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Immunomagnetic beads for gathering purification of zearalenone and preparing method and application of immunomagnetic beads

The technology of zearalenone and immunomagnetic beads is applied in the field of enrichment and purification processing technology of zearalenone samples, which can solve the hidden dangers of safety, low separation efficiency, complicated purification and separation operations of zearalenone samples, etc. It can eliminate the interference of impurities, improve the lower limit of detection, and improve the accuracy and reliability of detection.

Inactive Publication Date: 2016-02-03
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to make up for the deficiencies in the prior art, to provide a kind of immunomagnetic beads for the enrichment and purification of zearalenone and its preparation method and application, so as to solve the complicated operation of purification and separation of zearalenone samples , low separation efficiency, and high potential safety hazards

Method used

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  • Immunomagnetic beads for gathering purification of zearalenone and preparing method and application of immunomagnetic beads
  • Immunomagnetic beads for gathering purification of zearalenone and preparing method and application of immunomagnetic beads
  • Immunomagnetic beads for gathering purification of zearalenone and preparing method and application of immunomagnetic beads

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of immunomagnetic beads for zearalenone enrichment and purification

[0029] This example presents the preparation method of the conjugate obtained by coupling the zearalenone monoclonal antibody and the carboxyl-containing immunomagnetic beads as the immunomagnetic beads for enriching and purifying zearalenone. The method includes:

[0030] 1. Preparation of Zearalenone Monoclonal Antibody

[0031] 1. Synthesis of zearalenone hapten (see attached for the synthetic route figure 1 ) and identification

[0032]Dissolve 110mg of p-phenylenediamine in 15ml of 0.2mol / L HCl aqueous solution, add 70mg of NaNO dropwise at 0-4°C 2 2ml of aqueous solution, protected from light for 0.5-1 hour, and then slowly dropwise added a mixture of 110mg of zearalenone in 2ml of 0.5mol / L sodium acetate aqueous solution. Protect from light for 1-3 hours. Add an appropriate amount of concentrated hydrochloric acid to acidify, produce a precipitate, centrifuge, wash wi...

Embodiment 2

[0061] Example 2 Characteristic Detection of Immunomagnetic Beads

[0062] Take 0.1 mL of zearalenone-enriched immunomagnetic beads prepared according to Example 1 (concentration: 10 mg / mL) in a 10 mL centrifuge tube, wash the magnetic beads twice with 5 mL of deionized water, and remove them after magnetic separation Supernatant; then add 1mL of the sample to be tested (the zearalenone standard substance is prepared respectively with PBS buffer solution to a concentration of 10ng / mL, 20ng / mL, 30ng / mL, 40ng / mL, 50ng / mL, 60ng / mL , 70ng / mL, 80ng / mL zearalenone solution as the sample to be tested, and PBS buffer as the blank sample to be tested), mix well, capture at 25°C for 20min, and mix the magnetic beads for 5min during the period; magnetic separation Then remove the supernatant, and wash the magnetic beads twice with 5 mL deionized water to remove interfering impurities. Finally, add 1mL of methanol to elute, collect the eluate, and use HPLC method to detect zearalenone in...

Embodiment 3

[0066] The usage method of embodiment 3 immunomagnetic beads

[0067] 1. Sample pretreatment

[0068] Homogenize the sample with a homogenizer; weigh 5.0±0.05g of the sample into a sample bottle, add 1.0±0.05g of sodium chloride, 25ml of 60% methanol solution, vortex for 5min with a vortexer, or shake on a shaker for 20min, 3000g Above, centrifuge at room temperature (20-25°C / 68-77°F) for 5 minutes; (if the centrifugation conditions are not available, this step can also be replaced by the following operation: take 10ml of supernatant after standing and filter) absorb 5ml (equivalent to 1g sample) Centrifuge the supernatant / filtrate, add 5ml deionized water, mix well, and set aside. (for magnetic bead capture).

[0069] 2. Immunomagnetic bead capture

[0070] Take 0.2ml of zearalenone immunomagnetic beads in a 10ml centrifuge tube, wash the beads twice with 5ml of deionized water, and separate the washing solution with a magnetic separation rack each time (stand still on the...

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Abstract

The invention relates to immunomagnetic beads for gathering purification of zearalenone and a preparing method and application of the immunomagnetic beads. The immunomagnetic beads coupled with zearalenone monoclonal antibodies are prepared through the process of activation, coupling, washing and enclosing with carboxyl magnetic beads as carriers and zearalenone monoclonal antibodies as recognition midbodies, the immunomagnetic beads are incubated in a proper buffer solution under a certain condition and can efficiently capture and gather zearalenone in a detection sample. The immunomagnetic beads for gathering purification of zearalenone have the advantages that the concentration of zearalenone in the detection is increased, the detecting lower limit is improved, interference of impurities is eliminated, the detection accuracy and reliability are improved, the sample treating time is shortened, and quick detection is achieved.

Description

technical field [0001] The invention relates to an enrichment and purification process for zearalenone samples, in particular to an immunomagnetic bead used for enrichment and purification of zearalenone, and a preparation method and application thereof. Background technique [0002] Zearalenone (Zearalenone, ZEN) is a white crystal, also known as F-2 toxin, which is a toxic metabolite produced by Fusarium graminearum and other strains. ZEN mainly pollutes corn, sorghum, wheat, barley and other grain crops and their products. Toshitsugu investigated ZEN contained in grains, food and feed in 19 countries and regions and found that samples from many countries and regions, including China, Argentina, Canada, Poland and Yemen, were contaminated to varying degrees. With the help of contaminated dairy products, meat products and other animal-derived foods, ZEN and its metabolites can enter the human body, posing a threat to human health. The harm of ZEN to animals and humans is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
CPCG01N33/577G01N33/531
Inventor 罗晓琴曹东山赵正苗贾芳芳魏力杰朱亮亮何方洋冯月君
Owner BEIJING KWINBON BIOTECH
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