Tacrolimus monoclonal antibody hybridoma cell strain and application thereof
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A hybridoma cell line and monoclonal antibody technology, which is applied in the field of immunoassay, can solve the problems of cumbersome steps, rapid detection, and high cost, and achieve good detection sensitivity and specificity
Inactive Publication Date: 2020-12-04
JIANGNAN UNIV
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[0003] Tacrolimus content analysis methods include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and other instrumental methods. These detection methods have the disadvantages of time-consuming, cumbersome steps, inability to perform rapid on-site detection, and high cost. Therefore, it is of great significance to establish a rapid and simple detection method for tacrolimus.
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Embodiment 1
[0022] Example 1 Preparation of hybridoma cell line ICP2B3
[0023] (1) Preparation of complete antigen:
[0024] a. The synthetic route of hapten is as follows:
[0025]
[0026] Weigh 19 mg of tacrolimus, dissolve it in 0.6 mL of methanol:water:pyridine (volume ratio 4:1:1), add 13 mg of carboxymethoxylaminehemihydrochloride (CMO), and stir in a water bath at 70°C 6 h, and stand at room temperature overnight in the dark. Subsequently, the solution was dried by nitrogen gas, resuspended in 1 mL of chloroform, and extracted three times with ultrapure water. The organic phase was collected and dried again with nitrogen gas, and the final product was dissolved in 1 mL of anhydrous N,N-dimethylformamide (DMF), namely the tacrolimus hapten.
[0027] b. Weigh 10 mg of tacrolimus hapten and dissolve it in 800 μL DMF, then add 6.3 mg 1-ethylcarbodiimide hydrochloride (EDC), 5 mg N-hydroxysuccinimide (NHS), the mixture was stirred at room temperature for 6 h; 5 mg keyhole limpe...
Embodiment 2
[0037] Example 2 IC of tacrolimus monoclonal antibody 50 Determination of
[0038] Carbonate buffer solution (CBS): Weigh out Na 2 CO 3 1.59g, NaHCO 3 2.93g, respectively dissolved in a small amount of double distilled water and mixed, add double distilled water to about 800mL and mix evenly, adjust the pH value to 9.6, add double distilled water to make up to 1000 mL, store at 4°C for later use;
[0039] Phosphate buffered saline (PBS): 8.0 g NaCl, 0.2 g KCl, 0.2 g KH 2 PO 4 , 2.9 g Na 2 HPO 4 • 12 H 2 O, dissolve in 800mL pure water, adjust the pH to 7.2-7.4 with NaOH or HCl, and set the volume to 1000mL;
[0040] Washing solution (PBST): add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L PBS solution with pH 7.4;
[0043] TMB Chromogenic Solution: Solution A: Na 2 HPO 4 •12H 2 O 18.43 g, citric acid 9.33 g, dilute to 1000 mL with pure water; solution B:...
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Abstract
The present invention discloses a tacrolimus monoclonal antibody hybridoma cell strain and an application thereof, and belongs to the technical field of immunodetection. The tacrolimus monoclonal antibody hybridoma cell strain ICP2B3 is classified and named as a monoclonal cell strain, the preservation date is November 28, 2019, and the preservation number is CGMCC No.19172. Hapten is synthesizedthrough tacrolimus oximation, and tacrolimus complete antigen is prepared and completely mixed with an equal amount of Freund's adjuvant for complete emulsifying. By immunizing BALB / c mice through back subcutaneous multipoint injection, high-titer and low-IC50 mouse spleen cells are selected to fuse with myeloma cells, screening is conducted through an indirect competitive enzyme-linked immunosorbent assay, subcloning for three times is also conducted, the monoclonal antibody secreted by the cell strain has relatively good specificity and detection sensitivity on tacrolimus (IC50 is 0.996 ng / mL) . The result can be used for establishing an immunodetection method for tacrolimus content in a clinical blood sample and has practical application value.
Description
technical field [0001] The invention relates to a tacrolimus monoclonal antibody hybridoma cell line and application thereof, belonging to the technical field of immunoassay. Background technique [0002] Tacrolimus is a macrolide immunosuppressant isolated from Streptomyces. It belongs to calcineurin inhibitors and is mostly used in the clinical treatment of organ transplantation and nephrotic syndrome. Tacrolimus mainly binds to the protein FKBP-12 to form a drug-protein complex, inhibits the expression of interleukin-2 (IL-2), and inhibits the function of T lymphocytes. Although compared with cyclosporine, tacrolimus has stronger immunosuppressive intensity and less clinical adverse reactions, but it still has low bioavailability, many influencing factors, and neurotoxicity, nephrotoxicity, hematological toxicity, etc. Adverse reactions. In addition, tacrolimus has a narrow therapeutic window and a large difference in pharmacokinetics. A low concentration of tacrolimus...
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