Vomitoxin hybridoma, monoclonal antibody, and preparation method and application of monoclonal antibody

A technology of monoclonal antibody and DON, applied in biochemical equipment and methods, microorganism-based methods, anti-fungal/algae/lichen immunoglobulin, etc., can solve the problems of high harm, poor accuracy, and cumbersome processing for experimenters , to achieve the effect of easy popularization and application, high specificity and high sensitivity

Inactive Publication Date: 2013-03-06
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection methods of vomitoxin include high performance liquid chromatography, gas chromatography, thin layer chromatography, etc., the equipment is expensive, the technical level is high, and the samples need to be purified, which i...

Method used

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  • Vomitoxin hybridoma, monoclonal antibody, and preparation method and application of monoclonal antibody
  • Vomitoxin hybridoma, monoclonal antibody, and preparation method and application of monoclonal antibody
  • Vomitoxin hybridoma, monoclonal antibody, and preparation method and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Deoxynivalenol complete antigen synthesis

[0018] (1) Deoxynivalenol hapten synthesis (synthetic technical route see figure 2 )

[0019] 20-100mg vomitoxin (purchased from Shanghai AMP Scientific Instrument Co., Ltd., article number CDFV-ALX-630-115-M001), with 1-3mol maleic anhydride, catalytic amount of DMAP, in 2-5ml toluene, at room temperature Stir and react at 80°C. After 8-20 hours, evaporate the solvent to dryness, acidify, extract, and recrystallize to obtain the vomitoxin hapten.

[0020] (2) Deoxynivalenol immune antigen synthesis

[0021] The immunogen was obtained by coupling the deoxynivalenol hapten with bovine serum albumin.

[0022] Dissolve 60mg of bovine serum albumin in 9.5ml of PBS (0.01mol / L, pH5.0) solution, add 12.5mg of EDC, add 10mg of vomitoxin hapten dissolved in 1ml of DMF, stir at room temperature for 1 hour, add 6mg of EDC, and place in the dark Stir the reaction overnight, dialyze with 0.01mol / LPBS for 3 days, change the d...

Embodiment 2

[0026] Example 2 Preparation of Deoxynivalenol Monoclonal Antibody

[0027] Mix the immune antigen obtained above with Freund's adjuvant (purchased from Sigma, product number F5881) at a ratio of 1:3, and immunize Balb / c mice aged 8-10 weeks, and inject subcutaneously at multiple points on the back of the neck. 150 μg / rat, two weeks after the second immunization, Freund's incomplete adjuvant (purchased from Sigma, product number F5506) was used, and the immunization dose was the same as above; 28 days later, the third immunization was used, Freund's incomplete adjuvant, and the immunization dose was the same as above, 31 Immunization was boosted two days later, and the immunogen was used directly for immunization.

[0028] Three days after the booster immunization, one immunized Balb / c mouse was taken, sacrificed by dislocation after orbital blood collection, the spleen was sterilized in 75% alcohol, and the spleen cell suspension was prepared, transferred to a 50ml centrifuge...

Embodiment 3

[0034] Example 3 Establishment of indirect ELISA method

[0035] (1) Antigen antibody titer determination

[0036] Dilute the vomitoxin-ovalbumin-coated antigen with PBS (pH7.4, 0.01mol / L) at 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, and pack at 100 μl per well Incubate at 37°C for 2 hours, pour off the coating solution, wash twice with PBST, add 200 μl of blocking solution to each well, incubate at 37°C for 2 hours, pour off the liquid in the well, and set aside.

[0037] Add 50 μl / well of deoxynivalenol standard solution to the wells of the plate, and then add goat anti-mouse enzyme-labeled secondary antibody (purchased from Beijing Jibiai Biotechnology Co., Ltd.) diluted 1:4000 with PBS (pH7. Company) 50 μl / well, and then add the deoxynivalenol monoclonal diluted with PBS (pH7.4, 0.01mol / L) 1:3000, 1:6000, 1:12000, 1:24000, 1:48000, 1:96000 Antibody 50μl / well, react at 25°C for 30min, wash the plate 3 times with PBST, and pat dry.

[0038] After adding the chromogenic subs...

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Abstract

The invention discloses vomitoxin hybridoma, a monoclonal antibody, and a preparation method and application of the monoclonal antibody. The preparation method comprises the steps of: immunizing a Balb/c mouse with a vomitoxin and bovine serum albumin conjugate as an immunizing antigen; preparing a hybridoma by the spleen cells and the myeloma cell SP2/0 of the mouse; obtaining a monoclonal antibody hybridoma strain D-2-1CGMCC No. 5161 through an indirect ELISA (Enzyme-Linked Immunosorbent Assay) screening and a limiting dilution method; and injecting the hybridoma strain into the abdominal cavity of the mouse to produce a monoclonal antibody, wherein the monoclonal antibody is used for indirect competitive ELISA testing of the vomitoxin. The monoclonal antibody is capable of specifically detecting the vomitoxin, and high in sensitivity; and when an ELISA kit prepared by the monoclonal antibody is used for detecting the vomitoxin, the cost is low, large equipment is not needed, the operation is simple and convenient, and the preparation method is convenient for popularization and utilization.

Description

technical field [0001] The invention relates to a hybridoma, a monoclonal antibody and its preparation and use, in particular to a deoxynivalenol hybridoma, a deoxynivalenol monoclonal antibody and a preparation method and use thereof. technical background [0002] Deoxynivalenol (chemical structure see figure 1 ), chemically known as deoxyfusarium enol, belongs to the trichothecene family of molds, has high cytotoxicity and immunosuppressive properties, Fusarium graminearum, Fusarium pseudocladide, Fusarium pirosporum and other Fusarium Bacterial strains and strains of the genus Cephalosporin, Lacternium, and Trichoderma can all produce the toxin, and the pollution of grains and cereals by vomitoxin is very common. DON can cause severe vomiting reactions in ducklings, pigs, cats, dogs and other animals, and severe cases can cause death. Animals with acute poisoning are mainly manifested as unsteady standing, unresponsiveness, piloerection, loss of appetite, vomiting, etc....

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/14G01N33/577C12R1/91
Inventor 何方洋韩黎吴鹏冯才伟罗贵昆韩雪琳孙震赵正苗李勇陈勇冯才茂罗晓琴
Owner BEIJING KWINBON BIOTECH
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