CN114395533B - 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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Abstract
The invention belongs to the technical field of immunology and in-vitro diagnosis branching, and particularly relates to a hybridoma cell strain secreting an anti-human Cys C monoclonal antibody, the 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 cysC-1H9 monoclonal antibody and 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 labeled antibody, and the Cysc-5F1 monoclonal antibody is used as a coated antibody, and 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 branching, and particularly relates to a hybridoma cell strain secreting an anti-human Cys C monoclonal antibody, the monoclonal antibody secreted by the hybridoma cell strain and application of the hybridoma cell strain.
Background
Human Cys C is abbreviated as cystatin C. Cystatin C is a low molecular weight, basic non-glycosylated protein consisting of 122 amino acids with a molecular weight of 13.3 kDa. Almost all cells of the human body produce cystatin C, and the concentration of human Cys C in human serum (less than or equal to 1.03 mg/L) is constant under normal conditions, and is irrelevant to the differences of sex, weight, age and the like.
Cystatin C is of great importance in clinical detection. Cystatin C is only removed through glomerular filtration, is an endogenous marker reflecting the change of glomerular filtration rate, and can efficiently and accurately evaluate kidney function. Cystatin C concentration increases when kidney function is impaired. It has been thought by researchers that cystatin C also rapidly reflects the recovery of renal function, especially in the clinical application of renal transplantation where acute rejection or drug therapy may cause damage to the kidneys, giving a rapid diagnosis.
Although there are some methods in the art for detecting cystatin C, the sensitivity and accuracy thereof have been unsatisfactory. There is therefore an urgent need in the art to develop antibodies (particularly monoclonal antibodies that can be used in pairs) and methods for the accurate, specific, rapid, sensitive detection of cystatin C.
Disclosure of Invention
In order to solve the problems, the invention provides two hybridoma cell strains secreting the 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 labeled antibody and a coated antibody, and are used for preparing a kit for detecting the content of human Cys C in human serum.
The first object of the present invention is to provide two hybridoma cell lines secreting anti-human Cys C monoclonal antibodies, which address the deficiencies of the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme: the method comprises the steps of selecting a prokaryotic expressed human Cys C antigen to immunize a mouse, taking spleen lymphocytes of the immunized mouse and mouse myeloma cells to carry out cell fusion, and carrying out positive screening and subcloning to obtain a cell strain, namely a hybridoma cell strain 1H9 and a hybridoma cell strain 5F1, wherein the cell strain can stably secrete an anti-human Cys C monoclonal antibody and is preserved in China center for type culture collection, and the preservation numbers are CCTCC NO. C2021188 and CCTCC NO. C2021189 in sequence.
A second object of the present invention is to provide two anti-human Cys C monoclonal antibodies, which address the deficiencies of the prior art.
In order to achieve the above purpose, the invention is realized by the following technical scheme: selecting Balb/c female mice, injecting liquid paraffin into the abdominal cavity of the mice, injecting hybridoma cells with the preservation number of CCTCC NO. C2021188 into the abdominal cavity after two weeks, collecting ascites after the abdomen of the mice swells obviously, centrifuging, collecting supernatant, and purifying to obtain the Cysc-1H9 monoclonal antibody; in the same way, another Cysc-5F1 monoclonal antibody is obtained by injecting hybridoma cells with the preservation number of CCTCC NO. C2021189 into the abdominal cavity of another Balb/c female mouse.
The subtype of the two anti-human Cys C monoclonal antibodies provided by the invention is IgG1, and the antibodies have specificity to 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 object of the present invention is to provide the use of two anti-human Cys C monoclonal antibodies in the preparation of an in vitro diagnostic kit, in view of the deficiencies of 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 coated antibody.
The kit is an enzyme-linked immunosorbent assay kit, a chemiluminescent immunoassay kit, a fluorescent 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 strain thereof can efficiently and stably secrete the two anti-human Cys C monoclonal antibodies; the specificity of the anti-human Cys C monoclonal antibody is strong. When the two antibodies are paired, the specificity is good and the sensitivity is high in detecting the human Cys C. Is widely applicable to the application detection field of human Cysc.
Biological preservation description 1:
culture name: hybridoma cell line 1H9 was deposited with the China Center for Type Culture Collection (CCTCC) at 2021, 8 and 19, accession number: the preservation number of the Chinese university of Wuhan is CCTCC NO. C2021188.
