Immunomagnetic beads enriched with t-2 toxin and its preparation method and application

An immunomagnetic bead and toxin technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of low separation efficiency, complicated purification and separation of T-2 toxin samples, etc., to improve the detection limit, improve detection accuracy and Reliability, the effect of eliminating the interference of impurities

Active Publication Date: 2017-07-21
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 enriched with T-2 toxin and its preparation method and application, so as to solve the problem of complex purification and separation of T-2 toxin samples, low separation efficiency, Technical issues with major security risks

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  • Immunomagnetic beads enriched with t-2 toxin and its preparation method and application
  • Immunomagnetic beads enriched with t-2 toxin and its preparation method and application
  • Immunomagnetic beads enriched with t-2 toxin and its preparation method and application

Examples

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Effect test

Embodiment 1

[0029] Example 1 Preparation of immunomagnetic beads enriched for T-2 toxin

[0030] This example provides a method for preparing the conjugate obtained by coupling the T-2 toxin monoclonal antibody with carboxyl-containing immunomagnetic beads as the immunomagnetic beads enriched in T-2 toxin. The method includes:

[0031] 1. Preparation of T-2 toxin monoclonal antibody

[0032] 1. Synthesis of T-2 toxin hapten (synthetic route see appendix figure 1 ) and identification

[0033] Dissolve 100mg of T-2 toxin in acetonitrile, then add 200μL of acetic acid and 200mg of pyridinium dichromate, react at 70°C for 4 hours, stop the reaction, rotary evaporate, remove acetonitrile, add water-ethyl acetate extraction, dry, evaporate to dryness, and apply silica gel The column was separated by elution with chloroform / methanol (10:1) to obtain an intermediate product.

[0034] Add 3 mL of ethanol to dissolve the above intermediate product, then add 50 mg of potassium carbonate and 5 mL...

Embodiment 2

[0065] Example 2 Characteristic Detection of Immunomagnetic Beads

[0066] Take 0.1 mL of T-2 toxin-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 the upper beads after magnetic separation. clear; then add 1mL of the sample to be tested (the T-2 toxin standard was prepared with PBS buffer solution to T -2 toxin 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 for 5 minutes during the period; remove the supernatant after magnetic separation, and wash with 5mL deionized water Rinse the magnetic beads twice to remove interfering impurities. Finally, 1 mL of methanol was added for elution, the eluate was collected, and the content of T-2 toxin in the sample to be tested was detected by HPLC according to GB / T28718-2012. The results are s...

Embodiment 3

[0070] The usage method of embodiment 3 immunomagnetic beads

[0071] 1. Sample pretreatment

[0072] 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, and vortex with a vortex for 5min, or Shake for 20 minutes, over 3000g, and centrifuge at room temperature (20-25°C / 68-77°F) for 5 minutes; absorb 5mL of centrifuged supernatant, add 5mL of deionized water, mix well, and set aside.

[0073] 2. Immunomagnetic bead capture

[0074]Take 0.2 mL of T-2 toxin immunomagnetic beads in a 10 mL centrifuge tube, wash them 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 All adsorption); Add 5mL of the processed sample to the rinsed T-2 toxin immunomagnetic beads, mix well, capture a...

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Abstract

The invention relates to a T-2 toxin-enriched immunomagnetic bead as well as a preparation method and application thereof. The T-2 toxin-enriched immunomagnetic bead is characterized in that a carboxyl magnetic bead is taken as a carrier and a T-2 toxin monoclonal antibody is used as an identifying intermediate, an immunomagnetic bead coupled with the T-2 toxin monoclonal antibody is prepared by the processes of activating, coupling, washing and closing; the immunomagnetic bead is incubated in a proper buffer solution under certain conditions for efficiently capturing and enriching the T-2 toxin in a detection sample. The T-2 toxin-enriched immunomagnetic bead disclosed by the invention has the advantages of concentrating the T-2 toxin in an to-be-detected sample, improving a lower detection limit, eliminating impurity interference, improving the accuracy and reliability of detection, shortening treatment time of the sample and achieving quick detection.

Description

technical field [0001] The invention relates to a process for enriching and purifying a T-2 toxin sample, in particular to an immunomagnetic bead enriched with T-2 toxin and its preparation method and application. Background technique [0002] T-2 toxin (T-2toxin) is the most toxic type A trichothecene mycotoxins, and it exists widely in nature. Penicillium and other molds mainly pollute wheat, barley, corn and other grains and their products. T-2 toxin metabolizes slowly in animals, mainly harms hematopoietic tissues and immune organs, can cause immune system damage and blood toxicity, can inhibit animal cell DNA, RNA and protein synthesis, induce cell apoptosis, and cause symptoms such as leukocyte deficiency. After humans accidentally eat a large amount of grains contaminated with this toxoid, in addition to acute symptoms such as vomiting, abdominal pain, and diarrhea, it can also cause leukocyte deficiency, necrotizing angina, bone marrow dysplasia, esophageal and gast...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/531
CPCG01N33/531G01N33/577
Inventor 罗晓琴张颖龙光宗马腊腊崔海峰何方洋徐念琴崔廷婷
Owner BEIJING KWINBON BIOTECH
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