CN115340985A - Hybridoma cell strain secreting anti-cat A-type blood specific monoclonal antibody, monoclonal antibody and application of monoclonal antibody - Google Patents
Hybridoma cell strain secreting anti-cat A-type blood specific monoclonal antibody, monoclonal antibody and application of monoclonal antibody Download PDFInfo
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- CN115340985A CN115340985A CN202211025611.2A CN202211025611A CN115340985A CN 115340985 A CN115340985 A CN 115340985A CN 202211025611 A CN202211025611 A CN 202211025611A CN 115340985 A CN115340985 A CN 115340985A
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
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Abstract
The invention discloses a hybridoma cell strain secreting anti-cat A type blood specific monoclonal antibody, monoclonal antibody and application thereof, wherein cat A type red blood cell suspension is used as antigen to immunize BALB/c mice, and a hybridoma cell strain A17H secreting anti-cat A type blood specific monoclonal antibody is obtained through immunization, cell fusion, screening and cloning, and the preservation number is CCTCC NO: c2022269, the A17H monoclonal antibody secreted can agglutinate with the red blood cell of A type of cat, but does not agglutinate with the red blood cell of other blood types of cat, it is a kind of specificity anti cat A type blood type that stereotypes, detect accurately high-efficiently.
Description
Technical Field
The invention relates to a hybridoma cell strain secreting a cat A-type specific monoclonal antibody, a monoclonal antibody and application thereof, and belongs to the technical field of biology.
Background
The blood group system is a system for typing blood according to different types of antigens on erythrocyte membranes, and the cat recognized blood group system is an AB blood group system and comprises three blood groups of A, B and AB. Because the serum of the B-type blood cat contains high-titer anti-A antibodies, the prior art adopts the standard serum of the B-type blood cat to carry out agglutination test to identify the A-type blood.
With the maturation of hybridoma technology and the development of cell engineering, the development and production of monoclonal antibody blood typing reagents aiming at specific blood group antigens become the international trend, and the problems of complex operation and low identification accuracy still exist in the prior art for cat blood group identification. Cat a type erythrocyte monoclonal antibodies have been developed by france Alvedia company, american DMSlabortors and the like, but due to different titers of anti-A serum of different batches, the serum needs to be subjected to various pretreatments, and the identification steps are complicated and the accuracy is not high; and the defects that B-type cats need to be raised for blood sampling for a long time and the like, and the application of the method to the blood grouping of the cats is limited.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a hybridoma cell strain secreting a cat A-type blood type specific monoclonal antibody, the monoclonal antibody and application thereof, which are convenient for performing blood type detection on cats.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a hybridoma cell strain secreting a cat A-resistant blood type specific monoclonal antibody, which has a preservation number of CCTCC NO: C2022269.
further, BALB/c mice were immunized with feline type A erythrocyte ghosts or type A haemofeline erythrocytes as antigens.
Further, the anti-cat type A blood type specific monoclonal antibody is prepared from a monoclonal antibody with the preservation number of CCTCC NO: c2022269 hybridoma cell line secretion.
Further, the anti-cat A blood type specific monoclonal antibody is an IgM monoclonal antibody, and the anti-cat A blood type specific monoclonal antibody agglutinates with cat A blood red cells.
The invention provides a blood type detection reagent, test paper or kit, which comprises the anti-cat type A blood type specific monoclonal antibody.
The invention provides a blood type detection device which is characterized by comprising the blood type detection reagent, test paper or kit.
The invention provides an application of the anti-cat type A blood type specific monoclonal antibody, or a blood type detection reagent, test paper or kit in cat blood type detection.
The invention provides a blood type detection method, which comprises the step of contacting a sample to be detected with the anti-cat type A blood type specific monoclonal antibody to determine the blood type of the sample to be detected.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, a feline A-type erythrocyte ghost or feline A-type erythrocyte is used as an antigen to immunize a BALB/c mouse to obtain a hybridoma cell strain A17H secreting the feline A-type hemagglutination-resistant monoclonal antibody, the secreted A17H monoclonal antibody can agglutinate with feline A-type erythrocytes, but does not agglutinate with feline other hemagglutination reactions, and the convenience and accuracy of feline blood type detection are obviously improved by applying the determination of specific feline A-type hemagglutination resistance.
