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Immunochromatographic colloidal gold test strip for detecting aflatoxin M1

A technology of aflatoxin and colloidal gold test paper, which is applied in the direction of measuring devices, analysis materials, instruments, etc., can solve the problems of high cost, inability to realize on-site detection, long operation time and other problems of liquid chromatography, and achieve simple production process and reduce The effect of cumbersome operation steps and convenient on-site operation

Inactive Publication Date: 2016-04-06
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High-performance liquid chromatography is costly, takes a long time to operate, and has many and complicated steps, so it cannot achieve real on-site detection

Method used

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  • Immunochromatographic colloidal gold test strip for detecting aflatoxin M1
  • Immunochromatographic colloidal gold test strip for detecting aflatoxin M1
  • Immunochromatographic colloidal gold test strip for detecting aflatoxin M1

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Experimental program
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Effect test

Embodiment 1

[0038] Preparation and titer detection of embodiment 1 whole antigen and monoclonal antibody

[0039] 1.1 Preparation of No. 1 Whole Antigen of AFB1

[0040] 1.1.1 Preparation of aflatoxin B1 oxime 2mgAFB1 (6.4×10 -6 mol) and 4mg carboxymethylhydroxylamine hemihydrochloride (AOAHCl) were dissolved in 4mL pyridinemethanol double distilled water mixture (volume ratio 4:1:1). Reflux reaction at 86°C for 5-6h. Monitor the progress of the reaction by thin layer chromatography (TLC) every 1h. Leave it at room temperature overnight. Vacuum dry or add 0.5mL triple distilled water to dissolve the obtained residue, and use 0.1mol / L HCl Adjust the pH to 3.0 to precipitate AFB1O, then extract the precipitate and the solution part with ethyl acetate for 2-3 times, collect the ethyl acetate part of the upper layer, and dry it with a gentle nitrogen flow. Both yellow and creamy oxime were obtained.

[0041] 1.1.2 Preparation of Competitive Immunization Antigen AFB was prepared by EDC activ...

Embodiment 2

[0051] Embodiment 2 Preparation of goat (rabbit) anti-mouse IgG in the present invention

[0052] Select healthy male New Zealand white rabbits or goats, use OVA-hapten as the immunogen and the same amount of Freund's complete adjuvant to mix into a water-in-oil emulsion by syringe pumping method, and carry out the first immunization at the amount of 1mg / kg body weight , Take multi-point injections under the skin on the back. Booster immunization every two weeks, with Freund's incomplete adjuvant instead of Freund's complete adjuvant, the dose and method are the same as the first immunization. From the third immunization, 10 days after each immunization, 1 mL of blood was collected from the ear vein for antibody titer detection. When the antibody titer no longer increased, the last (7th) immunization was performed without adjuvant. Thigh intramuscular injection, carotid artery bloodletting after 7 days, coagulated at room temperature for 2 hours and overnight at 4°C, centrifu...

Embodiment 3

[0053] The preparation of colloidal gold test paper strip in the present invention in embodiment 3

[0054] 3.1 Preparation of colloidal gold

[0055] The basic principle of immune colloidal gold technology is that chloroauric acid can be polymerized into gold particles of a certain size under the action of a reducing agent to form a negatively charged hydrophobic colloid solution that is stable due to electrostatic interaction. The present invention adopts trisodium citrate reducing agent reduction method to prepare colloidal gold, and the specific process is as follows: take 100mL aqueous solution of 0.01% chloroauric acid and heat it to boiling, accurately add 0.7mL of 1% trisodium citrate aqueous solution under stirring, golden yellow Chlorauric acid turns purple within 2 minutes, continue to boil for 15 minutes, and restore the original volume with distilled water after cooling, which is the prepared colloidal gold solution. Whether this colloidal gold solution meets the...

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Abstract

The invention belongs to the field of food safety monitoring, and discloses an immunochromatographic colloidal gold test strip for detecting aflatoxin M1. The test strip includes a sample pad, a nitrocellulose membrane (NC), an absorbent pad and a PVC backing. The nitrocellulose membrane is adhered to the on PVC backing; and the sample pad and the absorbent pad are adhered to the ends of the nitrocellulose membrane. The colloidal gold strip uses a one-step indirect competitive immunochromatographic technology for rapid detection of whether the aflatoxin M1 residue in milk meets the EU limit standard (less than 0.05ng / mL) and the limit standards of China (the United States and other countries) (less than 0.5n / mL), and has sensitivity up to 0.05ng / mL.

Description

technical field [0001] The invention belongs to the field of detection of immune colloidal gold and mycotoxin residues in the field of food safety. Specifically, the present invention relates to an immunocolloidal gold test strip suitable for detecting residual aflatoxin M1 in milk. technical background [0002] The molecular formula of aflatoxin M1 is C 17 h 12 o 7 , one of the structurally similar compounds of aflatoxin, which is a metabolite produced by the common Asperillus Flavus and Asperillus Parasiticus, and appears yellow in food and feed in hot and humid areas Aspergillus has the highest probability. The physical and chemical properties are quite stable and will not be destroyed by pasteurization. After mammals ingest feed or food contaminated with aflatoxin B1, it is converted into aflatoxin M1 through hydroxylation. The hazards of aflatoxin M1 are mainly carcinogenic and mutagenic, which can damage the liver tissue of humans and animals, and can lead to liv...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/531
CPCG01N33/558G01N33/531
Inventor 赖卫华吴成辉刘道峰
Owner NANCHANG UNIV
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