
- 210000001728 clone cell Anatomy 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims 1
- 210000001519 tissue Anatomy 0.000 abstract description 5
- 230000007365 immunoregulation Effects 0.000 abstract description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 14
- 108010048043 Macrophage Migration-Inhibitory Factors Proteins 0.000 description 5
- 102100037791 Macrophage migration inhibitory factor Human genes 0.000 description 5
- 230000008569 process Effects 0.000 description 4
- 238000004806 packaging method and process Methods 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 239000003862 glucocorticoid Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000010398 acute inflammatory response Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000012085 chronic inflammatory response Effects 0.000 description 1
- 230000007646 directional migration Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for preparing an immunoregulation molecule MIF antibody in auditory development, which comprises the following steps: animal immunization: preparing high-purity specific antigen, then selecting a proper immunization scheme according to the characteristics of the antigen, immunizing a mouse, selecting a mouse with high serum titer, and fusing cells: preparing fresh feeder cells, immunized mouse spleen cells and SP2/0 myeloma cells, fusing the myeloma cells with the spleen cells, and screening hybridoma cells: culturing and screening hybridoma cells by using a specific culture medium, detecting the titer of the supernatant of the hybridoma cells by using an antigen-coated plate ELISA method, and selecting positive clones (P/N ≧ 2.1) for continuous cloning. According to the preparation method of the MIF antibody as the immunoregulation molecule in auditory development, during preparation, the MIF antibody can be prepared by regulating tissue concentration, so that MIF antibody molecules with better titer can be obtained.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a preparation method of an immunoregulation molecule MIF antibody in auditory development.
Background
Mif (microphage migration inhibition factor) is a protein of about 12.5kDa, mainly secreted by activated lymphocytes, monocytes, macrophages, etc. Macrophage migration inhibitory factor has various functions including inhibition of macrophage directed migration, regulation of glucocorticoid (glucocorticoid) -mediated immunosuppressive effect, etc., but its main function is to participate in inflammatory reaction. In mammals, macrophage migration inhibitory factor is stored in the cytoplasm, and upon exogenous stimulation (e.g., pathogen infestation), the macrophage migration inhibitory factor in the cytoplasm is rapidly released extracellularly. The released macrophage migration inhibitory factor acts as a cytokine to regulate acute and chronic inflammatory responses, thereby affecting the immune response of the body. Therefore, macrophage migration inhibitory factor plays an important role in the process of the body's antibacterial and viral infection reaction.
Disclosure of Invention
The invention aims to provide a method for preparing MIF antibody as an immunoregulatory molecule in auditory development, so as to solve the problem that the MIF antibody is low in concentration and low in cost when prepared.
In order to achieve the purpose, the invention provides the following technical scheme: the preparation method of the MIF antibody as the immunoregulation molecule in auditory development comprises the following steps:
(1) animal immunization: preparing high-purity specific antigen, then selecting a proper immunization scheme according to the characteristics of the antigen, immunizing the mouse, and selecting the mouse with high serum titer.
(2) Cell fusion: fresh feeder cells, immunized mouse spleen cells, and SP2/0 myeloma cells were prepared, and the spleen cells were fused with the myeloma cells.
(3) Screening hybridoma cells: culturing and screening hybridoma cells by using a specific culture medium, detecting the titer of the supernatant of the hybridoma cells by using an antigen-coated plate ELISA method, and selecting positive clones (P/N ≧ 2.1) for continuous cloning.
(4) Hybridoma cell cloning: cloning hybridoma cells by limiting dilution method, enlarging and culturing positive clone, freezing and storing cell strain, and determining IgG subclass of positive clone supernatant antibody.
(5) Ascites production: the collected hybridoma cells are injected into a mouse body, and ascites is generated and collected.
(6) Antibody purification: the antibody is purified by selecting an appropriate purification method according to the subclass of the antibody.
(7) And (3) antibody identification: and (4) identifying the subclass, the titer, the concentration (BCA method) and the like of the purified antibody, labeling and warehousing.
Preferably, the subclasses of the antibody clone cells are ZMIF-6B9, ZMIF-5C11 and ZMIF-1F12 respectively.
Preferably, the antibody is purified by a purification method, Protein a or G column purification, ion exchange column purification, or the like.
Preferably, the antibody is packaged immediately after purification and frozen at-20 ℃ to avoid repeated freezing and thawing.
Preferably, the concentration of the antibody after purification is required to be between 3 and 5 mg/ml.
Compared with the prior art, the invention has the beneficial effects that: according to the preparation method of the MIF antibody, during preparation, the MIF antibody can be prepared by adjusting the tissue concentration, so that MIF antibody molecules with better titer can be obtained;
(1) the subclasses of the antibody monoclonal cells are respectively ZMIF-6B9, ZMIF-5C11 and ZMIF-1F12, the concentrations of the identified ZMIF-6B9, ZMIF-5C11 and ZMIF-1F12 are respectively 2.43mg/m, 0.94mg/ml and 3.28mg/ml, the titer of the ZMIF-6B9 reaches 64 ten thousand, and the titer of the ZMIF-5C11 reaches 256 thousand, so that the MIF antibody molecule with very high titer can be achieved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
This example 1 provides a method for preparing MIF antibody, an immunoregulatory molecule used in auditory development, and the method is described in detail below.