Drawings
FIG. 1 is a gel electrophoresis image of an anti-feline type A blood specific monoclonal antibody provided in an embodiment of the present invention;
FIG. 2 is a schematic diagram showing the agglutination result of the anti-feline type A blood specific monoclonal antibody provided by the embodiment of the present invention, wherein FIGS. A1-A3 are schematic diagrams showing the agglutination of the anti-feline type A blood specific monoclonal antibody with type A erythrocytes, and FIGS. B1-B3 are schematic diagrams showing the non-agglutination of the anti-feline type A blood specific monoclonal antibody with type B erythrocytes;
FIG. 3 is a schematic diagram showing the subtype identification results of the anti-feline type A blood specific monoclonal antibody provided in the embodiment of the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
A hybridoma cell strain secreting the monoclonal antibody against cat type A blood type is named as A17H, and is preserved in China Center for Type Culture Collection (CCTCC) at 2022, 8 months and 18 days, wherein the preservation address is Wuhan university in China, and the preservation number is CCTCC NO: C2022269.
The hybridoma cell strain or the passage cell strain thereof secreting the anti-cat A blood type specific monoclonal antibody secretes the anti-cat A blood type specific monoclonal antibody as an IgM monoclonal antibody, the anti-cat A blood type specific monoclonal antibody agglutinates with cat A blood erythrocytes, but does not agglutinate with other cat blood type red blood cells, the measurement result precision is high, and the hybridoma cell strain or the passage cell strain thereof is applied to cat blood type detection.
The first embodiment is as follows:
obtaining hybridoma cells
1. Preparation of immunogen and detection antigen
(1) A-type hemocat erythrocytes anticoagulated by EDTA are collected, washed three times by physiological saline, the erythrocytes are lysed by 5mM Na2HPO4 (pH 7.4), washed three times by PBS, and then resuspended by PBS to obtain erythrocyte ghost (ghost) suspension as immunogen.
In practice, the immunogen of this embodiment may also be prepared by washing fresh EDTA-anticoagulated feline hemoglobin type a with physiological saline at least three times, and then preparing a 3% erythrocyte suspension with physiological saline as the immunogen.
(2) The detection antigen is fresh cat A and B type red blood cells, and the red blood cells are washed three times by normal saline and prepared into red blood cell suspension with 3 percent of concentration by the normal saline.
2. Immunizing animals
(1) BALB/c female mice weighing about 20g at 6 weeks of age were immunized with the immunogen, and 0.5ml of the immunogen was directly injected intraperitoneally.
(2) Two weeks later, the immunization was boosted and 0.5ml of immunogen was injected directly intraperitoneally.
(3) Three days after the boosting, splenocytes are taken and are subjected to cell fusion test with mouse myeloma cells SP 2/0.
3. Cell fusion
(1) And (3) uniformly mixing the immune mouse spleen cells and mouse myeloma cells SP2/0 according to the proportion of 5-10.
(2) Serum-free RPMI-1640 medium was used for washing once, and the medium was removed by centrifugation at 1000rpm for 5 minutes.
(3) The fusion was carried out using 50% PEG (polyethylene glycol, purchased from Sigma, molecular weight 1500) as a fusion agent, and the fusion was terminated at room temperature for 2min, followed by centrifugation at 1000rpm for 5 minutes using serum-free RPMI-1640 medium.
(4) The cell pellet after centrifugation was suspended in complete medium of HAT-containing RPMI-1640, dispensed into 96-well cell culture plates, and cultured at 37 ℃ in a 5% CO2 cell incubator.
4. Screening and cloning of hybridoma cells
(1) The fused cells are statically cultured in an incubator for 10-14 days, half the amount of the culture solution is changed for 2 times, and culture supernatant is taken for screening when the fused cells grow to cover about 1/3 of the bottom of the hole.
