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Immunomagnetic bead used for vomitoxin enrichment purifying and preparation method and application thereof

A technology of vomitoxin and immunomagnetic beads, which is applied in the field of enrichment and purification process of vomitoxin samples, can solve the problems of complicated purification and separation operation and low separation efficiency of vomitoxin samples, so as to improve detection accuracy and reliability, improve The lower limit of detection and the effect of rapid detection

Active Publication Date: 2016-01-27
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to make up for the deficiencies in the prior art, to provide an immunomagnetic bead for the enrichment and purification of deoxynivalenol and its preparation method and application, so as to solve the problem of complex purification and separation of deoxynivalenol samples, low separation efficiency, and the existence of Technical issues with major security risks

Method used

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  • Immunomagnetic bead used for vomitoxin enrichment purifying and preparation method and application thereof
  • Immunomagnetic bead used for vomitoxin enrichment purifying and preparation method and application thereof
  • Immunomagnetic bead used for vomitoxin enrichment purifying and preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0029] Example 1 Preparation of Immunomagnetic Beads for Deoxynivalenol Enrichment and Purification

[0030] This example provides a method for preparing the conjugate obtained by coupling the deoxynivalenol monoclonal antibody with carboxyl-containing immunomagnetic beads as immunomagnetic beads for enrichment and purification of deoxynivalenol. The method includes:

[0031] 1. Preparation of monoclonal antibody against vomitoxin

[0032] 1. Synthesis of vomitoxin hapten (synthetic route see appendix figure 1 ) and identification

[0033] Dissolve 50mg of vomitoxin in acetonitrile, add 200μL of acetic acid, stir, then add 40mg of mercaptopropionic acid, and stir at 80°C for 12h. Stop the reaction, add water, add aqueous sodium hydroxide solution to adjust the pH to 13, add ethyl acetate to extract, remove the organic phase, add hydrochloric acid to adjust the pH to 6, add ethyl acetate to extract, evaporate the organic phase to dryness, wash crystals with petroleum ether, ...

Embodiment 2

[0064] Example 2 Characteristic Detection of Immunomagnetic Beads

[0065] Take 0.1 mL of the immunomagnetic beads enriched with vomitoxin prepared according to Example 1 (concentration is 10 mg / mL) in a 10 mL centrifuge tube, wash the magnetic beads twice with 5 mL of deionized water, and remove the supernatant after magnetic separation; Then add 1mL of the sample to be tested (the deoxynivalenol standard was prepared with PBS buffer to a concentration of 10ng / mL, 20ng / mL, 30ng / mL, 40ng / mL, 50ng / mL, 80ng / mL, 100ng / mL, 120ng / mL vomitoxin 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 during 5min; remove the supernatant after magnetic separation, and use 5mL to remove Wash the magnetic beads twice with ionic water to remove interfering impurities. Finally, add 1 mL of methanol to elute, collect the eluate, and use HPLC method to detect the content of deoxynivalenol in the s...

Embodiment 3

[0069] The usage method of embodiment 3 immunomagnetic beads

[0070] 1. Sample pretreatment

[0071] Grain and feed samples: Homogenize the sample with a homogenizer; weigh 5g (accurate to 0.01g) of the sample into a sample bottle, add 1.5g of sodium chloride and 30mL of 60% methanol solution, vortex with a vortex for 5min, or shake Shake the bed for 20 minutes, centrifuge at room temperature (20-25°C / 68-77°F) for 5 minutes at 3000g or more; absorb 5mL of centrifuged supernatant, add 5mL of deionized water, mix well, and set aside.

[0072] 2. Immunomagnetic bead capture

[0073] Put 0.2 mL of vomitoxin immunomagnetic beads into a 10 mL centrifuge tube, wash twice with 5 mL of deionized water, and separate the washing solution with a magnetic separation rack each time (stand still on the magnetic separation rack for 3 minutes each time to ensure that the magnetic beads are completely adsorbed. ); Add 5 mL of the processed sample to the rinsed DON immunomagnetic beads, mix w...

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Abstract

The present invention relates to an immunomagnetic bead used for vomitoxin enrichment purifying and a preparation method and application thereof, a carboxyl magnetic bead is used as a carrier, vomitoxin monoclonal antibody is used as an identifying intermediate, the immunomagnetic bead coupled with the vomitoxin monoclonal antibody is prepared by activation-coupling-washing-closed process, vomitoxin in a detection sample can be efficiently captured and enriched by incubation in certain conditions in a suitable buffer. The immunomagnetic bead used for vomitoxin enrichment purifying has the advantages of enrichment of concentration of vomitoxinin the to-be-tested sample, improvement of the lower detection limit, elimination of impurity interference, improvement of detection accuracy and reliability and reduction of sample processing time to achieve the rapid detection.

Description

technical field [0001] The invention relates to a process for enriching and purifying vomitoxin samples, in particular to immunomagnetic beads for enriching and purifying vomitoxin, a preparation method and application thereof. Background technique [0002] Vomitoxin (vomitoxin), also known as deoxynivalenol (deoxynivalenol, DON), belongs to the trichothecene compound, is a kind of compound produced by Fusarium genus, Cephalosporium genus, Urushiban genus, Trichoderma genus The metabolites produced by strains such as bacteria are highly toxic and can cause intestinal dysfunction in animals, causing symptoms such as vomiting, diarrhea, and abdominal pain, delaying growth, and reducing immune function. As a natural biotoxin, it widely exists in corn, wheat, flour, DDGS and other feed materials and feed products. In recent years, survey results show that the proportion of mycotoxins in some feed and raw materials in my country exceeds the standard as high as 60% to 70%. However...

Claims

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Application Information

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IPC IPC(8): G01N1/40
Inventor 刘玉梅罗晓琴徐念琴李行常平平何方洋冯才伟王建霞
Owner BEIJING KWINBON BIOTECH
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