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Enzyme linked immunoassay kit for detecting T-2 toxin, and application thereof

An enzyme-linked immunosorbent assay, T-2 technology, used in the determination of T-2 toxins in batches, concentrates), enzyme-linked immunosorbent assay kits, feed (raw material fields, can solve the problem of being unsuitable for conventional toxin analysis, lack of sensitivity, Expensive and other problems, to achieve the effect of simple structure, high sensitivity and high accuracy

Active Publication Date: 2014-02-12
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some methods such as TCL and chromatography are too complicated, some lack sensitivity, and some are expensive, so they are not suitable for routine toxin analysis

Method used

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  • Enzyme linked immunoassay kit for detecting T-2 toxin, and application thereof
  • Enzyme linked immunoassay kit for detecting T-2 toxin, and application thereof
  • Enzyme linked immunoassay kit for detecting T-2 toxin, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 Preparation of kit components

[0034] 1. Preparation of T-2 toxin hapten

[0035] A mixture of 24mg T-2 toxin, 30mg phthalic anhydride and 0.1ml pyridine in 2ml dimethyl sulfoxide (DMSO) was stirred and reacted at 80°C for 15 hours, the solvent was evaporated, and purified by column chromatography to obtain ortho Phthalic acid mono-T-2 toxin ester, the synthetic route diagram is as follows figure 1 .

[0036] Get above-mentioned product and measure through proton nuclear magnetic resonance spectrum, such as figure 2 As shown, the nuclear magnetic spectrum shows that two groups of aromatic ring signal peaks increased at about 8.0ppm and carboxyl signal peaks increased at about 13.3ppm, indicating that the hapten was successfully synthesized.

[0037] 2. Antigen preparation

[0038] Immunogen preparation——T-2 toxin hapten was coupled with bovine serum albumin (BSA) to obtain immunogen.

[0039] Dissolve 4.5mg of hapten in 0.5ml of dimethylformamide (DM...

Embodiment 2

[0055] Example 2 Construction of an enzyme-linked immunosorbent assay kit for detecting T-2 toxin

[0056] An enzyme-linked immunosorbent assay kit for detecting T-2 toxin was set up to include the following components:

[0057] (1) ELISA plate coated with T-2 toxin-conjugated antigen;

[0058] (2) 6 bottles of T-2 toxin standard solution, the concentrations are 0μg / L, 1μg / L, 3μg / L, 9μg / L, 27μg / L, 81μg / L;

[0059] (3) Concentrate the enzyme conjugate;

[0060] (4) Enzyme conjugate diluent;

[0061] (5) The substrate chromogenic solution is composed of substrate solution A and substrate solution B, the substrate solution A is carbamide peroxide, and the substrate solution B is tetramethylbenzidine;

[0062] (6) The stop solution is 2mol / L sulfuric acid.

Embodiment 3

[0063] The detection of T-2 toxin in the sample of embodiment 3

[0064] 1. Sample pretreatment

[0065] Homogenize the feed sample with a homogenizer; weigh 5.0g±0.05g of the homogenized feed sample into a 50ml polystyrene centrifuge tube, add 25ml of 50% methanol, shake vigorously with a shaker for 5min, keep at room temperature (20 Centrifuge at -25°C / 68-77°F) for 5 min; take 500 μl of the supernatant to a 2ml polystyrene centrifuge tube, add 500 μl of 10% sodium chloride aqueous solution and shake with a shaker for 1 min, mix well; take 20 μl for analysis.

[0066] 2. Detection with kit

[0067] Add 20 μl of T-2 toxin standard solution / sample to the microwells of the microtiter plate coated with T-2 toxin-coupled antigen, and then add 100 μl of enzyme conjugate working solution (use enzyme conjugate diluent for concentrated enzyme conjugate Dilute according to the volume of 1:10), seal the plate with a cover plate, react in the dark at 25°C for 10 minutes, pour out the l...

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Abstract

The present invention provides an enzyme linked immunoassay kit for detecting T-2 toxin. The kit contains a T-2 toxin conjugated antigen coated enzyme label plate, a T-2 toxin standard substance solution, a concentrated enzyme conjugate, an enzyme conjugate working solution, a substrate coloration solution and a termination solution. The invention further discloses a method for detecting T-2 toxin in a sample by using the enzyme linked immunoassay kit. The method comprises: carrying out a sample pretreatment, detecting by using the kit, and analyzing the detection result. The enzyme linked immunoassay kit can be used for detecting T-2 toxin residue in feed (raw materials, matching materials and concentrated materials) samples, has characteristics of simple operation, low cost, high sensitivity and on-site monitoring, and is suitable for screening of mass samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting T-2 toxin, which is suitable for the determination of T-2 toxin in feed (raw materials, batch materials, concentrated materials). technical background [0002] T-2 toxin is the most toxic of the trichothecene toxins produced by Fusarium fungus. It is a secondary metabolite that can cause poisoning reactions in animals. It is widely distributed in nature and often Found in contaminated field crops and grain stocks. No matter whether the content of T-2 toxin in the feed is high or low, it will lead to decreased feed intake, slow weight gain, immune system damage and so on. Studies have shown that T-2 toxin exhibits various toxicities such as genotoxicity, immunotoxicity, and blood toxicity to humans and animals, and can induce apoptosis of various cells. In 1973, at the joint meeting held in Geneva by the...

Claims

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Application Information

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IPC IPC(8): G01N33/577
CPCG01N33/531G01N33/535G01N33/577
Inventor 何方洋万宇平罗晓琴冯静郝士元齐向武聂雯莹马腊腊
Owner BEIJING KWINBON BIOTECH
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