CN114395533A - Hybridoma cell strain secreting anti-human Cys C monoclonal antibody, monoclonal antibody secreted by hybridoma cell strain and application of hybridoma cell strain - Google Patents
Hybridoma cell strain secreting anti-human Cys C monoclonal antibody, monoclonal antibody secreted by hybridoma cell strain and application of hybridoma cell strain Download PDFInfo
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- C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
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Abstract
The invention belongs to the technical field of immunology and in vitro diagnosis branches, and particularly relates to a hybridoma cell strain secreting an anti-human Cys C monoclonal antibody, a monoclonal antibody secreted by the hybridoma cell strain and application of the hybridoma cell strain. The hybridoma cell strain 1H9 and the hybridoma cell strain 5F1 have the preservation numbers of CCTCC NO. C2021188 and CCTCC NO. C2021189 in sequence, can stably secrete a cysc-1H9 monoclonal antibody and a cysc-5F1 monoclonal antibody, and have the characteristics of high titer, strong affinity and good specificity. In the preparation of the in vitro diagnostic kit, the Cysc-1H9 monoclonal antibody is used as a marker antibody, and the Cysc-5F1 monoclonal antibody is used as a coating antibody, so that the kit has the characteristics of good specificity and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of immunology and in vitro diagnosis branches, and particularly relates to a hybridoma cell strain secreting an anti-human Cys C monoclonal antibody, a monoclonal antibody secreted by the hybridoma cell strain and application of the hybridoma cell strain.
Background
Human Cys C is short for cystatin C. Cystatin C is a low molecular weight, basic, non-glycated protein consisting of 122 amino acids with a molecular weight of 13.3 kDa. Almost all cells of a human body can produce cystatin C, and the concentration of human Cys C in human serum (less than or equal to 1.03mg/L) is constant under normal conditions and is irrelevant to differences of sex, weight, age and the like.
Cystatin C has important significance in clinical detection. Cystatin C is cleared only by glomerular filtration, is an endogenous marker reflecting glomerular filtration rate change, and can efficiently and accurately evaluate kidney function. Cystatin C concentrations increase when kidney function is impaired. It is believed by researchers that cystatin C also rapidly reflects the recovery of renal function, especially acute rejection in renal transplant clinical applications or the possible damage to the kidney caused by drug therapy, giving a rapid diagnosis.
Although there are some methods for detecting cystatin C in the art, the sensitivity and accuracy are still unsatisfactory. Therefore, there is a strong need in the art to develop antibodies (especially monoclonal antibodies that can be used in pairs) and methods for detecting cystatin C accurately, specifically, rapidly and sensitively.
Disclosure of Invention
In order to solve the problems, the invention provides two hybridoma cell strains secreting anti-human Cys C monoclonal antibodies, and the two hybridoma cell strains are respectively cultured by an in-vivo ascites method to prepare the two anti-human Cys C monoclonal antibodies. The two anti-human Cys C monoclonal antibodies obtained by the method are respectively used as a marker antibody and a coating antibody and are used for preparing a kit for detecting the content of human Cys C in human serum.
The first purpose of the invention is to provide two hybridoma cell strains secreting anti-human Cys C monoclonal antibodies aiming at the defects in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme: the mouse is immunized by using prokaryotic expression human Cys C antigen, spleen lymphocytes of the immunized mouse are taken to be fused with mouse myeloma cells, the cell strain is obtained through positive screening and subcloning and is named as hybridoma cell strain 1H9 and hybridoma cell strain 5F1, the cell strain can stably secrete anti-human Cys C monoclonal antibody and is preserved in China center for type culture collection with the preservation numbers of CCTCC NO. C2021188 and CCTCC NO. C2021189 in sequence.
The second purpose of the invention is to provide two anti-human Cys C monoclonal antibodies aiming at the defects in the prior art.
In order to achieve the purpose, the invention is realized by the following technical scheme: selecting a Balb/c female mouse, injecting liquid paraffin into the abdominal cavity of the mouse, injecting hybridoma cells with the preservation number of CCTCC NO. C2021188 into the abdominal cavity after two weeks, collecting ascites after the abdominal cavity is obviously expanded, centrifuging, collecting supernate, and purifying to obtain a Cysc-1H9 monoclonal antibody; in the same way, another female Balb/c mouse is injected with hybridoma cell with the preservation number of CCTCC NO. C2021189 to obtain another Cysc-5F1 monoclonal antibody.
The two anti-human Cys C monoclonal antibodies provided by the invention have the subtype of IgG1 and have specificity on human Cys C.