Biological preservation description 2:
culture name: hybridoma cell line 5F1 was deposited with the chinese collection center (CCTCC) at 2021, 8 and 19, accession number: the preservation number of the Chinese university of Wuhan is CCTCC NO. C2021189.
Drawings
FIG. 1 is a graph showing the results of the test for the stability of antibody secretion by hybridoma cells of example 1;
FIG. 2 shows the results of the detection of antibody titers and affinities of example 3;
FIG. 3 is a graph showing the results of detection of the antibody subtype of example 3;
FIG. 4 shows the results of the performance test of the antibodies 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 monoclonal antibodies against human Cys C
1. Immunization of animals
Female balb/C mice 6 weeks old were immunized with human Cys C prokaryotic expression antigen. Primary immunization, mixing and emulsifying the antigen and Freund's complete adjuvant in equal volume, and subcutaneously immunizing mice via back at multiple points, wherein the immunization dose is 100 mug/mouse. Three weeks later, boost was performed every 15 days, the antigen was mixed and emulsified with equal volumes of incomplete Freund's adjuvant, and mice were subcutaneously multipoint immunized via the back at a dose of 50 μg/mouse. On day 7 of each boost, mice were subjected to tail-end blood sampling to measure serum titers, and the serum titers reached 5 ten thousand or more for fusion, and the detection results are shown in table 1. Mice were immunized by intraperitoneal injection 3 days prior to fusion, without adjuvant, at a dose of 100 μg/mouse.
TABLE 1 serum titers of immunized mice detection results
2. Cell fusion
Taking spleen of immunized mice under aseptic condition, fully grinding and dispersing, selecting a 200-mesh screen to filter grinding liquid, removing undispersed tissue blocks, centrifuging at 1000rpm/min for 10min, collecting mouse lymphocytes, mixing with mouse myeloma cells sp2/0 according to the ratio of 10:1, and centrifuging to wash the cells to ensure that no fetal bovine serum exists in the mixed cells; cell fusion was performed by dropwise adding 1ml of PEG (molecular weight 1450) from slow to fast in a water bath at 37℃for 1min, gently mixing, and standing for 1min. The fusion was stopped with 25ml serum free DMEM, the addition rate was from slow to fast, and the reaction was stopped within 6min at volumes of 1ml, 2ml, 4ml, 6ml, 12ml per minute. The fused cells were incubated at 37℃in a 5% carbon dioxide incubator for 15min, centrifuged at 800rpm/min for 10min, resuspended in HAT medium, and sub-packed in 96-well cell culture plates, and incubated at 37℃in a 5% carbon dioxide incubator.
3. Hybridoma cell screening and subcloning
On day seven of cell fusion, the culture supernatant was assayed by indirect ELISA by study test on day nine with complete exchange of HAT medium.
The specific detection method comprises the following steps:
human Cys C eukaryotic expression antigen was diluted to 0.5. Mu.g/ml with coating solution, 100. Mu.l/well coated in ELISA plates, coated overnight at 4 ℃. The plate was washed 3 times. Mu.l of blocking solution was added to each well of the ELISA plate and incubated at 37℃for 2h. The plate was washed 3 times. The cell supernatant to be detected was added to the ELISA plate at 100. Mu.l/well, and positive control was performed with mouse immune serum, and incubated at 37℃for 1h. The plate was washed 3 times. HRP-labeled goat anti-mouse antibody was diluted 4-fold, added to the ELISA plate, 100. Mu.l/well, and incubated at 37℃for 1h. The plate was washed 5 times. Adding 100 μl TMB color developing solution into each hole of the ELISA plate, and incubating at 37deg.C for 10min; the 2M sulfuric acid was stopped, 50. Mu.l/well, and the data were read at a wavelength of 450 nm.
And selecting a cell hole with strong positive reaction for subcloning, strictly requiring 3 times, and determining that the monoclonal cell strain is a stable cell strain with 100% of positive rate.
4. Hybridoma cell secretion antibody stability assay
Subculturing hybridoma cells, co-culturing for 30 generations, collecting culture supernatants at 1, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 generations respectively, detecting quality control by ELISA method, and analyzing stability of antibody secreted by cells. The hybridoma cell strains of the two strains (1H 9 and 5F 1) secreting the anti-human Cys C monoclonal antibodies and the passage cell strains can efficiently and stably secrete the anti-human Cys C monoclonal antibodies.
The detection results are shown in FIG. 1.
5. Hybridoma cell line preservation
Selecting hybridoma cell strain with stable growth, and performing expansion culture. When the cell density reached 80% of the specified value, cells were collected and frozen with cell frozen stock.
