CN110655580B - Hybridoma cell strain and application thereof - Google Patents
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Abstract
The invention relates to a hybridoma cell strain and application thereof, in particular to a hybridoma cell strain 1C12/1G12 and application thereof. Hybridoma cell strain 1C12/1G12 secreting anti-CD 317 monoclonal antibody is prepared by using hybridoma technology, and the anti-CD 317 monoclonal antibody 1C12/1G12 produced by the hybridoma cell strain can be used for immunoblotting, flow cytometry, immunohistochemistry and ELISA.
Description
Technical Field
The invention relates to the field of immunology, and particularly relates to a hybridoma cell strain 1C12/1G12 and application thereof. Hybridoma cell strain 1C12/1G12 secreting anti-CD 317 monoclonal antibody is prepared by using hybridoma technology, and the anti-CD 317 monoclonal antibody 1C12/1G12 produced by the hybridoma cell strain can be used for immunoblotting, flow cytometry, immunohistochemistry and ELISA.
Background
CD317, namely Differentiation Antigen Cluster 317(Cluster of Differentiation 317), also called Bone Marrow Stromal Cell Antigen 2(Bone Marrow Stromal Cell Antigen 2), Tetherin or HM1.24, is a finally differentiated B Cell surface marker protein discovered by monoclonal antibodies in 1994 and may play an important role in the development of B cells. Because of its high expression in multiple myeloma cells, it is considered as myeloma by tumor immunologists
Targeting antigens for immunotherapy has been followed by a number of targeted therapy-related studies. Meanwhile, CD317 is found to be highly expressed on various cancer cells such as lung cancer, breast cancer and the like, in addition to the surface of bone marrow stromal cells, mature B cells and myeloma cells. It was first identified in 2008 as an interferon-inducible host antiviral factor, and more scientists were added to the search in this field since then. Through research in the last decade, the problems of CD317 structure, antiviral property and immunological property are described, and some new functions such as participation in tumor development and exosome release are discovered successively, and the research heat is not reduced in the current year.
The position of antibody products as the most widely used tool in the field of biological research is undoubted. The specificity and range of application of antibodies has long plagued the antibody industry and is a significant crisis in the entire field of biological research. The poor quality of the antibody product directly leads to errors in experimental results, and the results of the study cannot be repeated and reproduced. The progress of the research project is often delayed by the problems associated with antibodies, and the resulting losses are also surprising. According to 2011 statistics, an average of $ 3.5 billion per year is wasted on these ineffective antibodies in the United states alone. In the world, 8 billion dollars are wasted every year, accounting for 50% of the total antibody cost in global research.
Disclosure of Invention
The CD317 monoclonal antibody prepared by the technology specifically targets and binds to CD317 molecules of human and mouse cells, can be applied to experiments such as immunoblotting, flow cytometry, immunohistochemistry, ELISA and the like, and is a powerful tool for researching the function of CD 317.
In one aspect of the present invention, there is provided an isolated monoclonal antibody or antigen binding portion thereof that specifically binds to CD317, which is a monoclonal antibody or antigen binding portion thereof produced by a monoclonal antibody having a collection number of CCTCC NO: hybridoma cell line 1C12/1G12 of C2019156.
In another aspect, the present invention provides a hybridoma cell line, wherein the preservation information is China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: C2019156.
in another aspect of the invention, there is provided a composition comprising a monoclonal antibody or antigen binding portion as described above.
In a further aspect, the invention provides the use of an isolated anti-CD 317 monoclonal antibody or antigen binding portion thereof of the invention to detect the presence or expression level of CD317 in a sample, or to prepare a kit or detection reagent for detecting the presence or expression level of CD317 in a sample.
In the technical scheme of the invention, the samples include, but are not limited to, blood samples, tissue samples and cell samples.
In embodiments of the invention, the antibody or antigen-binding portion thereof may further comprise a detectable label.
In the technical scheme of the invention, the method for detecting the existence or the expression level of the CD317 in the sample comprises an ELISA detection method, a Western blot detection method, a flow cytometry detection method and an immunohistochemical detection method.
In the technical scheme of the invention, the kit comprises an ELISA detection kit or a Western blot detection kit, and the detection reagent comprises a flow cytometry detection reagent and an immunohistochemical detection reagent.
In yet another aspect, the invention provides the use of an isolated anti-CD 317 monoclonal antibody or antigen binding portion thereof of the invention in an ELISA assay.
In still another aspect, the invention provides an application of the isolated anti-CD 317 monoclonal antibody or the antigen binding part thereof in Western blot detection.
In yet another aspect, the invention provides the use of an isolated anti-CD 317 monoclonal antibody, or antigen binding portion thereof, of the invention in a flow cell assay.
In yet another aspect, the invention provides the use of an isolated anti-CD 317 monoclonal antibody, or antigen binding portion thereof, of the invention in an immunohistochemical assay.