(2) Adding 50ul of cell culture supernatant into a U-shaped 96-hole enzyme label plate, respectively mixing with 3% of cat A and B type red blood cells prepared by 50ul of normal saline, reacting at room temperature for half an hour, centrifuging at 1000rpm for 1min, gently shaking, observing and recording the agglutination test result.
(3) Screening cell pores agglutinated with cat A-type erythrocytes but not agglutinated with B-type erythrocytes as positive pore cells, performing cell amplification culture and monoclonality to obtain a hybridoma cell strain A17H capable of stably secreting monoclonal antibodies against cat A-type blood types, performing amplification culture, cryopreserving the cells, and storing in liquid nitrogen.
Example two:
preparation of monoclonal antibody reagent for resisting cat type A blood group
(1) The hybridoma cell line A17H obtained was subjected to amplification culture in RPMI-1640 medium containing 10% fetal bovine serum. PBS washing 2 times, then PBS heavy suspension, adjusting cell density to 5x10 6 /ml。
(2) 6 weeks old BALB/c mice were injected intraperitoneally with 0.5ml Freund's incomplete adjuvant (Sigma), one week later with 0.5ml hybridoma cell suspension, and 7-10 days later with ascites.
(3) Carrying out 12000rpm on ascites containing the monoclonal antibody, centrifuging for 10 minutes, taking supernatant, and adding 10 times of deionized water to separate out the antibody; after centrifugation at 12000rpm for 15 minutes, the antibody precipitate was dissolved in PBS, and the A280 absorbance was measured to calculate the antibody concentration A280/1.18. The antibody concentration was adjusted to 1 mg/ml by dilution with PBS, and 5 ug, 10ug, and 20ug of the antibodies were added to 1 Xreduction type loading buffer, followed by electrophoresis on 10% SDS-PAGE and staining with Coomassie Brilliant blue, and as a result, an IgM heavy chain having a molecular weight of 70 kDa and a 25 kDa light chain were observed, as shown in FIG. 1.
Example three:
specific identification of monoclonal antibodies secreting anti-cat type A blood group
And (3) carrying out specificity identification on the prepared monoclonal antibody for resisting the cat type A blood group by adopting a test tube method. The method comprises the following specific steps:
(1) 3 cases of A and B blood type red blood cells are taken, washed 3 times by physiological saline, centrifuged at 2000rpm for 5 minutes, and finally prepared into 3 percent red blood cell suspension by physiological saline.
(2) 2 groups of 3 test tubes were prepared, wherein A1, A2 and A3 in FIG. 2 are blood red cells of type A, B1, B2 and B3 are blood red cells of type B, and 0.1ml of 20 ug/ml A17H monoclonal antibody was added to each test tube, and then 3% of A and 0.1ml of blood red cell suspension of type B were added to each test tube, and the mixture was allowed to stand at room temperature for reaction for 15 minutes, centrifuged at 1000r/min for 1min, and gently shaken to observe the presence or absence of agglutination.
Fig. 2 is a schematic diagram of the agglutination result of the anti-feline type a blood specific monoclonal antibody provided in this example, in which fig. A1-A3 are schematic diagrams of the agglutination of the anti-feline type B blood specific monoclonal antibody and type a erythrocytes, and fig. B1-B3 are schematic diagrams of the non-agglutination of the anti-feline type B blood specific monoclonal antibody and type B erythrocytes. It is clear from the figure that all 3B-type erythrocytes agglutinate, while 3A-type erythrocytes do not agglutinate, and have remarkable specificity.
Example four:
antibody subtype identification
According to the requirements of the operating procedures of the mouse monoclonal antibody subtype identification kit of Proteintetech, 50ul of hybridoma culture supernatant is added into an ELISA plate bar, 50ul of HRP-labeled secondary antibody of each subtype is added, incubation is carried out for 1 hour at room temperature, and TMB is developed after PBST is washed for 3 times.
FIG. 3 is a schematic diagram showing the subtype identification results of the anti-feline blood type-A specific monoclonal antibody provided in this example, and the results show that the A17H monoclonal antibody is IgM and the light chain is Kappa type.