2 hybridoma cells capable of stably secreting anti-human Cys C monoclonal antibodies are obtained through cell fusion, and the antibodies secreted respectively 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 the 8 th month 19 of 2021, with a preservation address: 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 antibodies
1. Ascites preparation
Selecting a balb/c mouse with a designated age of about 12 weeks, injecting 500 μl of liquid paraffin into the abdominal cavity, and injecting 10 into the abdominal cavity after 2 weeks 6 The injection volume was 100. Mu.l/mouse hybridoma cells. After 5-7 days, continuously observing the mice, collecting ascites after the abdomen of the mice swells obviously, centrifuging at 12000rpm/min for 15min, removing impurities, and storing in a refrigerator at-20 ℃.
2. Ascites purification
The ascites fluid was centrifuged at 12000rpm/min for 10min, filtered with a coarse filter paper and precipitated with 50% ammonium sulfate. Purification was then performed with reference to the Bogurone protein-A column instructions. The purified antibodies were collected, dialyzed against 10mM PBS, collected, filtered through a 0.22 μm filter, and then sub-packaged, and stored in a-80℃refrigerator for subsequent detection and validation.
EXAMPLE 3 monoclonal antibody identification
1. Antibody titer and affinity assays
The potency and affinity of the antibodies were detected by indirect ELISA. The specific detection method comprises the following steps:
human Cys C eukaryotic expression antigen was diluted to 2. Mu.g/ml with coating solution, 100. Mu.l/well coated into the ELISA plate, and coated overnight at 4 ℃. The plate was washed 3 times. Mu.l of blocking solution was added to each well of the ELISA plate and incubated at 37℃for 2h. The plate was washed 3 times. The antibody to be detected is diluted at an initial concentration of 1mg/ml, added to the ELISA plate at a certain ratio, and incubated at 37℃for 1h at 100. Mu.l/well. The plate was washed 3 times. HRP-labeled goat anti-mouse antibody was diluted 4-fold, added to the ELISA plate, 100. Mu.l/well, and incubated at 37℃for 1h. The plate was washed 5 times. Adding 100 μl TMB color developing solution into each hole of the ELISA plate, and incubating at 37deg.C for 10min; the 2M sulfuric acid was stopped, 50. Mu.l/well, and the data were read at a wavelength of 450 nm.
As shown in FIG. 2, the 1H9 titer was 10 ten thousand, and the 5F1 titer was 100 ten thousand or more.
2. Antibody subtype detection
The detection was performed according to the instructions using a SBA subtype detection kit from Southern Biotech, both anti-human Cys C monoclonal antibodies subtype IgG1.
The detection results 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 labeling antibody, 20 parts of positive serum and 40 parts of negative serum are detected by a double-antibody sandwich ELISA method, and P/N >2 is used as positive. The test results are shown in Table 2.
TABLE 2 antibody pair screening results
5. Antibody performance detection
On the fluorescent immunization platform, compared with the outsourcing control antibody, the Cys C-1H9 monoclonal antibody and the Cys C-5F1 monoclonal antibody are subjected to pairing test, and the signal value, the linearity and the like are superior to those of the outsourcing control antibody.
The detection results are shown in FIG. 4.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (8)
1. A hybridoma cell strain secreting anti-human Cys C monoclonal antibody has a preservation number of CCTCC NO. C2021188.
2. A hybridoma cell strain secreting anti-human Cys C monoclonal antibody has a preservation number of CCTCC NO. C2021189.
3. An anti-human Cys C monoclonal antibody, secreted by the hybridoma cell strain of claim 1.
4. An anti-human Cys C monoclonal antibody, secreted by the hybridoma cell line of claim 2.
5. The anti-human Cys C monoclonal antibody of claim 3 or 4, wherein the subtype is IgG1.
6. Use of the anti-human Cys C monoclonal antibody of claim 3 or 4 for the preparation of a kit for detecting human Cys C, said use being based on a double antibody sandwich method.
7. The use according to claim 6, wherein the anti-human Cys C monoclonal antibody of claim 3 is used as a labeling antibody and the anti-human Cys C monoclonal antibody of claim 4 is used as a coating antibody.
8. The use according to claim 6, wherein the kit is an enzyme-linked immunosorbent assay 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| 胱抑素 C 抗体制备及化学发光免疫检测方法的建立;王云龙 等;生物技术通报;第第34卷卷(第第8期期);第75-79页 * |
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