Hybridoma cell strain capable of secreting vitamin B6 monoclonal antibody and application thereof

A hybridoma cell line and monoclonal antibody technology, applied in the direction of microorganisms, analytical materials, biological testing, etc., can solve the problems of long incubation time, step restrictions, etc., and achieve the effect of good detection sensitivity

Active Publication Date: 2021-04-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the high sensitivity and specificity of microbiological methods, their long incubation times (typically 20-24 hours) and complicated steps limit the rapid detection of target species

Method used

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  • Hybridoma cell strain capable of secreting vitamin B6 monoclonal antibody and application thereof
  • Hybridoma cell strain capable of secreting vitamin B6 monoclonal antibody and application thereof
  • Hybridoma cell strain capable of secreting vitamin B6 monoclonal antibody and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation of embodiment 1 hybridoma cell line MTB

[0039] (1) Preparation of complete antigen:

[0040] Vitamin B 6 Hapten preparation:

[0041] The hapten synthesis route is as follows:

[0042]

[0043] a. Compound 2: Compound 1 (20.0 g, 118 mmol) and 2,2-dimethoxypropane (200 mL) were dissolved in 200 mL of acetone, then p-toluenesulfonic acid (81.4 g, 473 mmol) was added, and stirred overnight at 130°C. The reaction mixture was cooled and poured into ice water, the aqueous layer was extracted with dichloromethane, the organic phases were combined, washed with brine, washed with Na 2 SO 4 After drying and concentration, the desired product was recrystallized from ether, and the mixture was filtered to obtain compound 2 (13.0 g) as a white solid.

[0044] b. Compound 3: under nitrogen, NaH solution (6.88g, 287mmol, 60% oil content) was added to 70.0mL THF at 0°C, and compound 2 (15.0 g, 71.7 mmol). The resulting mixture was refluxed for 30 min, during...

Embodiment 2

[0064] Embodiment 2 vitamin B 6 IC of monoclonal antibodies 50 Determination of

[0065] Carbonate buffer solution (CBS): weigh 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;

[0066] Phosphate buffered saline (PBS): 8.0g NaCl, 0.2g KCl, 0.2g KH 2 PO 4 , 2.9 g Na 2 HPO 4 12H 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;

[0067] Washing solution (PBST): 0.5 mL of Tween-20 was added to 1000 mL of 0.01 mol / L PBS solution with pH 7.4;

[0068] PBST: PBS containing 0.05% Tween–20;

[0069] Antibody diluent: wash buffer containing 0.1% gelatin;

[0070] TMB Chromogenic Solution: Solution A: Na 2 HPO 4 12H 2 O 18.43g, citric acid 9.33g, dilute to 1000mL with pure water; solution B: dis...

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Abstract

The invention discloses a hybridoma cell strain capable of secreting a vitamin B6 monoclonal antibody and application thereof, and belongs to the technical field of food safety immunodetection. The hybridoma cell strain MTB capable of secreting the vitamin B6 monoclonal antibody is preserved in China General Microbiological Culture Collection Center (CGMCC), is classified and named as a monoclonal cell strain, is preserved on November 28, 2019, and has a preservation number of CGMCC No.19183. Firstly, a vitamin B6 hapten is synthesized, then a vitamin B6 complete antigen is prepared, a BALB / c mouse is immunized, high-titer and low-IC50 mouse splenocytes are taken and fused with myeloma cells through a PEG method, and the hybridoma cell strain MTB is obtained through indirect competitive enzyme-linked immunosorbent assay screening and three times of subcloning. The monoclonal antibody secreted by the cell strain has good specificity and detection sensitivity to vitamin B6.

Description

technical field [0001] The present invention relates to a strain that secretes vitamin B 6 The invention relates to a monoclonal antibody hybridoma cell line and an application thereof, belonging to the technical field of food safety immunoassay. Background technique [0002] Vitamin B 6 Represents a group of highly bioavailable 3-hydroxy-2-picoline derivatives, including pyridoxine, pyridoxal, pyridoxamine and their phosphorylated forms. Vitamin B 6 Involved in various enzymatic reactions in amino acid, carbohydrate and lipid metabolism. In addition vitamin B 6 Plays an important role in nervous system function, hormone function, hemoglobin synthesis. Interestingly, it was recently discovered that vitamin B 6 Conjugates as Potential Drug Carriers in Targeted Synergistic Cancer Therapy Humans Cannot Synthesize Vitamin B in Vivo 6 . Therefore, obtain B vitamins only from diet, supplements and gut flora 6 . Vitamin B 6 Found in a variety of foods, including poultry,...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44C07K14/795C07K14/77C07D213/65G01N33/82G01N33/577C12R1/91
Inventor 宋珊珊胥传来曾露匡华徐丽广孙茂忠刘丽强吴晓玲胡拥明郝昌龙
Owner JIANGNAN UNIV
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