Reagent box for detecting clenbuterol hydrochloride and its detection method

A technology of clenbuterol hydrochloride and a kit, which is applied in the directions of measuring devices, instruments, scientific instruments, etc., can solve the problems of high false positive rate, instability, time-consuming and other problems, and achieve the effects of high sensitivity, simple structure and convenient use.

Inactive Publication Date: 2006-06-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chromatographic techniques (HPLC, GC-MS, LC-MS) are time-consuming and costly, and are limited in practical application. They are usually used as deterministic quantitative testing methods; immunoassay techniques (ELISA) are commonly used on-site sampling rapid screening tests Methods, at present, my country's import and export inspection and quarantine, veterinary health departments and slaughterhouses mostly use clenbuterol hydrochloride ELISA detection kits produced by German R-Biopharm, British Randox and other companies, but due to the high price of imported kits, to a certain extent limits its use to
Domestic ELISA kits generally have many problems such as insufficient sensitivity, high false positive rate, and instability

Method used

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  • Reagent box for detecting clenbuterol hydrochloride and its detection method
  • Reagent box for detecting clenbuterol hydrochloride and its detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1 preparation kit and detection liver sample

[0023] CBL is a typical hapten, so it has reactogenicity in the immune reaction, but it needs to be combined with macromolecular substances to be immunogenic. The carboxyl group in CBL is an active group that can combine with the amino group of the protein. Therefore, the diazo coupling method can be used to combine CBL with macromolecular protein bovine serum albumin (BSA), and the synthetic CBL-BSA can be used as an artificial immune antigen for animal immunization.

[0024] Preparation of CBL-BSA antigen: Dissolve 3-6 mg of CBL in 100 mmol / L pre-cooled HCl, slowly add 1 mol / L sodium nitrite solution dropwise, test with KI starch test paper until the test paper turns dark purple, and obtain diazotization derivative. Weigh 10-20 mg BSA and dissolve in carbonate buffer solution with pH 9.650 mmol / L. Slowly drop the diazotized CBL into the BSA solution, adjust the pH with 1mol / L NaOH and maintain it at 9.0-9.5, ...

Embodiment 2

[0066] Embodiment 2 prepares kit

[0067] Preparation of CBL-BSA antigen: Same as Example 1.

[0068] Preparation of polyclonal clenbuterol hydrochloride antibody: same as Example 1.

[0069] Eu 3+ - Preparation of goat anti-rabbit antibody:

[0070] Take 1ml of 5g / L goat anti-rabbit antibody dissolved in 50mmol / L PBS pH7.0, change the buffer condition through PD-10 column, and the eluent is 50mmol / L NaCl containing 0.155mol / L NaCl 2 CO 3 -NaHCO 3 pH9.0 buffer. The protein peaks were collected and quantified by UV absorption analysis (1.46A 280 -0.74A 260 ), dilute the goat anti-rabbit antibody to 2g / L with the eluent above. Take 500μl and add Eu containing 0.2mg 3+ -N 2 -[p-isocyanate-benzyl]-diethylenetriaminetetraacetic acid (Eu 3+ -DTTA) in a vial with magnetic stirring at 28°C for 16 hours. The reaction solution was chromatographed on a Sephadex-G50 column (1×40 cm) equilibrated with 80 mmol / L Tris-HCl pH 7.8 buffer solution to collect protein peaks, and dilut...

Embodiment 3

[0086] Embodiment 3 prepares kit

[0087] Preparation of CBL-BSA antigen: Dissolve 6 mg of CBL in 100 mmol / L pre-cooled HCl, slowly add 1 mol / L sodium nitrite solution dropwise, test with KI starch test paper until the test paper turns dark purple, and obtain diazotized derivatives . Weigh 20 mg BSA and dissolve it in carbonate buffer solution with pH 9.650 mmol / L. Slowly drop the diazotized CBL into the BSA solution, adjust the pH with 1mol / L NaOH to maintain it at 9.0-9.5, continue the reaction for 4 hours after the addition, and dialyze with PBS with a pH of 7.410mmol / L in a refrigerator at 4°C Two days later, the CBL-BSA conjugates were obtained for UV scanning detection, and were distributed for use after passing the test.

[0088] The preparation of the polyclonal clenbuterol hydrochloride antibody was the same as that in Example 1, omitted.

[0089] Eu 3+ - Preparation of goat anti-rabbit antibody:

[0090] Take 2ml of 5g / L goat anti-rabbit antibody dissolved in 50...

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Abstract

A method for detecting CBL includes competing CBL antibody by tree CBL with CBL ¿C OVA on micro hole board, washing off unconnected CBL antibody, adding EU3+ - sheep resisting rabbit antibody and washing off unconnected EU3+ - sheep resisting rabbit antibody, adding intensified liquid and using time ¿C identification luminoscope to determine its fluorescence intensity, presenting fluorescence intensity to CBL concentration in sample to be inverse ratio, comparing with standard curve to obtain CBL content in sample. The reagent kit for realizing said method is also disclosed.

Description

technical field [0001] A kit for detecting clenbuterol hydrochloride and its detection method, which belong to the technical field of time-resolved fluorescent immunoassay (TRFIA), and are used for detecting the content of clenbuterol hydrochloride (CBL for short) in animal food, blood and urine detection. Background technique [0002] Clenbuterol hydrochloride (CBL) is a β-2 adrenergic receptor agonist, clinically used as a bronchial spasmolytic drug, which has an excitatory effect on bronchial smooth muscle β-2 receptors. Studies have shown that clenbuterol hydrochloride can enhance lipolysis and slow down protein catabolism, so if it is used in large doses in animal husbandry, it can significantly improve feed conversion rate and lean meat rate, so it is also called lean meat powder. However, if the dosage is too large, it will have a considerable toxic effect on the liver, kidney and other organs of animals and humans (indirectly), and there will be symptoms of poisonin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N21/76G01N33/532
Inventor 金坚时瑾黄飚张莲芬许赣荣宓晓黎
Owner JIANGNAN UNIV
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