The invention provides a blood type detection reagent, test paper or kit, which comprises a monoclonal antibody against cat type A blood type specificity; a blood type detection device comprises a blood type detection reagent, test paper or a kit, which can be applied to the blood type detection of cats.
The invention also provides a blood type detection method, which comprises the step of contacting a sample to be detected with the anti-cat type A blood type specific monoclonal antibody so as to determine the blood type of the sample to be detected.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make various improvements and modifications without departing from the technical principle of the present invention, and those improvements and modifications should be considered as the protection scope of the present invention.
Claims (8)
1. A hybridoma cell strain secreting a feline A-type blood specific monoclonal antibody is characterized in that the preservation number of the hybridoma cell strain is CCTCC NO: C2022269.
2. the hybridoma cell line secreting a monoclonal antibody specific for feline type a blood according to claim 1, wherein a BALB/c mouse is immunized with feline type a erythrocyte ghosts or feline type a erythrocytes as an antigen.
3. The monoclonal antibody against cat A type blood type specificity is characterized in that the monoclonal antibody against cat A type blood type specificity is prepared from a monoclonal antibody with a preservation number of CCTCC NO: c2022269 hybridoma cell line secretion.
4. The monoclonal antibody specific to feline type A blood cells according to claim 3, wherein the monoclonal antibody specific to feline type A blood cells is an IgM monoclonal antibody that agglutinates with feline type A blood cells.
5. A blood group testing reagent, strip or kit comprising an anti-feline type a blood specific monoclonal antibody of claims 3 or 4.
6. A blood typing device comprising the blood typing reagent, test strip or kit according to claim 5.
7. Use of a monoclonal antibody specific to cat type a blood type according to claim 3 or 4 or a blood type test reagent, strip or kit according to claim 5 for testing cat blood type.
8. A method for testing blood type, which comprises contacting a test sample with the monoclonal antibody specific to cat type A blood type of claim 3 or 4 to determine the blood type of the test sample.
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| CN202211025611.2A CN115340985A (en) | 2022-08-25 | 2022-08-25 | Hybridoma cell strain secreting anti-cat A-type blood specific monoclonal antibody, monoclonal antibody and application of monoclonal antibody |
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| CN202211025611.2A CN115340985A (en) | 2022-08-25 | 2022-08-25 | Hybridoma cell strain secreting anti-cat A-type blood specific monoclonal antibody, monoclonal antibody and application of monoclonal antibody |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117805402A (en) * | 2024-03-01 | 2024-04-02 | 北京纳百生物科技有限公司 | Cat blood typing detection method and kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030092088A1 (en) * | 2001-07-26 | 2003-05-15 | Andrews Gordon A. | Method and kit for typing feline blood |
| US20080220427A1 (en) * | 2006-12-21 | 2008-09-11 | Regents Of The University Of California | Test for determining blood type in the cat |
| EP3476948A1 (en) * | 2017-10-25 | 2019-05-01 | LABOklin Labor für klinische Diagnostik GmbH & Co. KG | Method and device for determining the blood group of a cat in ab blood group system |
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- 2022-08-25 CN CN202211025611.2A patent/CN115340985A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030092088A1 (en) * | 2001-07-26 | 2003-05-15 | Andrews Gordon A. | Method and kit for typing feline blood |
| US20080220427A1 (en) * | 2006-12-21 | 2008-09-11 | Regents Of The University Of California | Test for determining blood type in the cat |
| EP3476948A1 (en) * | 2017-10-25 | 2019-05-01 | LABOklin Labor für klinische Diagnostik GmbH & Co. KG | Method and device for determining the blood group of a cat in ab blood group system |
Non-Patent Citations (1)
| Title |
|---|
| J. L. GREEN,等: "Production and characterisation of murine monoclonal antibodies to feline erythrocyte A and B antigens", vol. 10, pages 33 - 35 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117805402A (en) * | 2024-03-01 | 2024-04-02 | 北京纳百生物科技有限公司 | Cat blood typing detection method and kit |
| CN117805402B (en) * | 2024-03-01 | 2024-05-28 | 北京纳百生物科技有限公司 | Cat blood typing detection method and kit |
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