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Aflatoxin sterigmatocystin hybridoma cell strain, antibody, immunoadsorbent, immunoaffinity column, kit and applications of immunoadsorbent, immunoaffinity column and kit

A hybridoma cell line and aflatoxin technology, applied in the field of immunoaffinity columns and kits, to achieve stable performance

Inactive Publication Date: 2013-06-19
北京中检维康技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The hybridoma cell line was deposited in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microorganisms on November 24, 2011

Method used

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  • Aflatoxin sterigmatocystin hybridoma cell strain, antibody, immunoadsorbent, immunoaffinity column, kit and applications of immunoadsorbent, immunoaffinity column and kit
  • Aflatoxin sterigmatocystin hybridoma cell strain, antibody, immunoadsorbent, immunoaffinity column, kit and applications of immunoadsorbent, immunoaffinity column and kit
  • Aflatoxin sterigmatocystin hybridoma cell strain, antibody, immunoadsorbent, immunoaffinity column, kit and applications of immunoadsorbent, immunoaffinity column and kit

Examples

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preparation example Construction

[0024] Specifically, the preparation method of hybridoma cell line and monoclonal antibody in the present invention may comprise the following steps:

[0025] (1) Preparation of aflatoxin immunogen

[0026] 4 mg aflatoxin B 1 Dissolve in 2 mL of acetone, add 40 μL of 10% HO 2 SO 4 , Stir the reaction at 56°C for 4h, evaporate the reaction product to dryness, add 5mL H 2 O, extracted twice with 25 mL chloroform, then with 20 mL H 2 O washed the organic layer, retained the organic layer, and evaporated the organic solvent to obtain a yellow solid product.

[0027] Take 1.0 mg of the yellow solid product, add 2 mL of 0.5% BSA solution (dissolve 20 mg of BSA in 4 mL of PBS), react at 37 ° C for 30 min; add 100 μL of 6.5 mM NaHB 4 , react at 4°C for 30min; add 50μL of 0.1N HCl to remove excess NaHB 4 . At 4°C, dialyze with PBS solution for 3 days to obtain the immunogen, aflatoxin B 1 -BSA (AflatoxinB 1 -BSA).

[0028] (2) Preparation of hybridoma cells and monoclonal ant...

Embodiment 1

[0069] This example is used to prepare aflatoxin immunogen: aflatoxin B 1 -BSA (Aflatoxin B 1 -BSA)

[0070] 4 mg aflatoxin B 1 Dissolve in 2 mL of acetone, add 40 μL of 10% HO 2 SO 4 , Stir the reaction at 56°C for 4h; evaporate the reaction product to dryness, add 5mL H 2 O, extracted twice with 25 mL chloroform, then with 20 mL H 2 The organic layer was washed with O, and the organic layer was retained; the organic solvent was evaporated to obtain a yellow solid product.

[0071] Take 1.0 mg of the yellow solid product, add 2 mL of 0.5% BSA solution (20 mg of BSA dissolved in 4 mL of PBS) to it, react at 37° C. for 30 min; add 100 μL of 6.5 mM NaHB 4 , react at 4°C for 30 min; add 50 μL of 0.1N HCl to remove excess NaHB 4 . Dialyze with PBS solution for 3 days at 4°C. aflatoxin B 1 -SA.

Embodiment 2

[0073] This embodiment is used for preparing anti-aflatoxin B 1 , B 2 , G 1 , G 2 , M 1 , M 2 and Aspergillus versicolor monoclonal antibodies

[0074] Animal immunization: The immunized animals are about 6-8 weeks old, female BALB / c mice. with immunogen, aflatoxin B 1 -BSA immunization of 3 mice. Take an appropriate amount of immunogen (100 μg / rat) and add an equal amount of Freund's complete adjuvant to make an emulsifier for immunization, and then change the adjuvant to incomplete adjuvant for 6 times of immunization, with an interval of 2 weeks between each time. Except for the first multi-point subcutaneous injection on the back of the neck, the rest were intraperitoneal injections. After the immunization, the mice were sacrificed, and splenocytes were collected.

[0075] Cell fusion: Splenocytes and hybridoma cells were subjected to cell fusion experiments at a ratio of 10:1.

[0076] Hybridoma cell cloning: screening of hybridoma cells by limiting dilution met...

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Abstract

The invention discloses a hybridoma cell strain CGMCC NO. 5506 and a monoclonal antibody obtained by secretion of the cell strain. The invention also provides an immunoadsorbent comprising a solid phase vector and an antibody coupled with the solid phase vector, wherein the antibody is the above monoclonal antibody; and an immunoaffinity column loaded with the immunoadsorbent. The invention also provides a kit containing the above immunoadsorbent or the above immunoaffinity column, and applications of the above immunoadsorbent, the above immunoaffinity column and the kit in detecting aflatoxins and sterigmatocystins. Besides, the invention provides a separation method and a detection method specifically. The monoclonal antibody with stable and specific performance is developed by the invention, so that purification and detection for six aflatoxins and sterigmatocystins at the same time are realized.

Description

technical field [0001] The present invention relates to a hybridoma cell line, a monoclonal antibody secreted by the hybridoma cell line, an immunoadsorbent prepared from the antibody, an immunoaffinity column and a test kit equipped with the immunoadsorbent, and other We are purifying and detecting aflatoxin B 1 , B 2 , G 1 , G 2 , M 1 , M 2 and versicolor application. Background technique [0002] Aflatoxins are a group of structurally similar secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, etc., and are a group of compounds with difuranocoumarin as the basic structure. At present, 12 species have been isolated and identified, especially aflatoxin B 1 , B 2 , G 1 , G 2 , M 1 and M 2 It is a strong pollutant, widely present in grains, feed and its processed products. Among the more than 200 known mycotoxins, it is the most toxic and has the highest pollution frequency. Aflatoxins have the effects of inducing mutations, suppress...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/14B01J20/286B01D15/22C07D493/14C07D493/22G01N30/02C12R1/91
Inventor 王雄鲍蕾梁成珠李为喜吕宁许艳丽果旗江帆雷丰华弓全全戚大海吴兆广
Owner 北京中检维康技术有限公司
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