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Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same

An enzyme-linked immunosorbent reagent, aflatoxin technology, applied in the direction of microorganism-based methods, anti-fungal/algae/lichen immunoglobulin, microorganisms, etc., can solve the problems of complicated experimental process, poor accuracy, and high technical level requirements. To achieve the effect of increasing immunogenicity

Active Publication Date: 2013-03-06
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thin-layer chromatography is a classic method for the determination of aflatoxin, and it is also one of the standard methods for the determination of aflatoxin B1 in food and feed in my country. However, the sample pretreatment of thin-layer chromatography is cumbersome, the experimental process is complicated, and the time required is long. The accuracy is poor, and it is harmful to the experimenters; the high-performance liquid chromatography equipment is expensive, the technical level is high, and the sample needs to be purified, which is not conducive to on-site screening. Sampling Rapid Screening Test Method

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same
  • Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same
  • Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The preparation of embodiment 1 kit components

[0059] 1. Antigen Synthesis

[0060] a. Synthesis of hapten

[0061] Aflatoxin B1 was condensed with hydroxylamine hydrochloride to introduce a new hydroxyl group, and then reacted with succinic anhydride to obtain a hapten with a carboxyl functional group.

[0062] The specific steps of the hapten: put 156mg of aflatoxin B1 and 105mg of hydroxylamine hydrochloride in 5ml of pyridine solution and stir at room temperature for 12-24 hours, then rotary evaporate to remove pyridine and unreacted hydroxylamine hydrochloride to obtain a crude product, and add the obtained crude product to 5ml of pyridine and 100mg of succinic anhydride were reacted at 60°C for 10-20 hours, the pyridine was removed by rotary evaporation, 10ml of water was added, extracted with ethyl acetate, evaporated to dryness and recrystallized in a mixed system of ethanol and water to obtain the aflatoxin B1 hapten.

[0063] b. Immunogen synthesis

[006...

Embodiment 2

[0095] Embodiment 2 detects the formation of the ELISA kit of aflatoxin B1

[0096] An enzyme-linked immunosorbent assay kit for detecting aflatoxin B1 was set up to include the following components:

[0097] (1) A microtiter plate coated with aflatoxin B1-coupled antigen;

[0098] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0099] (3) Aflatoxin B1 monoclonal antibody working solution;

[0100] (4) 6 bottles of aflatoxin B1 standard solution, the concentrations are 0μg / L, 0.05μg / L, 0.1μg / L, 0.2μg / L, 0.6μg / L, 1.8μg / L;

[0101] (5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A liquid is carbamide peroxide, and the substrate chromogenic liquid B liquid tetramethylbenzidine;

[0102] (6) The stop solution is 2mol / L hydrochloric acid;

[0103] (7) The concentrated washing solution is a 0.2 mol / L phosphate buffer solution with a pH value of 7.4, containing 1.0% Tween-20 and 0.4‰ sodium azi...

Embodiment 3

[0105] The detection of aflatoxin B1 in the actual sample of embodiment 3

[0106] Sample pretreatment

[0107] (1) Oil (peanut oil, blended oil, corn oil, soybean oil, etc.)

[0108] Weigh 2.0±0.05g of edible oil sample into a 50ml polystyrene centrifuge tube; add 4ml of methanol-water solution, shake for 5min, above 3000g, centrifuge at room temperature (20-25°C / 68-77°F) for 5min; pipette 2.0ml Put the supernatant into a 50ml polystyrene centrifuge tube, add 2.0ml deionized water, then add 6ml chloroform, shake for 5min, centrifuge at room temperature (20-25°C / 68-77°F) for 5min; remove the upper layer For liquid, take 3ml of the lower organic phase into a 10ml clean glass test tube, and dry it in a water bath at 50-60°C under nitrogen flow; add 1ml of n-hexane, vortex for 30s with a vortex instrument to dissolve the dry residue, add 1ml of complex solution working solution, and use Vortex for 30s, over 3000g, centrifuge at room temperature (20-25°C / 68-77°F) for 5min; remov...

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Abstract

The invention provides an enzyme-linked immunosorbent assay kit for detecting an aflatoxin B1-containing medicine and an application for the same. The enzyme-linked immunosorbent assay (ELISA) kit comprises an ELISA plate coated with a coating antigen, an enzyme label, aflatoxin B1 specific antibody working solution (contained in the case that the coating antigen on the ELISA plate and the enzyme label are enzyme-labelled antibodies or enzyme-labelled antigens), aflatoxin B1 standard substance solution, substrate developing solution, stopping solution, concentrated washing solution and concentrated compound solution. The method for detecting aflatoxin B1 by virtue of the kit provided by the invention comprises the following steps of: performing sample pre-treatment at first, and then detecting by virtue of the kit, and finally analysing the detected result. The enzyme-linked immunosorbent assay kit provided by the invention can be used for detecting the residual amount of aflatoxin B1 in samples such as oil, peanuts and grains, as well as is simple and convenient to operate, low in expense, high in sensitivity, capable of being monitored in the field, and suitable for screening lots of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay detection technology, in particular to an enzyme-linked immunoassay kit for detecting aflatoxin B1 drugs and an application thereof. Background technique [0002] Aflatoxin B1 (structural formula see figure 1 ) is a derivative of dihydrofuran oxinone, which contains a difuran ring and an oxinone, the former is a basic toxic structure, and the latter is related to carcinogenicity. Aflatoxins (B1, B2, G1, G2) are a group of highly toxic compounds with similar structures produced by Aspergillus flavus, Aspergillus parasiticus and A.nomius. Aflatoxins can cause cancer, mainly in the liver, intestines, lungs and chest of lesions. These fungi are produced in tropical and subtropical food, feed and their raw materials, mainly polluting grains, rice, corn, beans, tree nuts and peanuts, etc., posing a serious threat to human health. Aflatoxin exceeds the standard phenomenon, so aflatoxin has become an imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577C07K16/14C12N5/20C12R1/91
Inventor 韩黎何方洋吴鹏万宇平韩雪琳陈勇孙震赵正苗李勇冯才茂冯静罗晓琴
Owner BEIJING KWINBON BIOTECH
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