(1) Animal immunization: preparing high-purity specific antigen, then selecting a proper immunization scheme according to the characteristics of the antigen, immunizing the mice, and selecting the mice with high serum titer.
(2) Cell fusion: fresh feeder cells, immunized mouse spleen cells, and SP2/0 myeloma cells were prepared, and the spleen cells were fused with the myeloma cells.
(3) Screening hybridoma cells: culturing and screening hybridoma cells by using a specific culture medium, detecting the titer of the supernatant of the hybridoma cells by using an antigen-coated plate ELISA method, and selecting positive clones (P/N ≧ 2.1) for continuous cloning.
(4) Hybridoma cell cloning: hybridoma cells were cloned by limiting dilution method, and positive clones were cultured in an expanded state, and cell lines were frozen and simultaneously the IgG subclasses of the supernatant antibodies of the positive clones were determined, and the subclasses of the cells of the antibody clones were ZMIF-6B9, ZMIF-5C11, and ZMIF-1F12, respectively.
(5) Ascites production: the collected hybridoma cells are injected into a mouse body, and ascites is generated and collected.
(6) Antibody purification: the antibody is purified by selecting an appropriate purification method according to the subclass of the antibody, and the antibody purification method is a purification method, Protein A or G column purification, ion exchange column purification, or the like.
(7) And (3) antibody identification: and (3) identifying the subclass, the titer, the concentration (BCA method) and the like of the purified antibody, marking and warehousing, immediately packaging and freezing at-20 ℃ after the antibody is purified, avoiding repeated freezing and thawing, wherein the concentration of the purified antibody is required to be 3mg/ml, and the preparation work of the MIF antibody is realized by adjusting the tissue concentration, so that the MIF antibody molecule with better titer can be obtained.
Example 2
This example 2 provides a method for preparing MIF antibody, an immunoregulatory molecule used in auditory development, and the method is described in detail below.
(1) Animal immunization: preparing high-purity specific antigen, then selecting a proper immunization scheme according to the characteristics of the antigen, immunizing the mouse, and selecting the mouse with high serum titer.
(2) Cell fusion: fresh feeder cells, immunized mouse spleen cells, and SP2/0 myeloma cells were prepared, and the spleen cells were fused with the myeloma cells.
(3) Screening hybridoma cells: culturing and screening hybridoma cells by using a specific culture medium, detecting the titer of the supernatant of the hybridoma cells by using an antigen-coated plate ELISA method, and selecting positive clones (P/N ≧ 2.1) for continuous cloning.
(4) Hybridoma cell cloning: hybridoma cells were cloned by limiting dilution method, and positive clones were cultured in an expanded state, and cell lines were frozen and simultaneously the IgG subclasses of the supernatant antibodies of the positive clones were determined, and the subclasses of the cells of the antibody clones were ZMIF-6B9, ZMIF-5C11, and ZMIF-1F12, respectively.
(5) Ascites production: the collected hybridoma cells are injected into a mouse body, and ascites is generated and collected.
(6) Antibody purification: the antibody is purified by selecting an appropriate purification method according to the subclass of the antibody, and the antibody purification method is a purification method, Protein A or G column purification, ion exchange column purification, or the like.
(7) And (3) antibody identification: and (3) identifying the subclass, the titer, the concentration (BCA method) and the like of the purified antibody, marking and warehousing, immediately packaging and freezing at-20 ℃ after the antibody is purified, avoiding repeated freezing and thawing, wherein the concentration of the purified antibody is required to be 4mg/ml, and the preparation work of the MIF antibody is realized by adjusting the tissue concentration, so that the MIF antibody molecule with better titer can be obtained.
Example 3
This example 3 provides a method for preparing MIF antibody, an immunoregulatory molecule used in auditory development, and the method is described in detail below.
(1) Animal immunization: preparing high-purity specific antigen, then selecting a proper immunization scheme according to the characteristics of the antigen, immunizing the mouse, and selecting the mouse with high serum titer.
(2) Cell fusion: fresh feeder cells, immunized mouse spleen cells, and SP2/0 myeloma cells were prepared, and the spleen cells were fused with the myeloma cells.
(3) Screening hybridoma cells: culturing and screening hybridoma cells by using a specific culture medium, detecting the titer of the supernatant of the hybridoma cells by using an antigen-coated plate ELISA method, and selecting positive clones (P/N ≧ 2.1) for continuous cloning.
(4) Hybridoma cell cloning: hybridoma cells were cloned by limiting dilution method, and positive clones were cultured in an expanded state, and cell lines were frozen and simultaneously the IgG subclasses of the supernatant antibodies of the positive clones were determined, and the subclasses of the cells of the antibody clones were ZMIF-6B9, ZMIF-5C11, and ZMIF-1F12, respectively.
(5) Ascites production: the collected hybridoma cells are injected into a mouse body, and ascites is generated and collected.
