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Protein G coated Sundan red I immunoaffinity column and preparation method thereof

A technology of Sudan red and immunoaffinity, applied in the field of Sudan red Ⅰ immunoaffinity column and its preparation, can solve the problems of reducing antibody utilization rate and detection efficiency, reducing the elution speed of affinity chromatography column, etc., and achieving improved capture capacity, time and cost savings, and ease of use

Inactive Publication Date: 2017-05-17
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of protein G has high affinity, the use of protein G-treated carriers will reduce the elution speed of the affinity chromatography column, thereby reducing the utilization rate of antibodies and the efficiency of detection

Method used

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  • Protein G coated Sundan red I immunoaffinity column and preparation method thereof
  • Protein G coated Sundan red I immunoaffinity column and preparation method thereof
  • Protein G coated Sundan red I immunoaffinity column and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Preparation of Sudan I Immunoaffinity Purification Column

[0062] 1. Agarose Gel Activation

[0063] Take 2% agarose gel Sepharose 4B and wash it with 20 times the volume of distilled water to wash away the remaining ethanol. Use a funnel to filter out the water. Weigh 5g of the wet gel after filtering off the water, add 7.5mL of 0.8M NaOH, 2mL of 30% epichlorohydrin, and 2mg / mL of sodium borohydride NaBH 4 , 5mL, and reacted in a shaking table at 25°C for 8h. After the reaction, wash with 50 mL of distilled water to remove the mixed epichlorohydrin in the gel.

[0064] 2. Coupling of Recombinant Protein G to Activated Sepharose

[0065] Take 1 gram of activated agarose gel, and use coupling buffer (0.1M NaHCO 3 , 0.8M NaCl, pH8.9) and washed 3 times. Add 20 mL of 2 mg / mL protein G, and couple at room temperature for 4 h.

[0066] The coupled agarose carrier was washed 3 times with 20 mM, pH 7.4 phosphate buffer PBS. The agarose carrier Sepharose coup...

Embodiment 2

[0078] Example 2 Sudan Red Ⅰ Immunoaffinity Purification Column Recovery Test

[0079]

[0080] The results of the standard recovery rate determination (n=3)

[0081] The results of the three sample recovery experiments are shown in the table below:

[0082]

Embodiment 3

[0083] Example 3: Determination of Sudan Red I Immunoaffinity Column Capacity—Comparison of Performance of Purification Columns Prepared by the Method of the Invention and the Conventional Method

[0084] The preparation of the purification column processed by recombinant protein G was prepared according to Example 1

[0085] Preparation of Purification Column Without Recombinant Protein G Treatment Direct Coupling Method to Prepare Affinity Column

[0086] 1. Carrier activation

[0087] Select the agarose carrier, Sepharose 4B, and activate it with epichlorohydrin activation method.

[0088] Take 2% pre-swelled agarose gel Sepharose 4B, wash it with 20 times the volume of distilled water, wash away the remaining ethanol, and filter out the water with a funnel.

[0089] Weigh 5 g of the wet gel after filtering off the water, add 7.5 mL of 0.8 M NaOH, 2 mL of 30% epichlorohydrin, and 2 mg / mL of sodium borohydride NaBH 4 , 5mL, and reacted in a shaking table at 25°C for 8h. ...

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Abstract

The invention provides a Sundan red I immunoaffinity column which comprises a carrier, protein G and a Sundan red I antibody, wherein the protein G is coupled with the carrier through chemical bonds; the Sundan red I antibody is in cross-link with the protein G by using a cross-linking agent after being joined; the protein G is recombinant protein G which is based on GenBank CAA27638.1 and is subjected to gene recombinant expression. Under the condition that detection properties are not affected, the use amount of the Sundan red I antibody of a sepharose gel carrier pretreated with the protein G is about 3 / 4 of that of an antibody for preparing purification column packing by using a direct coupling method. The Sundan red I provided by the invention is very high in purity, can be directly used in high performance liquid chromatography detection without need of extra subsequent purification treatment, therefore, the time and the cost of operators can be saved.

Description

technical field [0001] The invention belongs to the field of food inspection, and in particular relates to a Sudan red I immunoaffinity column coated with protein G and a preparation method thereof. Background technique [0002] Sudan Red is a synthetic azo, oil-soluble chemical dye. In 1896, the scientist Dadi named it Sudan Red and it is still used today. Sudan red is widely used in the fields of biology and chemistry, and is used in industrial products such as motor oil, car wax and shoe polish. With the gradual understanding of the structure and toxicity of Sudan Red, the International Agency for Research on Cancer (IARC) classifies Sudan Red as a third type of carcinogen. Although this type of substance lacks sufficient evidence to directly cause human cancer, it has potential cancer risk. [0003] Sudan red is a lipophilic azo compound with dark red or dark yellow flaky crystals in appearance, insoluble in water, and mainly includes 4 types: Ⅰ, Ⅱ, III and Ⅳ. Wherein...

Claims

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Application Information

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IPC IPC(8): B01J20/286B01J20/30B01D15/20B01D15/22
CPCB01D15/20B01D15/22B01J20/286
Inventor 董颖超秦玉昌李军国刘士杰谷旭单耕柳家鹏
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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