The two anti-human Cys C monoclonal antibodies provided by the invention have the characteristics of high titer, strong affinity and good specificity.
The third purpose of the invention is to provide the application of two anti-human Cys C monoclonal antibodies in preparing in vitro diagnostic kits aiming at the defects in the prior art.
The application is based on a double antibody sandwich method.
In the kit, Cysc-1H9 monoclonal antibody is used as a labeled antibody, and Cysc-5F1 monoclonal antibody is used as a coating antibody.
The kit is an enzyme-linked immunoassay kit, a chemiluminescence immunoassay kit, a fluorescence immunoassay kit and a colloidal gold immunoassay kit.
The invention has the beneficial effects that:
the two hybridoma cell strains secreting the anti-human Cys C monoclonal antibodies and the passage cell strains thereof can efficiently and stably secrete the two anti-human Cys C monoclonal antibodies; the anti-human Cys C monoclonal antibody has strong specificity. The two antibodies are paired, and the specificity is good and the sensitivity is high when the human Cys C is detected. Is widely applicable to the application and detection field of human Cysc.
Biological preservation description 1:
culture name: the hybridoma cell strain 1H9 has been deposited in China Center for Type Culture Collection (CCTCC) at 19.8.2021, with the deposition address: china, Wuhan university, with the preservation number CCTCC NO. C2021188.
Biological preservation description 2:
culture name: the hybridoma cell strain 5F1, which has been deposited in China Center for Type Culture Collection (CCTCC) at 19/8/2021, with the deposition address: china, Wuhan university, with the preservation number CCTCC NO. C2021189.
Drawings
FIG. 1 shows the results of the measurement of the stability of antibody secretion from hybridoma cells according to example 1;
FIG. 2 shows the results of measurement of antibody titer and affinity in example 3;
FIG. 3 shows the results of detection of the antibody subtype of example 3;
FIG. 4 shows the results of the measurement of the antibody performance of example 3.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1 preparation of hybridoma cell lines secreting anti-human Cys C monoclonal antibodies
1. Animal immunization
A female balb/C mouse of 6 weeks of age is immunized with the antigen expressed from the human Cys C pronucleus. The primary immunization, the antigen and Freund's complete adjuvant are mixed and emulsified in equal volume, and the mice are immunized by subcutaneous multiple points on the back, and the immunization dose is 100 mu g/mouse. Three weeks later, every 15 days of booster immunization, antigen and incomplete Freund's adjuvant equal volume mixed emulsion, through the subcutaneous multiple immunization of mice on the back, the immunization dose is 50 ug/mouse. On the 7 th day of each boosting immunization, the mice are subjected to tail-cutting blood sampling to measure the serum titer, the serum titer can reach more than 5 ten thousand for fusion, and the detection results are shown in table 1. 3 days before fusion, mice were immunized by intraperitoneal injection without adjuvant, and the immunization dose was 100 μ g/mouse.
TABLE 1 serum titer test results for immunized mice
2. Cell fusion
Under the aseptic condition, taking the spleen of an immunized mouse, fully grinding and dispersing, selecting a 200-mesh screen to filter grinding fluid, removing undispersed tissue blocks, centrifuging at 1000rpm/min for 10min, collecting mouse lymphocytes, mixing the mouse lymphocytes with mouse myeloma cells sp2/0 according to the proportion of 10:1, centrifuging and washing the cells, and ensuring that no fetal calf serum exists in the mixed cells; dropping 1ml PEG (molecular weight 1450) slowly to rapidly in 37 deg.C water bath for cell fusion within 1min, mixing gently, and standing for 1 min. The fusion was stopped with 25ml serum free DMEM, added slowly and quickly, and stopped within 6min at volumes of 1ml, 2ml, 4ml, 6ml, 12ml per minute. The fused cells are placed into an incubator with 37 ℃ and 5% carbon dioxide for incubation for 15min, centrifuged at 800rpm/min for 10min, resuspended by HAT culture medium, subpackaged into 96-well cell culture plates, and placed into an incubator with 37 ℃ and 5% carbon dioxide for culture.
3. Hybridoma cell screening and subcloning
On day seven of fusion, all the HAT medium was changed and the culture supernatant was examined by indirect ELISA using research assays on day nine.