In yet another aspect, the invention provides an ELISA detection kit comprising an isolated anti-CD 317 monoclonal antibody or and or an antigen binding portion thereof of the invention. Preferably, the antibody or antigen-binding portion thereof may further comprise a detectable label. The kit may further comprise a second antibody that specifically recognizes the antibody or antigen-binding portion thereof; the second antibody-binding moiety may also comprise a detectable label.
In yet another aspect, the invention provides a Western blot detection kit comprising the isolated anti-CD 317 monoclonal antibody or the antigen binding part thereof. Preferably, the antibody or antigen-binding portion thereof may further comprise a detectable label. The kit may further comprise a second antibody that specifically recognizes the antibody or antigen-binding portion thereof; the second antibody-binding moiety may also comprise a detectable label.
In yet another aspect, the invention provides a flow cytometric assay reagent comprising an isolated anti-CD 317 monoclonal antibody, or antigen binding portion thereof, according to the invention. Preferably, the antibody or antigen-binding portion thereof may further comprise a detectable label. The cell detection reagent may further comprise a second antibody that specifically recognizes the antibody or antigen binding portion thereof; the second antibody-binding moiety may also comprise a detectable label.
In yet another aspect, the invention provides an immunohistochemical detection reagent comprising the isolated anti-CD 317 monoclonal antibody or antigen binding portion thereof of the present invention. Preferably, the antibody or antigen-binding portion thereof may further comprise a detectable label. The cell detection reagent may further comprise a second antibody that specifically recognizes the antibody or antigen binding portion thereof; the second antibody-binding moiety may also comprise a detectable label.
In the present invention, the term "antigen-binding portion" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (e.g., CD317) to which the antibody binds, competing with the intact antibody for specific binding to the antigen. See generally, Fundamental Immunology, ch.7(Paul, w., ed., 2 nd edition, Raven Press, n.y. (1989), which is incorporated herein by reference in its entirety for all purposes.
In some cases, the antigen-binding portion of the antibody is a single chain antibody (e.g., scFv) in which the VL and VH domains are paired to form a monovalent molecule by a linker that enables it to be produced as a single polypeptide chain (see, e.g., Bird et al, Science 242: 423-. Such scFv molecules can have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used (Holliger et al (1993), Proc. Natl. Acad. Sci. USA 90: 6444-. Other linkers useful in the present invention include those made by althan et al (1995), Protein eng.8: 725-731, Choi et al (2001), Eur.J.Immunol.31: 94-106, Hu et al (1996), Cancer Res.56: 3055-3061, Kipriyanov et al (1999), J.mol.biol.293: 41-56 and Rovers et al (2001), Cancer Immunol.
In some cases, the antibody is a diabody, i.e., a diabody in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and generating two antigen binding sites (see, e.g., Holliger P. et al, Proc. Natl. Acad. Sci. USA 90: 6444-.
The antigen-binding portion of an antibody (e.g., the antibody fragment described above) can be obtained from a given antibody (e.g., the C3 and C16 monoclonal antibodies) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation), and specifically screened for the antigen-binding portion of the antibody in the same manner as for an intact antibody.
As used herein, the terms "hybridoma", "hybridoma cell line" and "hybridoma cell line" are used interchangeably and when referring to the terms "hybridoma", "hybridoma cell line" and "hybridoma cell line", it also includes subclones and progeny cells of the hybridoma.
Description of biological Material preservation
The present invention relates to the following biological materials which have been preserved in the China center for type culture Collection (CCTCC, China, Wuhan, university of Wuhan):
the hybridoma cell strain 1C12/1G12 (abbreviated as 1C12/1G12 in the text) has the preservation number of CCTCCNO: C2019156 and the preservation time of 2019, 08 months and 07 days.
Drawings
FIG. 1 shows the application of 1C12/1G12 antibody in flow detection of CD317 expression of human Hela cells.
FIG. 2 shows the application of 1C12/1G12 antibody in ELISA experiments.
FIG. 3 shows the application of 1C12/1G12 antibody in immunoblotting (Western blot) experiments. 1: primary antibody was PBS, 2: the primary antibody was the 1C12/1G12 antibody, 3: primary antibody was serum from immunized mice diluted 10 ten thousand fold.
FIG. 4 shows the application of 1C12/1G12 antibody in immunohistochemical experiments. The left image is a tonsil tissue section, the middle image is a lung tissue section, and the right image is a parathyroid gland tissue section. The concentration of the 1C12/1G12 antibody was 0.25 ug/ml. The results showed that tonsils and lung tissue were positive for CD317 expression, and that parathyroid gland did not express CD 317.
