CN113402608A - Hybridoma cell strain, monoclonal antibody secreted by hybridoma cell strain and application of monoclonal antibody - Google Patents
Hybridoma cell strain, monoclonal antibody secreted by hybridoma cell strain and application of monoclonal antibody Download PDFInfo
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- CN113402608A CN113402608A CN202110705928.XA CN202110705928A CN113402608A CN 113402608 A CN113402608 A CN 113402608A CN 202110705928 A CN202110705928 A CN 202110705928A CN 113402608 A CN113402608 A CN 113402608A
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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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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- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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Abstract
本发明涉及食品安全技术领域，尤其涉及一种杂交瘤细胞株及其分泌的单克隆抗体与应用。本发明所述杂交瘤细胞株为抗猪骨骼肌肌钙蛋白T单克隆抗体的杂交瘤细胞株skTnT‑2E8，保藏于中国典型培养物保藏中心，地址为中国.武汉.武汉大学，保藏日期为2021年3月12日，保藏编号为CCTCCNO:C202158。采用本发明的杂交瘤细胞株分泌的单克隆抗体为IgG2a亚型，效价能达到1:4.1×105，亲和力常数Ka＝5.14×106L/mol。为特异性的检测猪骨骼肌肌钙蛋白T提供了依据。
The invention relates to the technical field of food safety, in particular to a hybridoma cell line and the monoclonal antibody secreted by the hybridoma cell line and its application. The hybridoma cell line of the present invention is the hybridoma cell line skTnT-2E8 of the anti-porcine skeletal muscle troponin T monoclonal antibody, which is preserved in the China Center for Type Culture Collection at the address of Wuhan University, China. The preservation date is: On March 12, 2021, the deposit number was CCTCCNO:C202158. The monoclonal antibody secreted by the hybridoma cell line of the present invention is of IgG2a subtype, the titer can reach 1:4.1×10 5 , and the affinity constant Ka=5.14×10 6 L/mol. It provides the basis for the specific detection of porcine skeletal muscle troponin T.
Description
Technical Field
The invention relates to the technical field of food safety, in particular to a hybridoma cell strain, a monoclonal antibody secreted by the hybridoma cell strain and application of the hybridoma cell strain.
Background
At present, many laboratories at home and abroad use molecular biology technical means to detect source components in meat products, and DNA detection methods are various and mainly comprise nucleic acid probe hybridization, DNA fingerprint analysis, PCR specific amplification, PCR-RFLP and the like. These methods have a certain sensitivity, but also have the disadvantages of high cost, high requirement on detection objects, large workload, incapability of on-site detection and the like, and particularly have great uncertainty on detection of cooked meat products, because the methods depend on the processing process of the cooked meat products to a great extent, and the diversity of the production process causes different levels of degradation of gene substances. Furthermore, this method only detects possible sources of animal DNA, and must be done at all times to prevent cross-contamination, and milk, blood, and fat are all possible sources of DNA. Therefore, other auxiliary methods are required to perform mutual authentication. The immunological method has the characteristics of high sensitivity, good specificity, low cost, convenient operation and the like, and is suitable for screening large-batch samples. However, in immunological analysis, it is important to prepare a monoclonal antibody with high specificity and good affinity. However, in the prior art, no report that an anti-porcine skeletal muscle troponin T monoclonal antibody with high affinity and good potency is used for the immunological analysis of porcine skeletal muscle troponin T is found.
Disclosure of Invention
The invention aims to provide a monoclonal antibody with high specificity and good affinity for use in immunological analysis of pig skeletal muscle troponin T.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a hybridoma cell strain which is a hybridoma cell strain skTnT-2E8 of a monoclonal antibody against porcine skeletal muscle troponin T and is preserved in China center for type culture collection, wherein the address is Wuhan university, Wuhan, China, the preservation date is 2021, 3 and 12 days, and the preservation number is CCTCCNO: C202158.
The invention also provides a preparation method of the hybridoma cell strain, which comprises the following steps: taking splenocytes of mice immunized by the immune antigen and SP2/0 cells according to the proportion of 4-6: 1 of the number ratio of the cells to carry out PEG fusion, and screening to obtain the hybridoma cell strain.
Preferably, the immunizing antigen is a porcine skeletal muscle troponin T antigen.
Preferably, the mice are female Balb/c mice aged 6-8 weeks.
