Aflatoxin B1 magnetic particle separation enzyme-linked immunoassay

A technology of aflatoxin and magnetic particles, which is applied in the direction of measuring devices, analytical materials, material excitation analysis, etc., can solve problems such as difficult to eliminate, and achieve the effects of high sensitivity, good repeatability, and simple processing methods

Inactive Publication Date: 2010-03-03
北京倍爱康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aflatoxin is very heat-resistant and can only be completely destroy...

Method used

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  • Aflatoxin B1 magnetic particle separation enzyme-linked immunoassay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Anti-FITC antigen coupled with surface amino (COOH-) magnetic particles to prepare magnetic separation reagent

[0028] Take 100 mg of magnetic particles containing carboxyl (COOH-) active groups on the surface and wash them three times with 0.1 M MES (2-[N-morpholino] ethanesulfonic acid), 10 ml of pH 4.5-5 solution. The magnetic particles were resuspended in 1ml of this solution, and 2mg of anti-FITC antibody was added, and mixed evenly. Add 100 μl of 10 mg / ml EDC solution, mix well and react at room temperature for 2 hours. After washing the magnetic beads 3 times with 10 ml of 0.01 M phosphate buffered saline (PBS) pH7.4 containing 1% bovine serum albumin (BSA), the solution was used to prepare a 2.5 mg / ml magnetic separation reagent working solution.

Embodiment 2

[0029] Example 2: AFB1-BSA linked alkaline phosphatase (ALP), preparation of enzyme-labeled antigen reagent

[0030] Take 5 mg of AFB1-BSA antigen, concentrate to 5 mg / ml, add 10 μl of 13.76 mg / ml activator 2-Iminothiolane HCl (2IT) solution, place at room temperature for 20 minutes, add glycine to terminate the activation reaction, and place at room temperature for 5 minutes. Use Sephadex G25 column to desalt and collect protein elution peaks.

[0031] Take 5 mg of ALP solution, add 50 μl of 6.69 mg / ml Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) solution, leave it at room temperature for 30 minutes, add glycine to terminate the activation reaction, and leave it at room temperature for 5 minutes. Use Sephadex G25 column to desalt and collect protein elution peaks.

[0032] The activated AFB1-BSA was mixed with the activated ALP, left at room temperature for 10 hours, then separated and purified using Supperdex200 gel chromatography column to remove unli...

Embodiment 3

[0035] Example 3: Anti-AFB1 antibody linked to FITC to prepare anti-reagent

[0036] Take 5mg of anti-AFB1 antibody solution and dialyze against 20mM pH9.0 carbonate buffer for more than 12h, and the concentration after dialysis is required to be greater than 1mg / ml. Add 500 μl of 0.5 mg / ml FITC solution (prepared with 20 mM pH9.0 carbonic acid buffer), mix well and react at room temperature for more than 12 hours. Use Sephadex G25 column to remove unbound FITC, and collect the protein elution peak.

[0037] The preparation method of the anti-reagent diluent is the same as that of the enzyme-labeled antigen reagent diluent. The above-mentioned AFB1 antibody FITC conjugate is diluted to 1-5 μg / ml with the diluent to prepare the anti-reagent working solution.

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Abstract

The invention provides an aflatoxin B1 (AFB1) magnetic separation enzyme-linked immunity quantitative detection method, belonging to the field of food safety immunoassay technique. The method adopts the immuno-detection principle of competition law; and AFB1 is connected with biological enzyme to prepare enzyme-labeled antigen reagent, anti-fluorescein isothiocyanate (FITC) antibody is absorbed onthe surfaces of magnetic particles to prepare magnetic separation reagent, and the FITC is connected with the AFB1 antibody to prepare anti-reagent. In a sample, the AFB1 competes with the enzyme-labeled AFB1 and is combined with a small amount of FITC-labeled anti-AFB1 antibody, so that antigen-antibody complex can be formed. After the magnetic separation reagent is added, the complex is caughtonto the surfaces of the magnetic particles by the anti-FITC antibody connected on the surfaces of the magnetic particles. After being washed, the product is finally added with substrate and detected.The method has the advantages that (1) the magnetic particles are used for replacing the traditional enzyme-labeled plate to be taken as a solid-phase carrier, so that immunoreaction is carried out under the approximate liquid phase condition; and the reaction is more complete and rapid, and has the characteristics of high specificity and good repeatability compared with the traditional enzyme-linked immuno sorbent assay (ELISA); furthermore, (2) by adopting one-step competition law principle, the time used for detection is short.

Description

technical field [0001] The invention belongs to the technical field of immune detection and analysis, relates to food safety-related immunological detection and analysis technology, and in particular provides a magnetic particle separation enzyme-linked immunoassay method for rapid detection of aflatoxin B1 (Aflatoxin B1, AFB1), which is suitable for peanuts, Qualitative detection of aflatoxin B1 in various foods and feeds such as corn. Background technique [0002] Aflatoxin (AF) is a metabolite of toxin-producing strains of Aspergillus flavus and Aspergillus parasiticus, which is commonly found in moldy food and feed, and is the most toxic mycotoxin found so far. It can pollute food and feed through various ways, directly or indirectly enter the human food chain, and threaten human health and life safety. After entering the body, aflatoxin not only inhibits the synthesis of DNA and RNA, but also inhibits the synthesis of liver protein, which seriously damages the internal...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/577G01N33/535G01N21/31G01N21/64
Inventor 仝文斌陈立杰韩子华张婉英
Owner 北京倍爱康生物技术有限公司
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