(6) Antibody purification: the antibody is purified by selecting an appropriate purification method according to the subclass of the antibody, and the antibody purification method is a purification method, Protein A or G column purification, ion exchange column purification, or the like.
(7) And (3) antibody identification: and (3) identifying the subclass, the titer, the concentration (BCA method) and the like of the purified antibody, marking and warehousing, immediately packaging and freezing at-20 ℃ after the antibody is purified, avoiding repeated freezing and thawing, wherein the concentration of the purified antibody is required to be between 5mg/ml, and the preparation work of the MIF antibody is realized by adjusting the tissue concentration, so that the MIF antibody molecule with better titer can be obtained.
It should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in the process, method, article, or apparatus that comprises the element.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (5)
1. A preparation method of an immune regulatory molecule MIF antibody in auditory development is characterized by comprising the following steps:
(1) animal immunization: preparing high-purity specific antigen, then selecting a proper immunization scheme according to the characteristics of the antigen, immunizing the mouse, and selecting the mouse with high serum titer.
(2) Cell fusion: fresh feeder cells, immunized mouse spleen cells, and SP2/0 myeloma cells were prepared, and the spleen cells were fused with the myeloma cells.
(3) Screening hybridoma cells: culturing and screening hybridoma cells by using a specific culture medium, detecting the titer of the supernatant of the hybridoma cells by using an antigen-coated plate ELISA method, and selecting positive clones (P/N ≧ 2.1) for continuous cloning.
(4) Hybridoma cell cloning: cloning hybridoma cells by limiting dilution method, enlarging and culturing positive clone, freezing and storing cell strain, and determining IgG subclass of positive clone supernatant antibody.
(5) Ascites production: the collected hybridoma cells are injected into a mouse body, and ascites is generated and collected.
(6) Antibody purification: the antibody is purified by selecting an appropriate purification method according to the subclass of the antibody.
(7) And (3) antibody identification: the purified antibody is identified in subclass, titer, concentration (BCA method) and the like, labeled and put in storage.
2. The method for preparing MIF antibody as immunoregulatory molecule in auditory development according to claim 1, wherein: the subclasses of the antibody clone cells are ZMIF-6B9, ZMIF-5C11 and ZMIF-1F12 respectively.
3. The method for preparing MIF antibody as immunoregulatory molecule in auditory development according to claim 1, wherein: the antibody purification method comprises a purification method, Protein A or G column purification, ion exchange column purification and the like.
4. The method for preparing the immune regulatory molecule MIF antibody in auditory development according to claim 1, wherein the immune regulatory molecule MIF antibody comprises the following components: the antibody is immediately subpackaged and frozen at-20 ℃ after being purified, and repeated freeze thawing is avoided.
5. The method for preparing the immune regulatory molecule MIF antibody in auditory development according to claim 1, wherein the immune regulatory molecule MIF antibody comprises the following components: the concentration of the antibody after purification is required to be between 3 and 5 mg/ml.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0977799A (en) * | 1995-09-13 | 1997-03-25 | Sapporo Immuno Diagnostic Lab:Kk | Monoclonal antibody against human macrophage migration-inhibiting factor (human mif) and hybridoma producing the antibody |
| CN101041820A (en) * | 2006-03-21 | 2007-09-26 | 胡川闽 | Macrophagocyte transfer inhibition factor monoclonal antibody and method for making same |
| WO2007134538A1 (en) * | 2006-05-24 | 2007-11-29 | Institute Of Biophysics Chinese Academy Of Sciences | Rat antihuman macrophage migration inhibitory factor monoclonal antibody and its application |
| US20090220521A1 (en) * | 2008-01-04 | 2009-09-03 | Randolf Kerschbaumer | Anti MIF Antibodies |
| CN109422808A (en) * | 2017-08-31 | 2019-03-05 | 翟乾 | The preparation method of monoclonal antibody |
-
2022
- 2022-04-25 CN CN202210439715.1A patent/CN114573693A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0977799A (en) * | 1995-09-13 | 1997-03-25 | Sapporo Immuno Diagnostic Lab:Kk | Monoclonal antibody against human macrophage migration-inhibiting factor (human mif) and hybridoma producing the antibody |
| CN101041820A (en) * | 2006-03-21 | 2007-09-26 | 胡川闽 | Macrophagocyte transfer inhibition factor monoclonal antibody and method for making same |
| WO2007134538A1 (en) * | 2006-05-24 | 2007-11-29 | Institute Of Biophysics Chinese Academy Of Sciences | Rat antihuman macrophage migration inhibitory factor monoclonal antibody and its application |
| US20090220521A1 (en) * | 2008-01-04 | 2009-09-03 | Randolf Kerschbaumer | Anti MIF Antibodies |
| CN101983207A (en) * | 2008-01-04 | 2011-03-02 | 巴克斯特国际公司 | Anti mif antibodies |
| CN109422808A (en) * | 2017-08-31 | 2019-03-05 | 翟乾 | The preparation method of monoclonal antibody |
Non-Patent Citations (1)
| Title |
|---|
| 宋静;张超;梁婷;陈富强;侯桂华;: "抗MIF单克隆抗体的制备及其放射性碘标记方法", 山东大学学报(医学版) * |
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