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Hybridoma cell lines and antibodies, immunosorbents, immunoaffinity columns and kits and their applications

A hybridoma cell line, immunoadsorbent technology, applied in solid adsorbent liquid separation, antifungal/algae/lichen immunoglobulin, other chemical processes, etc., to achieve the effect of stable performance

Active Publication Date: 2014-10-22
北京中检维康生物技术有限公司 +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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  • Hybridoma cell lines and antibodies, immunosorbents, immunoaffinity columns and kits and their applications
  • Hybridoma cell lines and antibodies, immunosorbents, immunoaffinity columns and kits and their applications
  • Hybridoma cell lines and antibodies, immunosorbents, immunoaffinity columns and kits and their applications

Examples

Experimental program
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preparation example Construction

[0028] Specifically, the preparation method of hybridoma cell line and monoclonal antibody in the present invention may comprise the following steps:

[0029] (1) Preparation of zearalenone immunogen

[0030] Dissolve 5 mg of zearalenone ZON in 4 mL of pyridine, add 3 mg of O-carboxymethyl hydroxylamine, stir at room temperature for 24 hours, then vacuum-dry, dissolve in water, adjust the pH to 8.0, and extract 3 times with ethyl acetate, Dehydration crystallization. The crystals were dissolved in dioxane to form a 5 mmol / L solution, and 30 mg of BSA was weighed and dissolved in 2 mL of 0.05 mol / L (pH 7.4) PBS. At 4°C, mix the two, and slowly add 3 mg of N-hydroxysuccinimide (NHS) and 6 mg of N, N'-dicyclohexyl in a solution of 0.5 mL of dioxane Carbodiimide (DCC), the resulting mixture was stirred and reacted at room temperature for 24 hours, dialyzed, centrifuged, and frozen for later use.

[0031] (2) Preparation of hybridoma cells and monoclonal antibodies against zeara...

Embodiment 1

[0080] This example is used to prepare zearalenone immunogen and aflatoxin immunogen

[0081] (1) Dissolve 5 mg of zearalenone ZON in 4 mL of pyridine, add 3 mg of O-carboxymethyl hydroxylamine, stir at room temperature for 24 hours, then vacuum-dry, dissolve in water, adjust the pH to 8.0, and extract with ethyl acetate 3 times, dehydration and crystallization. The crystals were dissolved in dioxane to form a 5 mmol / L solution, and 30 mg of BSA was weighed and dissolved in 2 mL of 0.05 mol / L (pH 7.4) PBS. At 4°C, mix the two, and slowly add 3 mg of N-hydroxysuccinimide (NHS) and 6 mg of N, N'-dicyclohexyl in a solution of 0.5 mL of dioxane Carbodiimide (DCC), the resulting mixture was stirred and reacted at room temperature for 24 hours, dialyzed, centrifuged, and frozen for later use. The zearalenone immunogen, ZON-BSA, was obtained.

[0082] (2) 4 mg of 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 ...

Embodiment 2

[0085] 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 (antibody A) and monoclonal antibodies against the zearalenone congeners α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, and ZON (antibody B)

[0086] (1) Animal immunization: the immunized animals are female BALB / c mice about 6-8 weeks old. Five mice were immunized with the zearalenone immunogen. The zearalenone immunogen (100 μg / cause) was added with an equal amount of Freund's complete adjuvant to make an emulsified agent for immunization, and then the adjuvant was changed to an incomplete adjuvant for a total of 6 immunizations 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.

[0087] Cell fusion: Splenocytes and hybridoma cells were subjec...

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Abstract

The invention discloses a hybridoma cell strain CGMCC NO. 5504 and a monoclonal antibody obtained by secretion of the cell strain, and an application of the antibody. The invention also provides an immunoadsorbent comprising a solid phase vector and antibodies coupled with the solid phase vector, wherein the antibodies is the above monoclonal antibody and a monoclonal antibody obtained by secretion of a cell strain CGMCC NO. 5506; 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, sterigmatocystins and zearalenone analogue. 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 and six zearalenone analogues 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 and its application, an immunoadsorbent prepared from the antibody, and an immunoaffinity column and kit equipped with the immunoadsorbent , and their role in the purification of aflatoxin B 1 , B 2 , G 1 , G 2 , M 1 , M 2 , Aspergillus versicolor and zearalenone congeners α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON. 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 pollu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/14B01J20/286B01D15/22C07D493/14C07D493/22C07D313/00G01N30/02C12R1/91
Inventor 王雄鲍蕾傅武胜梁成珠许艳丽吕宁果旗江帆刘卓王丹戚大海吴兆广
Owner 北京中检维康生物技术有限公司
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