The specific detection method comprises the following steps:
diluting the human Cys C eukaryotic expression antigen to 0.5 mu g/ml by using a coating solution, coating 100 mu l/hole into an enzyme label plate, and coating overnight at 4 ℃. The plate was washed 3 times. 150 μ l of blocking solution was added to each well of the microplate and incubated at 37 ℃ for 2 h. The plate was washed 3 times. Adding cell supernatant to be detected into an enzyme label plate at a concentration of 100 mu l/hole, using mouse immune serum as a positive control, and incubating at 37 ℃ for 1 h. The plate was washed 3 times. The goat anti-mouse antibody marked by HRP is diluted by 4 ten thousand times, added into an enzyme label plate, and incubated for 1h at 37 ℃ in a 100 mu l/hole way. The plate was washed 5 times. Adding 100 mul of TMB color development solution into each hole of the ELISA plate, and incubating for 10min at 37 ℃; the data were read at 450nm wavelength, 2M sulfuric acid stop, 50. mu.l/well.
And selecting the cell pores with strong positive reaction for subcloning, strictly performing 3 times, and determining the monoclonal cell strain with 100% positive rate as a stable cell strain.
4. Hybridoma cell secreted antibody stability detection
Subculturing the hybridoma cells, culturing for 30 generations, collecting culture supernatants at 1, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 generations, detecting quality control product by ELISA method, and analyzing stability of antibody secreted by the cells. The two hybridoma cell strains (1H9 and 5F1) secreting anti-human Cys C monoclonal antibodies and the subculture cell strains thereof can efficiently and stably secrete anti-human Cys C monoclonal antibodies.
The detection results are shown in FIG. 1.
5. Hybridoma cell line preservation
And (4) selecting a hybridoma cell strain with stable growth, and carrying out expanded culture. When the cell density reached 80% of the specified value, the cells were collected and frozen in a cell freezing medium.
2 hybridoma cells which can stably secrete anti-human Cys C monoclonal antibodies are obtained through cell fusion, and the respectively secreted antibodies can be paired by detecting CysC antigens through a double-antibody sandwich method and are preserved in China Center for Type Culture Collection (CCTCC) at 8 months and 12 days in 2021, wherein the preservation addresses are as follows: the preservation number of the hybridoma cell strain 1H9 is CCTCC NO. C2021188, and the preservation number of the hybridoma cell strain 5F1 is CCTCC NO. C2021189.
EXAMPLE 2 preparation of anti-human Cys C monoclonal antibody
1. Preparation of ascites
Selecting balb/c mice of about 12 weeks old, performing intraperitoneal injection of 500 mul of liquid paraffin, and performing intraperitoneal injection of 10 after 2 weeks6One hybridoma cell per one, injection volume was 100. mu.l per one. And after 5-7 days, continuously observing the mice, collecting ascites after the abdominal distension is obvious, centrifuging at 12000rpm/min for 15min, removing impurities, and storing in a refrigerator at the temperature of-20 ℃.
2. Purification of ascites
The ascites was centrifuged at 12000rpm/min for 10min, filtered through coarse filter paper and precipitated with 50% ammonium sulfate. Purification was then performed with reference to the Bogelong protein-A column instructions. The purified antibody was collected, dialyzed against 10mM PBS, collected, filtered through a 0.22 μm filter, dispensed, and stored in a-80 ℃ freezer for subsequent testing and validation.
EXAMPLE 3 monoclonal antibody identification
1. Antibody titer and affinity assay
The titer and the affinity of the antibody are detected by adopting an indirect ELISA method. The specific detection method comprises the following steps:
diluting the human Cys C eukaryotic expression antigen to 2 mu g/ml by using a coating solution, coating 100 mu l/well into an enzyme label plate, and coating overnight at 4 ℃. The plate was washed 3 times. 150 μ l of blocking solution was added to each well of the microplate and incubated at 37 ℃ for 2 h. The plate was washed 3 times. Diluting the antibody to be detected at an initial concentration of 1mg/ml according to a certain proportion, adding the diluted antibody into an ELISA plate, incubating at the temperature of 37 ℃ for 1h at 100 mu l/hole. The plate was washed 3 times. The goat anti-mouse antibody marked by HRP is diluted by 4 ten thousand times, added into an enzyme label plate, and incubated for 1h at 37 ℃ in a 100 mu l/hole way. The plate was washed 5 times. Adding 100 mul of TMB color development solution into each hole of the ELISA plate, and incubating for 10min at 37 ℃; the data were read at 450nm wavelength, 2M sulfuric acid stop, 50. mu.l/well.
As shown in FIG. 2, the titer of 1H9 was 10 ten thousand, and the titer of 5F1 was 100 ten thousand or more.
