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Chemiluminescence enzyme-linked immunosorbent assay kit for aflatoxin m1 and method of use

A technology of aflatoxin and chemiluminescence enzyme is applied in the field of chemiluminescence enzyme-linked immunosorbent assay, which can solve the problems of time-consuming, limited sensitivity and high detection cost, and achieve the requirements of improving precision and stability, accurate detection requirements and long storage time. Effect

Active Publication Date: 2015-07-29
广东标允生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TLC has poor specificity and relatively low sensitivity, so AFM is required 1 The concentration of the standard product is high, and there is a potential high operational pollution; the instrument method requires a large investment in equipment, high operating technical requirements, more consumption of purification columns, and higher detection costs
They all have their own advantages and disadvantages, but these methods are not easy to popularize due to the disadvantages of poor specificity, low sensitivity, cumbersome sample pretreatment, expensive precision instruments, time-consuming, and inability to detect a large number of samples at a time. Simple, fast, and sensitive, it is also suitable for large-scale screening work, but its sensitivity has certain restrictions

Method used

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  • Chemiluminescence enzyme-linked immunosorbent assay kit for aflatoxin m1 and method of use
  • Chemiluminescence enzyme-linked immunosorbent assay kit for aflatoxin m1 and method of use
  • Chemiluminescence enzyme-linked immunosorbent assay kit for aflatoxin m1 and method of use

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 Aflatoxin M 1 Preparation of monoclonal antibody and coated antigen

[0051] (1) Preparation of coated antigen

[0052] 0.3mg AFM 1 Dissolve in 400 μL of anhydrous pyridine, add 1 mg of hydroxymethoxyhydroxylamine hemihydrochloride (CMO), shake at 37°C in the dark, take out a small amount every 4 hours for qualitative analysis by thin-layer chromatography until AFM 1 Fully converted to AFM 1 Oxime; take 6mg AFM 1 Dissolve the oxime in 20mL of 25% (V / V) methanol aqueous solution, add 367mg of EDAC (ethyl dimethylamine propyl carbodiimide), then add 1mL of 10mg / mL BSA, shake at room temperature for 48h in the dark. During the reaction, 367 mg of EDAC was added in two separate additions. After the reaction, the mixture was dialyzed with deionized water for 5 days, and the water was changed every day. The dialyzed solution was taken and passed through a 0.45 μm filter membrane to obtain aflatoxin M 1 Conjugated with bovine serum albumin (coated antigen), ...

Embodiment 2

[0055] Embodiment 2 Establishment of chemiluminescent enzyme immunoassay

[0056] (1) Optimization of coating antigen and antibody concentration

[0057] 1) Dilute the coated antigen according to 2.5mg / L, 1.25mg / L, 0.833mg / L, 0.625mg / L, 0.5mg / L with coating solution (0.05mol / L pH5.0 carbonate buffer) And longitudinally coated opaque white luminescent plate, 100 μL / well, 37 ° C for 24 h, washed twice with washing solution, and patted dry on absorbent paper.

[0058] 2) Add 150 μL / well of the prepared blocking solution to seal, overnight at 37°C, spin dry and put in an oven to dry.

[0059] 3) Add 50 μL / well of aflatoxin M diluted with 0.01mol / L PBST 1 Standard series solution

[0060] 4) Add 50 μL / well of aflatoxin M serially diluted with 0.01mol / L PBST 1 Monoclonal antibody (1:4000, 1:5000, 1:6000, 1:7000, 1:8000), 37°C for 60 minutes, wash the plate 5 times, and pat dry on absorbent paper.

[0061] 5) Add 100 μL / well of diluted horseradish peroxidase-labeled goat anti-mo...

Embodiment 3

[0071] Embodiment 3 Aflatoxin M 1 Chemiluminescent ELISA Kit

[0072] (1) Composition of the kit

[0073] 1) Coated with aflatoxin M 1 Antigen chemiluminescent microtiter plate: the microtiter plate is a 96-well detachable opaque white luminescent plate, which has been coated with aflatoxin M 1 Antigen and blocking solution; aflatoxin M 1 Antigen is aflatoxin M 1 For conjugates with bovine serum albumin, the coating concentration is 0.625mg / L.

[0074] Preparation of ELISA plate: Take a 96-well detachable opaque white luminescent plate, dilute the coated antigen to 0.625 mg / L with coating solution, add 100 μL to each well, and overnight at 37°C, pour out the liquid in the well, wash with washing solution for 2 Once, pat dry on absorbent paper. Then add 150 μL of blocking solution to each well, incubate overnight at 37°C, pour off the liquid in the well, dry in an oven at 37°C, and store in a vacuum-sealed aluminum foil bag at 4°C.

[0075] 2) Aflatoxin M 1 A series of ...

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Abstract

The invention discloses a chemiluminescence enzyme-linked immune detection kit of aflatoxin M1 and a using method, belonging to the technical field of chemiluminescence enzyme-linked immune detection. The detection kit comprises a chemiluminescence enzyme label plate coated with aflatoxin M1 antigen, an aflatoxin M1 standard, an aflatoxin M1 antibody, an enzyme-labeled antibody, a chemiluminescent solution A and a chemiluminescent solution b. The using method of the kit comprises the following steps of: (1) pre-processing the samples to be detected; (2) orderly adding the aflatoxin M1 standard solutions or samples and the aflatoxin M1 antibody, adding the enzyme-labeled antibody after a competing reaction, finally adding the chemiluminescent solutions for quantitative detection of the aflatoxin M1 by a chemiluminescence immunoassay analyzer; and (3) processing and analyzing the result. The chemiluminescence enzyme-linked immune detection kit of aflatoxin M1 disclosed by the invention has advantages of high sensitivity, good stability and important practical application and development values, and is suitable for screening a large number of samples.

Description

technical field [0001] The invention relates to the technical field of chemiluminescent ELISA, in particular to a kind of aflatoxin M 1 A chemiluminescence enzyme-linked immunoassay kit and a method of use thereof. Background technique [0002] Aflatoxins are toxic metabolites produced by fungi, which are widely found in various grains, food and feed, and are highly toxic to humans and animals. Aflatoxin was classified as a Class I carcinogen by the Cancer Research Institute of the World Health Organization (WHO) in 1993. Aflatoxin M 1 (Aflatoxin M 1 , abbreviated AFM 1 ) is one of the structurally similar compounds of aflatoxins, which are metabolites produced by common Asperillus Flavus and Asperillus Parasiticus, and are found in food and feed in hot and humid areas Aflatoxins were most likely to be present. AFM 1 The physical and chemical properties are quite stable and will not be destroyed by pasteurization. Aflatoxin M 1 The harm is mainly manifested in carci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/543G01N21/76
Inventor 杨金易孙远明王弘雷红涛沈玉栋徐振林李萍
Owner 广东标允生物科技有限公司
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