The invention also provides the monoclonal antibody secreted by the hybridoma cell strain.
Preferably, the subtype of the antibody is IgG2 a.
The invention also provides application of the monoclonal antibody in preparation of a product for detecting the porcine skeletal muscle troponin T.
Preferably, the product is an enzyme linked immunosorbent assay kit or a colloidal gold chromatography test strip.
The invention provides a hybridoma cell strain, a monoclonal antibody secreted by the hybridoma cell strain and application of the hybridoma cell strain. The monoclonal antibody secreted by the hybridoma cell strain has high titer, high affinity and good specificity, and provides experimental basis for specific and sensitive immunoassay of porcine skeletal muscle troponin T.
Drawings
FIG. 1 is a graph showing the detection of titer of monoclonal antibodies against porcine skeletal muscle troponin T.
FIG. 2 is a graph showing the measurement of subtype of monoclonal antibody against porcine skeletal muscle troponin T.
FIG. 3 is a diagram showing the specific detection of a monoclonal antibody against porcine skeletal muscle troponin T.
Deposit description
The hybridoma cell strain skTnT-2E8 of the monoclonal antibody against the porcine skeletal muscle troponin T is preserved in China center for type culture Collection (CCTCC NO: C202158) with the preservation date of 3 months and 12 days in 2021 of university of Wuhan, China.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1 preparation of porcine skeletal muscle troponin T monoclonal antibody
And processing fresh pork leg meat to obtain the immune antigen. Female Balb/c mice of 6 weeks of age were immunized with the resulting immunizing antigen in the manner shown in Table 1. And (3) after the immunization is finished, cutting the tail and taking blood to measure the titer and the inhibition rate, and selecting the mouse with the best result for fusion. Collecting fused mouse eyeball, bleeding, taking serum as positive control, taking out spleen under aseptic condition after neck removing and killing, preparing splenocyte, performing PEG fusion with SP2/0 cell at ratio of 5:1, adding fused cell suspension into 96-well plate with laid feeder cells, placing at 37 deg.C and 5% CO2Cultured in an incubator. Checking whether pollution exists or not on the next day, changing HT culture medium on the 7 th day, continuing to culture for 2d, screening positive holes by indirect ELISA, selecting strong positive holes, single clone holes and holes with good cell state as much as possible, performing subcloning by a limiting dilution method, and simultaneously performing amplification culture and cryopreservation until a single monoclonal cell strain secreting the antibody is established. Then adopting a method of inducing ascites in a mouse to prepare a large amount of porcine skeletal muscle troponin T monoclonal antibodies.
TABLE 1 immunization protocol
| Time of immunization | Number of immunizations | Immune site | Immunization dose | Remarks for |
| 0 | First immunization | Injection for neck and back | 40 mu g/ |
|
| 2 weeks | Second immunization | Injection for neck and back | 40 mu g/ |
|
| 4 weeks | Third immunization | Injection for neck and back | 40 mu g/body | Determination of potency |
| 6 weeks | Boosting immunity | Injection for neck and back | 20 mu g/body |
Example 2 porcine skeletal muscle troponin T monoclonal antibody potency assay
The titer of the porcine skeletal muscle troponin T monoclonal antibody is determined by adopting an indirect ELISA method. The specific method comprises the following steps: coating: the coated antigen was diluted with carbonate buffer to a concentration of 2. mu.g/mL and coated on a 96-well plate at 100. mu.L/well overnight at 4 ℃. Washing: returning the coated plate to room temperature, pouring off the coating solution, adding 300 μ L of washing solution into each well, standing for L min each time, washing for 3 times, and finally patting dry. And (3) sealing: adding 200 μ L of lotion containing 10% calf serum into each well, and sealing at 37 deg.C for 1 hr; the blocking solution was decanted, washed 3 times and patted dry. Adding a primary antibody: adding 100 mu L of pig skeletal muscle troponin T monoclonal antibody with a certain dilution factor into each hole, taking 100 mu L of PBS solution in each hole as a blank control hole, taking 100 mu L of negative serum in each hole as a negative control, and standing at 37 ℃ for 45 min; pouring out the liquid, washing for 3 times, and patting dry; the pig skeletal muscle troponin T monoclonal antibody with a certain dilution multiple is a monoclonal antibody diluted by a multiple ratio from 1:2000 times. Adding an enzyme-labeled secondary antibody: adding 100 μ L of HRP enzyme-labeled goat anti-mouse IgG diluted by 10000 times into each well, and standing at 37 deg.C for 30 min; the liquid was decanted, washed 3 times and patted dry. Color development: adding 100 μ L of substrate developing solution into each well, and reacting at 37 deg.C in dark for 15 min. And (4) terminating: the reaction was stopped by adding 50. mu.L of stop solution to each well. And (3) detection: the absorbance value at a wavelength of 450nm was measured (A450 nm).
