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Magnetic particle chemical luminous kit for detecting clenbuterol and application thereof

A chemiluminescence detection and kit technology, which is applied in chemiluminescence/bioluminescence, analysis by making materials react chemically, and measurement devices, can solve the problems of low false positive rate, expensive detection cost, cumbersome operation, etc., and achieve High sensitivity, fast detection, and low detection time

Inactive Publication Date: 2012-07-04
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantages of the HPLC method are that the instrument is expensive, the operation is cumbersome, time-consuming, the detection cost is expensive, the professional requirements for the detection personnel are high, and the sample pre-treatment is complicated
[0005] 2. Gas chromatography-mass spectrometry (GC-MS) has high sensitivity and low false positive rate
The reaction time and temperature have a great influence on the enzyme activity, and the stability of the reagent is poor

Method used

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  • Magnetic particle chemical luminous kit for detecting clenbuterol and application thereof
  • Magnetic particle chemical luminous kit for detecting clenbuterol and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of specific components of the kit

[0037] 1. Preparation of Luminescent Labels

[0038] a) Synthesis of Clenbuterol Hapten

[0039] Clenbuterol is acylated with succinic anhydride to acylate the alcohol hydroxyl group on the molecular structure of clenbuterol into a clenbuterol hapten containing a 4-carbon carboxyl indirect arm.

[0040] b) Preparation of Luminescent Labels

[0041] Take 4.5mmol / L ABEI, dissolve it in 4ml distilled water, dissolve 5.0mmol / L N-hydroxysuccinimide in 0.5ml N,N-dimethylformamide, mix the two thoroughly and react at room temperature for 3-4h . Take 15 mg of the clenbuterol hapten prepared above, adjust the volume to 1.5 ml with pH 7.4 PBS, then add the above activated ABEI solution, mix well, react overnight at room temperature, and purify through G-25 gel column.

[0042] 2. Preparation of fluorescent markers

[0043] a) Preparation of immunogens:

[0044] The immunogen was obtained by coupling the clenbuterol h...

Embodiment 2

[0057] The formation of the second test kit

[0058] A magnetic particle chemiluminescence detection kit for clenbuterol was constructed to contain the following components:

[0059] Fluorescent label for FITC-labeled clenbuterol monoclonal antibody

[0060] Luminescent marker of ABEI-labeled clenbuterol hapten

[0061] Separation Reagent for Paramagnetic Nanospheres Coated with Goat Anti-FITC Monoclonal Antibody

[0062] Clenbuterol standard solution (0ng / ml, 0.01ng / ml, 0.03ng / ml, 0.09ng / ml, 0.27ng / ml, 0.81ng / ml), the standard dilution is pH7.4, containing 0.03% NaN 3 , 0.05mol / L TRIS buffer. The percentage content is the mass percentage content.

[0063] The concentration of Clenbuterol quality control solution is 0.02ng / ml and 0.5ng / ml, respectively, and the quality control solution is pH7.4, containing 0.03% NaN 3 , 0.05mol / L TRIS buffer. The percentage content is the mass percentage content.

[0064] Concentrated wash solution pH 7.6, 0.4% Tween-20, 0.02% NaN 3 ,...

Embodiment 3

[0065] Example 3 Detection of Clenbuterol in Actual Samples

[0066] 1. Sample pretreatment

[0067] (1) Urine

[0068] Take 20μl of clear urine sample for direct determination (if the urine sample is cloudy, it must be filtered or more than 3000g, centrifuged at 15°C for 10min until clear), the sample not used temporarily should be frozen and stored, the sample dilution ratio: 1

[0069] (2) Tissues with low-fat meat, liver and other tissues

[0070] Weigh 2.0±0.05g of homogenized sample into a 50ml polystyrene centrifuge tube; add 6ml of 4% NaCl-0.1M HCl-methanol mixture, shake with a shaker until uniform; let stand for 10 minutes, centrifuge above 3000g for 5 minutes ; Liver samples: Take 1ml of supernatant and add 20μl of 1M sodium hydroxide solution and mix well (after mixing, measure the pH value, it is about 8); Muscle samples: Take 1ml of supernatant and add 30μl of 1M sodium hydroxide solution and mix well (mixed After homogenization, measure the pH value, about 8)...

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Abstract

The invention relates to a magnetic particle chemiluminescence kit for detecting clenbuterol and application thereof. The magnetic particle chemiluminescence kit comprises the following reagents: a luminescence marker, a fluorescein marker, a standard product, a quality control product and a separating reagent. The luminescence marker is a clenbuterol hapten marked by an isoluminol luminescence marker; the fluorescein marker is a clenbuterol monoclonal antibody marked by fluorescein or derivates thereof; and the separating reagent is a paramagnetic nanometer micro-bead coated with an anti-goat FITC (fluorescein isothiocyanate) monoclonal antibody. The invention further relates to a method for detecting clenbuterol in animal-derived food by using the magnetic particle chemiluminescence kit, wherein the method has the characteristics of higher sensitivity, higher specificity and faster detection speed on clenbuterol detection.

Description

technical field [0001] The invention relates to a chemiluminescence detection kit and an application method thereof, in particular to a magnetic particle chemiluminescence detection kit for detecting Clenrol residues in animal tissue, urine, feed and other samples. technical background [0002] Clenbuterol is a beta-stimulant. In recent years, it has been illegally added to the feed to improve the lean meat rate of fatty animals and accelerate the growth of animals. It is widely added to animal feed and can remain in animals. . However, this drug has serious side effects in humans. From light to lead to abnormal heart rhythm, heavy can lead to heart disease. At present, my country and many countries have banned its use as a feed additive. [0003] At present, methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) and enzyme-linked immunosorbent assay (ELISA) are mainly us...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N33/577
Inventor 何方洋万宇平冯才伟朱亮张禹汤庆彩何丽霞徐恩宁李勇杨昌松
Owner BEIJING KWINBON BIOTECH
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