Dihydropyridine monoclonal antibody hybridoma cell strain and application thereof
A hybridoma cell line, monoclonal antibody technology, applied in the direction of animal/human protein, serum albumin, ovalbumin, etc., can solve the problem of lack of detection methods, and achieve the effect of good detection sensitivity and good specificity
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Embodiment 1
[0021] Embodiment 1 Preparation of hybridoma cell line ABC02
[0022] (1) Preparation of complete antigen:
[0023] a. The hapten synthesis route is as follows:
[0024] ;
[0025] Add 570 mg of dihydropyridine to 600 mg of succinic anhydride dissolved in anhydrous pyridine (5 mL), and react in an oil bath at 60°C for 6 h. Cool to room temperature after stopping heating, transfer the reactant to 20mL of 10% hydrochloric acid placed in an ice-water bath, a large amount of white precipitate precipitates, wash with deionized water several times, and dry to obtain a white solid, which is dihydro Pyridine hapten.
[0026] b. Weigh 1.46 mg of hapten and dissolve it in 600 μL of DMF, then add 2.62 mg of EDC and 1.52 mg of NHS, and stir the mixture at room temperature for 6 h to obtain reaction solution A1; weigh 5 mg of BSA, dissolve in 0.1 M borate buffer solution to obtain solution B1; then, add solution A1 to solution B1 dropwise, and react at room temperature for 8 h to obt...
Embodiment 2
[0036] Example 2 IC of dihydropyridine monoclonal antibody 50 Determination of
[0037] Carbonate buffer solution (CBS): Weigh out Na 2 CO 3 1.59 g, NaHCO 3 2.93 g, respectively dissolved in a small amount of double-distilled water and mixed, added double-distilled water to about 800 mL, mixed evenly, adjusted the pH value to 9.6, added double-distilled water to make up to 1000 mL, and stored at 4°C for later use;
[0038] 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 800 mL of pure water, adjust the pH to 7.2-7.4 with NaOH or HCl, and dilute to 1000 mL;
[0039] Washing solution (PBST): add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L PBS solution with pH7.4;
[0040] PBST: PBS containing 0.05% Tween-20;
[0041] Antibody diluent: wash buffer containing 0.1% gelatin;
[0042] 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 wate...
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