2. Antibody subtype detection
Detection was performed using the SBA subtype detection kit from Southern Biotech according to the instructions, and both subtypes of the two anti-human Cys C monoclonal antibodies were IgG 1.
The results of the detection are shown in FIG. 3.
3. Antibody pairing screening
The monoclonal antibody 5F1 is used as a coating antibody, the monoclonal antibody 1H9 is used as a labeled antibody, 20 positive sera and 40 negative sera are detected by a double-antibody sandwich ELISA method, and P/N >2 is used as the positive. The results are shown in Table 2.
TABLE 2 results of antibody pairing screening
5. Antibody Performance assays
On a fluorescence immune platform, compared with an externally purchased control antibody, the Cys C-1H9 monoclonal antibody and the Cys C-5F1 monoclonal antibody are superior to the externally purchased control antibody in the aspects of signal value, linearity and the like in a pairing test.
The results of the detection are shown in FIG. 4.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (7)
1. Two hybridoma cell strains secreting anti-human Cys C monoclonal antibodies are named as a hybridoma cell strain 1H9 and a hybridoma cell strain 5F1 respectively, and the preservation numbers are CCTCC NO. C2021188 and CCTCC NO. C2021189 respectively.
2. The hybridoma cell strain of claim 1, wherein the mouse is immunized with the recombinantly expressed human Cys C antigen, spleen lymphocytes from the immunized mouse are taken to be cell-fused with mouse myeloma cells, and the hybridoma cell strain is obtained by positive screening and subcloning.
3. Two anti-human Cys C monoclonal antibodies, which are secreted by the two hybridoma cell lines of claim 1 and are respectively named as: Cysc-1H9 monoclonal antibody and Cysc-5F1 monoclonal antibody.
4. The two anti-human Cys C monoclonal antibodies of claim 3, wherein subtype is IgG 1.
5. The use of two anti-human Cys C monoclonal antibodies of claim 3 in the preparation of a kit for the detection of human Cys C, said use being based on a double antibody sandwich method.
6. The use according to claim 5, wherein the Cysc-1H9 monoclonal antibody secreted by the antibody with the collection number of CCTCC NO. C2021188 is used as a labeled antibody, and the Cysc-5F1 antibody secreted by the antibody with the collection number of CCTCC NO. C2021189 is used as a coating antibody.
7. The use of claim 5, wherein the kit is an enzyme-linked immunoassay kit, a chemiluminescent immunoassay kit, a fluorescent immunoassay kit, or a colloidal gold immunoassay kit.
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Citations (4)
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|---|---|---|---|---|
| JP2011125267A (en) * | 2009-12-17 | 2011-06-30 | Kitasato Institute | Cystatin c and gene thereof, anti-cystatin c antibody, and kit and method for diagnosing cat nephropathy |
| CN102321176A (en) * | 2011-08-31 | 2012-01-18 | 北京利德曼生化股份有限公司 | Method for preparing CysC-paired monoclonal antibody |
| CN103509115A (en) * | 2012-06-21 | 2014-01-15 | 中国科学院上海生命科学研究院 | Nanometer antibodies to human cystatin C and application thereof |
| CN107828739A (en) * | 2017-09-28 | 2018-03-23 | 四川迈克生物新材料技术有限公司 | The hybridoma of the anti-bladder chalone C monoclonal antibody of mouse and its monoclonal antibody and purposes of secretion can be secreted |
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Patent Citations (4)
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| JP2011125267A (en) * | 2009-12-17 | 2011-06-30 | Kitasato Institute | Cystatin c and gene thereof, anti-cystatin c antibody, and kit and method for diagnosing cat nephropathy |
| CN102321176A (en) * | 2011-08-31 | 2012-01-18 | 北京利德曼生化股份有限公司 | Method for preparing CysC-paired monoclonal antibody |
| CN103509115A (en) * | 2012-06-21 | 2014-01-15 | 中国科学院上海生命科学研究院 | Nanometer antibodies to human cystatin C and application thereof |
| CN107828739A (en) * | 2017-09-28 | 2018-03-23 | 四川迈克生物新材料技术有限公司 | The hybridoma of the anti-bladder chalone C monoclonal antibody of mouse and its monoclonal antibody and purposes of secretion can be secreted |
Non-Patent Citations (1)
| Title |
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| 王云龙 等: "胱抑素 C 抗体制备及化学发光免疫检测方法的建立", 生物技术通报, vol. 34, no. 8, pages 75 - 79 * |
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