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

Inactive Publication Date: 2021-02-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For veterinary drug dihydropyridine residues, there is currently a lack of detection methods for its residues. In order

Method used

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  • Dihydropyridine monoclonal antibody hybridoma cell strain and application thereof

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Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a dihydropyridine monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunodetection. The dihydropyridine monoclonal antibody hybridoma cell strain ABC02 is preserved in China General Microbiological Culture Collection Center (CGMCC), is classified and named as a monoclonal cell strain, and is preserved on November 28,2019 with the preservation number being CGMCC No. 19177. A dihydropyridine hapten is synthesized, then a dihydropyridine complete antigen is prepared, and is mixed with an equivalent Freund's adjuvant for thorough emulsification, a BALB/c mouse is immunized through subcutaneous injection on the back, fusion with myeloma cells is realized through a PEG method, and the hybridoma cell strain ABC02 is obtained through screening of indirect competitive enzyme-linked immunosorbent assay and three times of subcloning. The monoclonal antibody secreted by the cell strain has good specificity and detection sensitivity (IC50 is 0.35 ng/mL) to dihydropyridine, can be prepared into a kit for detecting dihydropyridine, and has practical application value.

Description

technical field [0001] The invention relates to a dihydropyridine monoclonal antibody hybridoma cell line and an application thereof, belonging to the field of food safety immunoassay. Background technique [0002] Dihydropyridine is an analgesic chemical stabilizer, mainly used for sedation and analgesia in horses, cattle, sheep and other animals. Studies have shown that residues of dihydropyridine in food-derived animal tissues pose a great health risk to consumers, and long-term use of animal-derived foods containing residues of dihydropyridine may lead to drug resistance. Therefore, establishing an effective detection method for dihydropyridine residues has far-reaching significance for effectively controlling the residues of dihydropyridine in milk products and ensuring the health of the general public. [0003] For veterinary drug dihydropyridine residues, there is currently a lack of detection methods for its residues. In order to protect the interests of consumers, ...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44C07K14/765C07K14/77C07D211/90G01N33/577C12R1/91
CPCC07D211/90C07K14/765C07K14/77C07K16/44G01N33/577G01N33/948G01N33/9486
Inventor 胥传来晁梦佳匡华徐丽广孙茂忠刘丽强吴晓玲宋珊珊胡拥明郝昌龙
Owner JIANGNAN UNIV
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