Enzyme linked immunosorbent assay kit for detecting residual clenbuterol hydrochloride and a detection method thereof

A technology of clenbuterol hydrochloride and enzyme-linked immunosorbent reagents, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of limited application range, high price, and high cost, and achieve shortened detection time and simple operation , the effect of reducing the workload

Inactive Publication Date: 2010-08-11
BEIJING APIS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chromatographic technology is time-consuming and expensive, and is limited in practical application. It is usually used as a deterministic quantitative inspection method; enzyme-linked immunoassay (ELISA) is a commonly used on-site sampling and rapid screening inspection method. Veterinary health departments and slaughterhouses mostly use the clenbuterol hydrochloride ELISA test kit (article number: R1701) produced by R-Biopharm in Germany, but due to the high price of imported reagents, its scope of use is limited to a certain extent

Method used

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  • Enzyme linked immunosorbent assay kit for detecting residual clenbuterol hydrochloride and a detection method thereof
  • Enzyme linked immunosorbent assay kit for detecting residual clenbuterol hydrochloride and a detection method thereof
  • Enzyme linked immunosorbent assay kit for detecting residual clenbuterol hydrochloride and a detection method thereof

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Embodiment approach

[0033] According to a preferred embodiment of the present invention, the operating steps of the inventive method are:

[0034] Take the 48 or 96-well coated plate coated with rabbit anti-clenbuterol hydrochloride antibody, add 50 μL clenbuterol hydrochloride standard sample and the processed sample to the corresponding microwell, add 100 μL enzyme-labeled diluent (5 ) Dilute the 20 times concentrated enzyme-labeled clenbuterol hydrochloride (4) with a ratio of 1:20 to obtain the enzyme-labeled clenbuterol hydrochloride (concentration is 0.005 μg / ml~0.05 μg / ml), after shaking and mixing Incubate at 37°C in the dark for 30 minutes, wash 3 times with washing solution, pat dry on absorbent paper, add 100 μL of substrate solution, keep in dark at room temperature for 10 minutes, add 100 μL of reaction termination solution to the microwell After mixing, measure the absorbance value at 450nm or dual wavelength 450nm / 630nm as soon as possible, and calculate the content of clenbuterol ...

Embodiment 1

[0042] Example 1 Preparation of CBL-ELISA kit

[0043] CBL is a small molecular hapten, which only has reactogenicity but no immunogenicity. It needs to be combined with macromolecular substances before it can be used to immunize animals to prepare antibodies. The amino group on the benzene ring in CBL is an active group, which can be combined with the amino group of the protein by the diazotization-coupling method.

[0044] Preparation of CBL-HSA antigen:

[0045] Dissolve 5-10 mg of CBL in 0.1 mol / L pre-cooled HCl, slowly add 1 mol / L sodium nitrite solution dropwise, check with KI starch test paper until the test paper turns dark purple, and obtain diazotized CBL. Weigh 50-100 mg of human serum albumin (HSA) and dissolve it in 0.05 mol / L carbonate buffer solution with a pH value of 9.6, slowly drop the diazotized CBL into the HSA solution, and adjust the pH with 1 mol / L NaOH After the dropwise addition, the reaction was continued for 4 hours, and dialyzed with 0.15 mol / L P...

Embodiment 2

[0070] Example 2 Adding clenbuterol hydrochloride (CBL) medicine

[0071] Dilute the 1 mg / mL CBL stock solution to an appropriate concentration and add it to urine or pork samples that have been identified as having no CBL residue by HPLC (high performance liquid chromatography), so that the CBL concentration in these samples is 0.2 μg / kg, 0.4 μg / kg, 0.5μg / kg, 1μg / kg, 2μg / kg, 3μg / kg and 4μg / kg, there are 5 samples for each concentration, which are used for addition recovery experiments.

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Abstract

The invention discloses an enzyme linked immunosorbent assay kit for detecting residual clenbuterol hydrochloride and a detection method thereof. The kit comprises a micropore plate (1) for precoating staphylococcus protein A and coating CBL antibodies, CBL standard substance (2), concentrated washing liquid (3), concentrated enzyme labeled CBL (4), enzyme labeled diluents (5), a substrate solution (6) and a stopping solution (7). The kit adopts a direct competition method, i.e. CBL in standard substance or samples to be detected and enzyme labeled CBL compete CBL antibodies on the micropore plate. The method can be used for directly detecting clenbuterol hydrochloride in animal derived food, urine and serum samples, and has the advantages of convenient and rapid operation. The operation time only needs 50 minutes, and the sensibility can reach 0.02mug/kg.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent assay kit for detecting residual clenbuterol hydrochloride and a detection method thereof, belonging to the technical field of enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA). More specifically, the present invention relates to an enzyme-linked immunoimmunoassay kit for detecting residual clenbuterol hydrochloride (CBL) content in animal-derived food, blood and urine and a detection method using the kit. Background technique [0002] Clenbuterol hydrochloride (clenbuterol, CBL) is a β-2 adrenergic receptor agonist, clinically used as a bronchial spasmolytic drug, it has excitatory effect on bronchial smooth muscle β-2 receptors. Studies have shown that clenbuterol hydrochloride can enhance fat decomposition and slow down protein catabolism, and large doses in animal husbandry can significantly improve feed conversion rate and lean meat rate, so it is also called lean m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/557G01N33/52
Inventor 不公告发明人
Owner BEIJING APIS BIOTECH
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