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A kind of immunomagnetic beads for enrichment and purification of vomitoxin and its preparation method and application

A technology of vomitoxin and immune magnetic beads, which is applied in the field of enrichment and purification process of vomitoxin samples, can solve the problems of complex 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: 2017-12-15
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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  • A kind of immunomagnetic beads for enrichment and purification of vomitoxin and its preparation method and application
  • A kind of immunomagnetic beads for enrichment and purification of vomitoxin and its preparation method and application
  • A kind of immunomagnetic beads for enrichment and purification of vomitoxin and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Preparation of immunomagnetic beads for enrichment and purification of vomitin

[0030] This example provides the preparation method of the conjugate obtained by coupling the vomitin monoclonal antibody and the carboxyl-containing immunomagnetic beads as the immunomagnetic beads for enriching and purifying the vomitin. The method includes:

[0031] 1. Preparation of vomitin monoclonal antibody

[0032] 1. Synthesis of vomiting toxin hapten (see attached synthetic route figure 1 ) And identification

[0033] Dissolve 50 mg of vomitin in acetonitrile, add 200 μL of acetic acid and stir, then add 40 mg of mercaptopropionic acid, and stir at 80°C for 12 hours. Stop the reaction, add water, add aqueous sodium hydroxide to adjust the pH to 13, add ethyl acetate for extraction, remove the organic phase, add hydrochloric acid to adjust the pH to 6, add ethyl acetate for extraction, the organic phase is evaporated to dryness, and the crystals are washed with petroleum ether t...

Embodiment 2

[0064] Example 2 Detection of characteristics of immunomagnetic beads

[0065] Take 0.1 mL of the immunomagnetic beads enriched with vomitoxin prepared according to Example 1 (with a concentration of 10 mg / mL) in a 10 mL centrifuge tube, rinse the 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 vomitin standard is formulated into a concentration of 10ng / mL, 20ng / mL, 30ng / mL, 40ng / mL, 50ng / mL, 80ng / mL, 100ng / mL, 120ng / mL of vomitin solution as the sample to be tested, and PBS buffer as the blank sample to be tested), mix well, capture at 25°C for 20 minutes, and mix the magnetic beads during 5 minutes; remove the supernatant after magnetic separation and use 5 mL to remove Rinse the magnetic beads twice with ionized water to remove interfering impurities. Finally, 1 mL of methanol was added for elution, the eluate was collected, and the content of vomitin in the sample to be tested was d...

Embodiment 3

[0069] Example 3 Method of using immunomagnetic beads

[0070] 1. Sample pretreatment

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

[0072] 2. Immunomagnetic bead capture

[0073] Take 0.2mL of vomitin immunomagnetic beads in a 10mL centrifuge tube, rinse with 5mL deionized water twice, and separate the washing liquid with a magnetic separation stand each time (leave it on the magnetic separation stand for 3 minutes each time to ensure that the magnetic beads are fully absorbed ); Add 5mL of the processed sample to the rinsed vomitoxin immunomagnetic beads, mix well, capture at 25°...

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Abstract

The present invention relates to an immunomagnetic bead used for enrichment and purification of DON, and a preparation method and application thereof. The carboxyl magnetic bead is used as a carrier, a DON monoclonal antibody is used as a recognition intermediate, and after activation-coupling-washing- The blocking process prepares immunomagnetic beads coupled with DON monoclonal antibody, and incubation in a suitable buffer under certain conditions can efficiently capture and enrich DON in the detection sample. The immunomagnetic beads used for the enrichment and purification of DON has the advantages of concentrating the DON concentration in the sample to be tested, increasing the lower limit of detection, eliminating the interference of impurities, improving the detection accuracy and reliability, and reducing the sample processing time to achieve rapid detection.

Description

Technical field [0001] The invention relates to an enrichment and purification treatment process for vomitin samples, in particular to an immunomagnetic bead for enrichment and purification of vomitin, and a preparation method and application thereof. Background technique [0002] Vomitoxin (vomitoxin), also known as deoxynivalenol (DON), belongs to the trichothecenes group of compounds, is composed of Fusarium, Cephalosporium, Myrothecium, Trichoderma The metabolites produced by other strains are highly toxic, which can cause intestinal disorders in animals, cause vomiting, diarrhea, abdominal pain and other symptoms, delay growth and reduce immune function. As a natural biotoxin, it is widely present in feed materials and finished feed products such as corn, wheat, flour and DDGS. In recent years, the survey results show that the proportion of mycotoxin contamination in some feeds and raw materials in my country is as high as 60% to 70%. The excessive proportion of vomitin is c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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