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the embodiments of the present invention taken in conjunction with the accompanying drawings, which are included to illustrate and not to limit the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
EXAMPLE 1 production of CD317 monoclonal antibody
The hybridoma method (first proposed by Kohler et al, Nature,256:495 (1975)) was used. A female BALB/c mouse (6 weeks old) is immunized by a CD317 protein antigen (purchased from Beijing Yi Qiao Shenzhou company), the antigen is emulsified by Freund complete adjuvant for the first immunization, the antigen is emulsified by Freund incomplete adjuvant for the second immunization, the antigen is injected at 5-6 points subcutaneously, and the amount of the antigen injected by each mouse is-100 ug. 10 days after the 3 rd immunization, a small amount of blood is collected from the tail of the mouse and subjected to serum titer ELISA detection, and the 4 th immunization is performed by selecting the mouse with high antibody titer (>1:100000), and 100ug of antigen protein is injected into each mouse through the abdominal cavity. 3-5 days after 4 th immunization, the splenocytes of the killed mice are taken to be fused with SP2/0 cells, and the stable hybridoma cells are obtained by HAT culture medium culture. Screening hybridoma capable of secreting CD317 antibody by ELISA method, subcloning by limiting dilution method, screening monoclonal hybridoma cell strain 1C12/1G12 capable of secreting CD317 antibody by ELISA method, performing scale-up culture step by step, and preserving seed by liquid nitrogen freezing.
Preparing an ascites antibody: female BALB/c mice (8 weeks old) were intraperitoneally injected with Freund's incomplete adjuvant, 0.5ml of each mouse was injected, 3-5 days later, the hybridoma cells in logarithmic phase were intraperitoneally injected, and 1-5 × 10 cells were injected into each mouse5Individual cells (0.5ml), and mice were sacrificed after-11 days of hybridoma injection to obtain ascites fluid. Centrifuging at 3000rpm and 4 deg.C for 10min, removing precipitate, diluting ascites with 10 times volume of 1 XPB solution, mixing, and filtering with 0.45 μm filter membrane. Ascites fluid was affinity-purified by Protein G (Protein G Sepharose 4 Fast Flow, GE Healthcare) to obtain a purified CD317 antibody Protein.
Example 2 flow assay application of CD317 monoclonal antibody
1) Collecting Hela, blowing, counting, and packaging into 5 centrifuge tubes (each tube is 3 × 10)5Each cell was centrifuged at 500g for 5min, and the supernatant was removed, and 1ml of PBS was added thereto, and centrifuged at 500g for 5min, and the supernatant was removed.
2) PBS or 5ug/ml 1C12/1G12 antibody was added, placed on ice, and incubated for 30 min.
3) 1ml of PBS + 5% FBS was added, 500g was centrifuged for 5min, the supernatant was removed, 100ul of DMEM culture solution +1ul of PE goal anti-mouse lgG (minor x-reactivity) antibody (biolegend,405307) was added, and the mixture was incubated on ice for 20 min.
4) 1ml of PBS + 5% FBS was added to each, and the mixture was centrifuged at 500g for 5min to remove the supernatant.
5) 300ul PBS + 5% FBS was added to each tube, resuspended, and detected using a Beckman Cytoflex CM.
The 1C12/1G12 antibody can be combined with CD317 protein on the surface of Hela cells (figure 1) and is applied to flow cytometry detection.
Example 3 ELISA detection application of CD317 monoclonal antibody
100ng of CD317 protein is coated on each well of the ELISA plate, the mixture is coated overnight at 4 ℃, and after the plate is washed by sealing, 1C12/1G12 antibody diluted by 2 times is added, the highest concentration is 40ug/ml, the lowest concentration is 19.5ng/ml, each well is 100ul, and the incubation is carried out for 1h at 37 ℃. The plates were washed 5 times with PBST, and HRP-goat anti-mouse IgG antibody (purchased from Byunyan) diluted 1:5000 was added and incubated at 37 ℃ for 45 min. And (3) washing the PBST for 5 times, adding 100ul of TMB into each hole, reacting for 3min in a dark place at room temperature, adding 50ul of stop solution into each hole, and detecting OD450 by using an enzyme-linked immunosorbent assay.
As shown in FIG. 2, 1C12/1G12 can be used for ELISA detection applications.
Example 4 Western blot detection application of CD317 monoclonal antibody
After CD317 protein is mixed with 5 × loading buffer, boiling and denaturing are carried out for 6min, SDS-PAGE is carried out, 20ul of protein is loaded on each hole, 50ng of protein is loaded on each hole, 400mA is transferred to a membrane for 1h, 5% skimmed milk powder is sealed for 1h at room temperature, PBS or 2ug/ml 1C12/1G12 antibody or immune mouse serum diluted by 10 ten thousand times is added for incubation for 1h at room temperature, after PBST is washed for 3 times, goat anti-mouse IgG secondary antibody diluted by 1:5000 is added, incubation for 45min at room temperature is carried out, and after PBST is washed for 3 times, Western HRP substrate (DenluR 0500) is added for development.
