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Mycotoxin composite immunosorbent, immunoaffinity column and kit, and applications thereof

An immunoadsorbent and immunophile technology, which is applied in other chemical processes, chemical instruments and methods, purification/separation/stabilization of organic compounds, etc., can solve the problems of low recovery rate of addition and inability to purify aflatoxin at the same time , to achieve stable performance

Active Publication Date: 2013-06-19
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The key to the immunoaffinity chromatography method is to select an efficient and specific antibody. However, the addition recovery rate of the liquid-liquid extraction, solid-phase extraction, heating evaporation concentration and other methods in the prior art are all low, and the follow-up detection Step causes very strong matrix effect, and can not simultaneously purify 6 kinds of aflatoxins (B 1 , B 2 , G 1 , G 2 , M 1 and M 2 ), six zearalenone congeners (α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN and ZON), versicolor and ochratoxin A as immunoadsorption media, while being able to Simultaneous purification and analysis of multiple viral toxins can obviously improve the efficiency of detection

Method used

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  • Mycotoxin composite immunosorbent, immunoaffinity column and kit, and applications thereof
  • Mycotoxin composite immunosorbent, immunoaffinity column and kit, and applications thereof
  • Mycotoxin composite immunosorbent, immunoaffinity column and kit, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] This example is used to prepare zearalenone immunogen, aflatoxin immunogen and ochratoxin A immunogen

[0099] (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.

[0100] (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...

Embodiment 2

[0104] This example is used to prepare monoclonal antibodies (antibody A) against zearalenone congeners α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and monoclonal antibodies against ochratoxin A Antibody (antibody B) and anti-aflatoxin B 1 , B 2 , G 1 , G 2 , M 1 , M 2 and Aspergillus versicolor monoclonal antibody (Antibody C).

[0105] (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.

[0106]Ce...

Embodiment 3

[0115] This example is used to prepare immunoaffinity column

[0116] 1. Preparation of agarose gel

[0117] Weigh required 1 g of CNBr-activated agarose gel powder (commercially purchased from GE Company of the United States, each gram of lyophilized powder can form 3.5 ml of swelling agarose gel in final volume), and dissolve it in 1 mM HCl. The immediately swollen agarose gel was placed on a sintered glass filter and washed with 1 mM HCl for 15 min.

[0118] 2. Antibody Conjugation

[0119] (a) using coupling buffer (0.2M NaHCO 3 , pH8.3) to dissolve the antibody, the antibodies are Antibody A, Antibody B and Antibody C, wherein the concentrations of Antibody A, Antibody B and Antibody C are all 5.1mg / ml, the volume of the antibody solution is 35ml, and the antibody solution is placed on ice Staging in the bath. Add the above-mentioned antibody-containing coupling buffer into a fully sealable container with a lid, and quickly add the agarose gel washed in step 1 to it. ...

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Abstract

The invention discloses an immunosorbent comprising a solid-phase carrier and an antibody coupled with the solid-phase carrier. The antibody comprises a monoclonal antibody secreted by a hybridoma cell line CGMCC NO.5504 and a monoclonal antibody secreted by a hybridoma cell line CGMCC NO.5505. The invention also provides a kit comprising the immunosorbent or an immunoaffinity column. Also, the invention provides applications of the immunosorbent, immunoaffinity column, and kit in detecting aflatoxins, sterigmatocystin, zearalenone congeners and ochratoxin A. The invention specifically provides separation and detection methods. According to the invention, the specific monoclonal antibodies with stable performances are developed, such that simultaneous purification and detection of 6 aflatoxins, sterigmatocystin, 6 zearalenone congeners and ochratoxin A are realized.

Description

technical field [0001] The present invention relates to a kind of immunoadsorbent, and the immunoaffinity column and test kit that this immunosorbent is housed and they are used in purifying aflatoxin B 1 , B 2 , G 1 , G 2 , M 1 , M 2 , Versicolor, zearalenone congeners α-ZER, α-ZOL, β-ZER, β-ZOL, ZAN, ZON and ochratoxin A. 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, suppressing immunity and causing cancer. The target org...

Claims

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Application Information

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IPC IPC(8): B01J20/22G01N33/577C07B63/00C07D313/00C07D311/76C07D493/14C07D493/22
Inventor 鲍蕾吕宁许艳丽韩深刘岩梁成珠王雄果旗江帆王丽李峻峰戚大海吴兆广
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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