Immunomagnetic bead for aflatoxin B1 enrichment purification and preparation method and application thereof

A technology of aflatoxin and immunomagnetic beads, which is applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of hidden safety hazards, complicated purification and separation operations of aflatoxin B1 samples, low separation efficiency, etc. Improve the accuracy and reliability of detection and the effect of improving the lower limit of detection

Inactive Publication Date: 2016-02-10
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 a kind of immunomagnetic beads for the enrichment and purification of aflatoxin B1 and its preparation

Method used

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  • Immunomagnetic bead for aflatoxin B1 enrichment purification and preparation method and application thereof
  • Immunomagnetic bead for aflatoxin B1 enrichment purification and preparation method and application thereof
  • Immunomagnetic bead for aflatoxin B1 enrichment purification and preparation method and application thereof

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0027] Example 1 Preparation of immunomagnetic beads for enrichment and purification of aflatoxin B1

[0028] In this example, a conjugate obtained by coupling a monoclonal antibody to aflatoxin B1 and a carboxyl-containing immunomagnetic bead as a preparation method for enriching and purifying aflatoxin B1 is provided. The method includes:

[0029] 1. Preparation of aflatoxin B1 monoclonal antibody

[0030] 1. Synthesis of aflatoxin B1 hapten (see attached for synthetic route figure 1 ) And identification

[0031] 62mg aflatoxin B1 was dissolved in 1mL dimethyl sulfoxide (DMSO), slowly added dropwise 25mg p-phenylenediamine and 0.1mL pyridine in 1mL DMSO mixture at 60℃, after the addition was completed, the reaction was continued for 15h, rotary steaming The solvent was removed, and the p-phenylenediamine monocondensate of aflatoxin B1 was obtained after purification by column chromatography, and the aflatoxin B1 hapten was obtained with a yield of 90%.

[0032] The above hapten was...

Example Embodiment

[0060] Example 2 Characteristic detection of immunomagnetic beads

[0061] Take 0.1mL (concentration of 10mg / mL) of immunomagnetic beads enriched with aflatoxin B1 prepared in accordance with Example 1 in a 10mL centrifuge tube, rinse the beads twice with 5mL deionized water, and remove the magnetic beads after magnetic separation. Clear; then add 1mL of the sample to be tested (the aflatoxin B1 standard is prepared with PBS buffer to the concentration of 5ng / mL, 10ng / mL, 15ng / mL, 20ng / mL, 25ng / mL, 30ng / mL, 35ng / mL, 40ng / mL aflatoxin B1 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 after magnetic separation Supernatant, rinse the magnetic beads twice with 5 mL deionized water to remove interfering impurities. Finally, 1 mL of methanol was added for elution, and the eluate was collected and tested by HPLC in accordance with "Determination of Afla...

Example Embodiment

[0065] Example 3 Method of using immunomagnetic beads

[0066] 1. Sample pretreatment

[0067] Homogenize the sample with a homogenizer; weigh 5.0±0.05g sample into a sample bottle, add 1.0±0.05g sodium chloride, 25ml 60% methanol solution, vortex with a vortexer for 5min, or shake for 20min on a shaker, 3000g Above, centrifuge at room temperature (20-25℃ / 68-77℉) for 5min; (if the centrifugation conditions are not available, this step can also be replaced by the following operation: take 10ml supernatant and filter after standing still) absorb 5ml (equivalent to 1g sample) Centrifuge the supernatant / filtrate, add 5ml deionized water, mix well, and set aside. (For magnetic bead capture).

[0068] 2. Immunomagnetic bead capture

[0069] Take 0.2ml of aflatoxin B1 immunomagnetic beads in a 10ml centrifuge tube, rinse the beads twice with 5ml of deionized water, and separate the washing liquid with a magnetic separation stand each time (leave it on the magnetic separation stand for 3 mi...

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Abstract

The invention relates to an immunomagnetic bead for aflatoxin B1 enrichment purification and a preparation method and application thereof. A carboxyl carboxyl serves as a carrier, an aflatoxin B1 monoclonal antibody serves as a recognition midbody, the immunomagnetic bead coupled with the aflatoxin B1 monoclonal antibody is prepared through the process of activating, coupling, washing and closing, and aflatoxin B1 in a detecting sample can be efficiently captured and enriched through incubation of the immunomagnetic bead in proper buffer liquid under certain conditions. The immunomagnetic bead for aflatoxin B1 enrichment purification has the advantages of increasing the concentration of aflatoxin B1 in the sample to be detected, improving the lower limit of detection, eliminating interference of impurities, improving detecting accuracy and reliability, shortening sample treatment time and achieving fast detection.

Description

technical field [0001] The invention relates to a process for enriching and purifying aflatoxin B1 samples, in particular to an immune magnetic bead used for enriching and purifying aflatoxin B1 and its preparation method and application. Background technique [0002] Aflatoxin B1 (AflatoxinB1 is abbreviated as AFB1) is a derivative of dihydrofuran oxinone, which contains a difuran ring and an oxinone (coumarin). Aflatoxin B1 is one of the most carcinogenic chemicals known. Aflatoxin B1 is highly toxic to humans and some animals, and its toxic effect is mainly damage to the liver. In natural food, aflatoxin B1 is the most common and most harmful. AQSIQ stipulates that aflatoxin B1 is one of the mandatory inspection items for most foods. China's food hygiene standards stipulate the allowable amount of aflatoxin B1 in several major contaminated foods. The allowable amount of aflatoxin B1 in corn, peanuts, and peanut oil is ≤20 μg / kg; other edible oils are ≤10 μg / kg. kg; for...

Claims

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Application Information

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IPC IPC(8): G01N33/543
CPCG01N33/54326
Inventor 朱亮亮贾芳芳徐念琴罗晓琴何方洋冯静杨春艳吴小胜
Owner BEIJING KWINBON BIOTECH
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