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Preparation and application of miniature high-efficiency clenbuterol immuno-affinity chromatography column

A technology of immunoaffinity chromatography column, which is applied in the field of preparation of miniature high-efficiency clenbuterol immunoaffinity chromatography column, can solve the problems of poor detection sensitivity, low reliability, and low accuracy rate, and achieve strong chemical stability, Improved sensitivity and specificity

Inactive Publication Date: 2014-07-16
NANNING LANGUANG BLUE LIGHT BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] 4) There is no miniaturized affinity column
The above-mentioned reasons lead to poor enrichment efficiency and purification ability of clenbuterol in the sample to be tested by the prepared affinity chromatographic column, resulting in shortcomings such as poor detection sensitivity, low accuracy, and low reliability.

Method used

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  • Preparation and application of miniature high-efficiency clenbuterol immuno-affinity chromatography column
  • Preparation and application of miniature high-efficiency clenbuterol immuno-affinity chromatography column
  • Preparation and application of miniature high-efficiency clenbuterol immuno-affinity chromatography column

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Embodiment Construction

[0054] 1. Preparation of miniature high-efficiency clenbuterol immunoaffinity chromatography column

[0055] 1) Preparation of clenbuterol-specific antibody

[0056] 1) Preparation of clenbuterol bromoacetyl ester: 50 mg of clenbuterol hydrochloride was dissolved in 500 L of dimethyl fumarate (DMF) solution and reacted with 50 L of bromoacetyl chloride at 60° C. for 30 minutes. After the reaction, 500 L of 50% methanol was added. Separation and purification by reverse column chromatography with preparative high performance liquid chromatography (C-18HPLC). The peaks shifted backward relative to the standard clenbuterol peaks were collected separately (see figure 1 ), the main component (about 20 mg) with clenbuterol ultraviolet (UV) absorption spectrum characteristics, freeze-dried and used now.

[0057] 2) Clenbuterol immunogen and coated antigen preparation. Take 50 mg of hemocyanin (KLH) (10 mL), add 1% SDS to heat denature, then add 15-30 mg of excess dithiothreitol (D...

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Abstract

The invention relates to a preparation method and application of a miniature high-efficiency clenbuterol immuno-affinity chromatography column. The technical scheme is characterized by comprising the following steps of: preparing a high-quality antibody, synthesizing efficient prepared bromoacetyl chloride clenbuterol purified by a liquid chromatogram and thiolation hemocyanin into immunogen immune animals, and obtaining antiserum; reacting the purified bromoacetyl chloride clenbuterol with thiolation agarose to obtain clenbuterol-agarose padding, loading the padding into an antigen affinity chromatography column; and purifying a clenbuterol specific antibody in the antiserum by using the antigen affinity chromatography column. The preparation method of the clenbuterol immuno affinity chromatography column comprises the following steps of: coupling high-quality antibodies at high density to the agarose oxidized by periodic acid to prepare an antibody affinity padding, and placing 25 mul of padding in a small specially-made column to prepare the miniature immuno-affinity chromatography column. The immuno-affinity chromatography column provided by the invention can be used for specifically gathering clenbuterol in a sample to be tested at high efficiency and can increase the accuracy, reliability and sensitiveness of detection when being combined with an analytic instrument and colloidal gold test paper for use.

Description

technical field [0001] The invention relates to a biological separation and enrichment technology, in particular to a preparation technology of a miniature high-efficiency clenbuterol immunoaffinity chromatography column and its application. Background technique [0002] Clenbuterol hydrochloride is a β-stimulant, which can increase lean meat percentage, reduce fat deposition and promote animal growth. It is widely used as a growth promoter by farmers or breeding enterprises, resulting in a large amount of clenbuterol hydrochloride in animal products. residual. Human consumption of such animal products can cause muscle tremors, heart palpitations, nervousness, headache, dizziness, nausea, vomiting, fever, tremors and other symptoms, seriously endangering the health of consumers. Therefore, many countries (including my country) have now been banned from adding clenbuterol to feed to increase the lean meat percentage of animals, and the content of clenbuterol in livestock pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/08B01J20/286B01J20/30G01N1/34G01N1/40C07K16/06
Inventor 萧浩谢体三潘丽金宁欢欢李芝蓉张芬
Owner NANNING LANGUANG BLUE LIGHT BIOTECH
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