Sirolimus monoclonal antibody hybridoma cell strain and application thereof
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A hybridoma cell line and antibody cloning technology, which is applied in the field of immunoassay, can solve the problems of rapid detection, cumbersome steps, and high cost, and achieve good specificity and good detection sensitivity
Inactive Publication Date: 2020-12-01
JIANGNAN UNIV
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[0003] Sirolimus content analysis methods include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and other instrumental methods. These detection methods have t...
Method used
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Embodiment 1
[0022] Example 1 Preparation of hybridoma cell line ROX1C5
[0023] (1) Preparation of complete antigen:
[0024] a. The hapten synthesis route is as follows:
[0025]
[0026] Weigh 19 mg of sirolimus, 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 for 6 h , and stand overnight at room temperature 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 blown dry with nitrogen again, and the final product was dissolved in 1 mL of anhydrous N,N-dimethylformamide (DMF), namely the sirolimus hapten.
[0027] b. Preparation of immunogen Sirolimus-KLH: Weigh 10 mg of hapten and dissolve it in 800 μL DMF, then add 6.3 mg 1-ethylcarbodiimide hydrochloride (EDC), 5 mg N-hydroxy succinimide (NHS), the mixture was stirred at room temperatu...
Embodiment 2
[0037] Example 2 IC of sirolimus 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 well, adjust the pH value to 9.6, add double distilled water to make up to 1000mL, store at 4°C for later use;
[0039] Phosphate buffered saline (PBS): 8.0g NaCl, 0.2g KCl, 0.2g KH 2 PO 4 , 2.9 g Na 2 HPO 4 • 12 H 2 O, dissolve in 800 mL of pure water, adjust the pH to 7.2-7.4 with NaOH or HCl, and set the volume to 1000 mL;
[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 18.43g, citric acid 9.33g, dilute to 1000 mL with pure water; solution B: dis...
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Abstract
The invention discloses a sirolimus monoclonal antibody hybridoma cell strain and application thereof, and belongs to the technical field of immunodetection. The invention discloses a sirolimus monoclonal antibody hybridoma cell strain ROX1C5, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC) and is classified and named as the monoclonal cell strain, the preservation date is November 28, 2019, and the preservation number is CGMCC No.19171. Sirolimus hapten is synthesized through oximation reaction, sirolimus complete antigen is prepared, the sirolimus complete antigen and an equal amount of Freund's adjuvant are mixed and emulsified completely, and BALB/c mice are immunized through back subcutaneous multipoint injection; high-titer and low-IC50 mouse splenocytes are selected to be fused with myeloma cells, screening is conducted through an indirect competitive enzyme-linked immunosorbent assay, and subcloning is conducted three times so as to obtain the cell strain. The monoclonal antibody secreted by the cell strain has good specificity and detection sensitivity (IC50 is 3.90 ng/mL) on sirolimus, is used for establishing an immunodetectionmethod of sirolimus content, and has practical application value.
Description
technical field [0001] The invention relates to a sirolimus monoclonal antibody hybridoma cell line and application thereof, and belongs to the technical field of immune detection. Background technique [0002] Sirolimus, also known as rapamycin, is a low-toxic and potent macrolide immunosuppressant. It is a white crystalline solid with good fat solubility and almost insoluble in water. First discovered in 1975 from the soil of Easter Island, Chile. Sirolimus is an inhibitor of T cell activation and proliferation. Its mechanism of action is different from other immunosuppressants, and its immunosuppressive activity is superior to that of tacrolimus and cyclosporine. It can treat and reverse developing acute rejection , and prevent chronic rejection. However, sirolimus has the characteristics of long half-life, narrow therapeutic window, and large difference in pharmacokinetics. Therefore, it is necessary to monitor the blood drug concentration within the target concentrati...
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