And (4) judging a result: and (A sample well-A blank)/(A negative control-A blank) is not less than 2.1, and when A450nm of the negative control is less than 0.2, the dilution factor of the antibody is the antibody titer. The results are shown in FIG. 1.
Through detection, the titer of the monoclonal antibody against the porcine skeletal muscle troponin T at the concentration of 1mg/ml reaches 1: 4.1X 105。
Example 2 porcine skeletal muscle troponin T monoclonal antibody subtype assay
The monoclonal antibody prepared in example 1 was subjected to subtype determination using a murine monoclonal antibody subtype identification kit purchased from Sigma. The results are shown in FIG. 2.
The results show that: the porcine skeletal muscle troponin T monoclonal antibody has obvious difference with secondary antibodies of different subclasses, wherein the A450nm value of the secondary antibody with IgG2a is the highest, the secondary antibody with IgG3 has weak color development, and the secondary antibody with IgG2a, IgG2b, IgG3 and IgA hardly develops color. The subtype of the porcine skeletal muscle troponin T monoclonal antibody is IgG2 a.
Example 3 affinity assay for porcine skeletal muscle troponin T monoclonal antibody
The pig skeletal muscle troponin T monoclonal antibody obtained in example 1 was measured for affinity constant (Ka) by a non-competitive ELISA method. The method comprises the following specific steps: coating: diluting the coated antigen with carbonate buffer solution to the concentration of 1, 0.5, 0.25 and 0.125. mu.g/mL, adding the diluted coated antigen into a 96-well enzyme label plate according to the amount of 100. mu.L/well, and coating at 4 ℃ overnight; the coating solution was decanted, washed 3 times and patted dry. And (3) sealing: adding 200 μ L of lotion containing 10% calf serum into each well, and keeping the temperature at 37 deg.C for 1 h; the blocking solution was decanted, washed 3 times and patted dry. Adding a monoclonal antibody: adding pig skeletal muscle troponin T monoclonal antibody with a certain dilution ratio according to the amount of 100 mu L/hole, reacting at 37 ℃ for 45min, then pouring off the liquid, washing for 3 times, and patting dry; the pig skeletal muscle troponin T monoclonal antibody with a certain dilution multiple is a monoclonal antibody diluted by a starting multiple ratio of 100 mu g/mL. Adding an enzyme-labeled secondary antibody: adding 100uL of goat anti-mouse IgG labeled with HRP enzyme and diluted by 10000 times into each hole, and standing at 37 ℃ for 30 min; the liquid was decanted, washed 3 times and patted dry. Color development: adding 100 μ L of substrate developing solution into each well, and reacting at 37 deg.C in dark for 15 min. And (4) terminating: the reaction was stopped by adding 50. mu.L of stop solution to each well. And (3) detection: the absorbance value at a wavelength of 450nm was measured (A450 nm).
The formula for the calculation of the affinity Ka is:
Ka＝(n-1)/2(nAb’-Ab)
in the formula: ab is the antibody concentration which generates a half-absorbance value when the antigen concentration is a; ab' is the antibody concentration which generates a half-absorbance value when the antigen concentration is b; n is the dilution factor between a and b.