As shown in FIG. 3, 1C12/1G12 can be used for Western blot detection application.
Example 5 immunohistochemical application of CD317 monoclonal antibody
1. The slices were dewaxed conventionally to water.
2. Antigen Heat repair (Heat-directed Epitope repair, HIER): the sections were soaked in antigen retrieval solution (10mM Tris,1mM EDTA solution, 0.05% Tween 20, pH 9.0) at pH9.0 for 95, 5 min.
3. To reduce non-specific background staining by endogenous peroxidase, sections were incubated in a Hydrogen Peroxide Block (Abcam) for 10-15 minutes.
PBST washing 5min/2 times.
5. Ultra V Block (Thermo) was added and incubated at room temperature for 5min to Block non-specific background staining.
PBST washing 5min/2 times.
7. Add 1C12/1G12 antibody working solution at 37 ℃ for 2 hours.
PBST washing 5min/2 times.
9. Primary Antibody Enhancer (Thermo) was added and incubated at room temperature for 20 min.
PBST wash 5min/2 times.
11. HRP Polymer (enzyme-labeled secondary antibody, purchased from petunia) was added and incubated at room temperature for 30 minutes.
PBST wash 5min/2 times.
13. 1-2 drops of DAB Plus Chromogen (Thermo) were added dropwise to 1ml of DAB Plus Substrate (Thermo), mixed well and added dropwise to the sections, and incubated for 15 minutes.
14. Fully washing with running water, re-dyeing, dewatering, transparentizing, and sealing
The results are shown in FIG. 4, where 1C12/1G12 can be used for immunohistochemistry.
Example 5 affinity detection of antibody binding to antigen
The affinity (KD value) of the antibody was determined using the bio-layer interference (BLI) principle of the Octet K2 instrument (PALL forteBio). The antibody was diluted with SD buffer (1 × PBS, 0.05% BSA, 0.002% tween 20). The method is operated on a computer, a Protein A sensor or an avidin sensor is used for coupling the antibody, and then antigens with various concentrations are incubated. All binding data were collected at 30 ℃. The reaction procedure consisted of four steps: an antibody-coupled sensor; baseline acquisition is carried out for 60 s; calculating a Kon value in combination with the antigen; the antigen or antibody is dissociated and a Koff value is calculated. The ratio of Kon to Koff is the affinity KD value of the antibody.
The results show that: the 1C12/1G12 antibody had an affinity KD of 9.16X 10-11M, anti-CD 317 mab 6H2H5(KD ═ 3.83 × 10-10M, application No. 201711314176.4) and anti-CD 317 mab 1C12C6(KD ═ 5.69 × 10)-9M, application No. 201610554612.4) is stronger.
Claims (14)
1. An isolated monoclonal antibody or antigen binding portion thereof that specifically binds to CD317, the monoclonal antibody or antigen binding portion thereof is produced by a monoclonal antibody having a collection number of CCTCC NO: hybridoma cell line 1C12/1G12 of C2019156.
2. The hybridoma cell line has the preservation information of preservation number of China Center for Type Culture Collection (CCTCC) NO: C2019156.
3. a composition comprising the monoclonal antibody or antigen binding portion thereof of claim 1.
4. Use of the monoclonal antibody or antigen binding portion thereof of claim 1 for the preparation of a kit or detection reagent for detecting the presence or expression level of CD317 in a sample.
5. The use according to claim 4, wherein the method for detecting the presence or expression level of CD317 in a sample comprises ELISA detection, Western blot detection, flow cytometry detection, immunohistochemistry detection.
6. The use of claim 4, wherein the kit comprises an ELISA detection kit or a Western blot detection kit, and the detection reagent comprises a flow cytometry detection reagent or an immunohistochemistry detection reagent.
7. A kit comprising the isolated anti-CD 317 monoclonal antibody or antigen binding portion thereof of claim 1.
8. The kit of claim 7, wherein the antibody, or antigen-binding portion thereof, further comprises a detectable label.
9. The kit of claim 7, further comprising a second antibody that specifically recognizes the monoclonal antibody or antigen-binding portion thereof of claim 1.
10. The kit according to claim 7, which is an ELISA detection kit or a Western blot detection kit.
11. A detection reagent comprising the isolated anti-CD 317 monoclonal antibody or antigen binding portion thereof of claim 1.
12. The detection reagent of claim 11, the antibody or antigen-binding portion thereof further comprising a detectable label.
13. The detection reagent of claim 11, further comprising a second antibody that specifically recognizes the monoclonal antibody or antigen-binding portion thereof of claim 1.
14. The detection reagent of claim 11, which is an immunohistochemical detection reagent or a flow cytometric detection reagent.
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