The pig skeletal muscle troponin T monoclonal antibody affinity constant Ka is 5.14 multiplied by 106L/mol。
Example 4 porcine skeletal muscle troponin T monoclonal antibody specificity assay
The specificity of the porcine skeletal muscle troponin T monoclonal antibody prepared in example 1 was determined by an indirect competitive ELISA method. The method comprises the following specific steps: coating: coating the skeletal muscle troponin antigen of cattle, sheep, chicken, duck and pig with carbonate buffer solution at concentration of 2 μ g/mL and coating amount of 100 μ L/well at 4 deg.C overnight; the coating solution was decanted, washed 3 times and patted dry. And (3) sealing: adding 200 μ L of lotion containing 10% calf serum into each well, and keeping the temperature at 37 deg.C for 1 h; the blocking solution was decanted, washed 3 times and patted dry. Adding a primary antibody: adding pig skeletal muscle troponin T monoclonal antibody with a certain dilution ratio according to the amount of 100 mu L/hole, and standing at 37 ℃ for 45 min; pouring out the liquid, washing for 3 times, and patting dry; adding 100 mu L/hole PBS into each hole as a blank control, and taking negative serum as a negative control; the pig skeletal muscle troponin T monoclonal antibody with a certain dilution multiple is a pig skeletal muscle troponin T monoclonal antibody diluted by a ratio of 1:2000 times. Adding an enzyme-labeled secondary antibody: adding 100 μ L of HRP enzyme-labeled goat anti-mouse IgG diluted by 10000 times into each well, and standing at 37 deg.C for 30 min; the liquid was decanted, washed 3 times and patted dry. Color development: adding 100 μ L of substrate developing solution into each well, and reacting at 37 deg.C in dark for 15 min. And (4) terminating: the reaction was stopped by adding 50. mu.L of stop solution to each well. And (3) detection: the absorbance value at a wavelength of 450nm was measured (A450 nm).
And (4) judging a result: and (A sample well-A blank)/(A negative control-A blank) is not less than 2.1, and when A450nm of the negative control is less than 0.2, the dilution factor of the antibody is the antibody titer. The results are shown in FIG. 3.
Through detection, the porcine skeletal muscle troponin T monoclonal antibody has the antigen titer of 1:40 ten thousand with porcine skeletal muscle troponin, the titer of chicken and duck skeletal muscle troponin is lower than 1:10 ten thousand, and the titers of cattle and sheep skeletal muscle troponin are respectively 1:6 thousand and 1:2 ten thousand. The specificity of the pig skeletal muscle troponin T monoclonal antibody obtained by the invention is good.
The embodiments show that the invention provides a hybridoma cell strain, a monoclonal antibody secreted by the hybridoma cell strain and application of the hybridoma cell strain. The monoclonal antibody secreted by the hybridoma cell strain has high titer, high affinity and good specificity, and provides experimental basis for specific and sensitive immunoassay of porcine skeletal muscle troponin T.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118406658A (en) * | 2023-06-27 | 2024-07-30 | 河北交通职业技术学院 | Hybridoma cell strain, anti-4-chlorophenoxyacetic acid monoclonal antibody produced by hybridoma cell strain and application of antibody |
| WO2025133083A1 (en) * | 2023-12-22 | 2025-06-26 | F. Hoffmann-La Roche Ag | Antibody against skeletal troponin t and diagnostic uses thereof |
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| US6333397B1 (en) * | 1989-04-25 | 2001-12-25 | Roche Diagnostics Gmbh | Monoclonal antibodies to troponin T and their production |
| US6376206B1 (en) * | 1989-04-25 | 2002-04-23 | Roche Diagnostics Gmbh | Specific antibodies to troponin T, their production and use in a reagent for the determination of myocardial necrosis |
| CN109705216A (en) * | 2018-12-28 | 2019-05-03 | 河北省科学院生物研究所 | A kind of anti-bovine skeletal muscle troponin I monoclonal antibody and its application |
| CN109810191A (en) * | 2018-12-28 | 2019-05-28 | 河北省科学院生物研究所 | A kind of anti-sheep skeletal muscle troponin I monoclonal antibody and its application |
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| US6333397B1 (en) * | 1989-04-25 | 2001-12-25 | Roche Diagnostics Gmbh | Monoclonal antibodies to troponin T and their production |
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| Title |
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| A.G. KATRUKHA ET.AL: "Isolation of human cardiac troponin T and localization of epitopes recognized by monoclonal antibodies to cardiac troponin T", 《FEDERATION OF EUROPEAN BIOCHEMICAL SOCIETIES》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118406658A (en) * | 2023-06-27 | 2024-07-30 | 河北交通职业技术学院 | Hybridoma cell strain, anti-4-chlorophenoxyacetic acid monoclonal antibody produced by hybridoma cell strain and application of antibody |
| WO2025133083A1 (en) * | 2023-12-22 | 2025-06-26 | F. Hoffmann-La Roche Ag | Antibody against skeletal troponin t and diagnostic uses thereof |
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