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Monoclonal antibody of glycinin and application thereof

A technology of soybean globulin and monoclonal antibody, which is applied in the direction of anti-plant immunoglobulin and peptide preparation methods, biochemical equipment and methods, etc., and can solve the problems of non-reference, cumbersome processing process, difficult on-site detection of soybean products, etc. , to achieve the effect of low cost and broad application prospects

Inactive Publication Date: 2009-06-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

From the perspective of immunology, these methods still have certain defects. The former requires repeated preparation of multiple antisera, cannot be compared between different laboratories, and has low sensitivity and accuracy; the latter cannot reflect immunoreactivity, and due to pretreatment The process is cumbersome and requires expensive special instruments, so it is difficult to be widely used in the field detection of soybean products

Method used

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  • Monoclonal antibody of glycinin and application thereof
  • Monoclonal antibody of glycinin and application thereof
  • Monoclonal antibody of glycinin and application thereof

Examples

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Effect test

Embodiment 1

[0025] Embodiment 1, the preparation of anti-glycinin monoclonal antibody

[0026] 1. Extraction and purification of glycinin

[0027] It is prepared by combining fractional salting out and gel filtration. Specific steps are as follows:

[0028] 1.1. Dehulled soybeans were refined, filtered and extracted with ether to degrease, and the residual ether was removed by rotary evaporation to obtain defatted soybean powder.

[0029] 1.2. At room temperature, add 0.03M Tris-HCl (pH 8.0, 10mM β-mercaptoethanol (purchased from: Shanghai Aladdin, product number: 1093795)) buffer (1:20) to the defatted soybean powder, dissolve, 10,000 Centrifuge at rpm / min for 30 min, and collect the supernatant.

[0030] 1.3. Adjust the pH to 6.4 with HCl, centrifuge at 10,000rpm / min (4°C) for 30min, and collect the precipitate with phosphate buffer (0.01mol / L phosphate buffer (PBS) with 10mmol / L β-mercaptoethanol added, pH8. 0) (0.01mol / L phosphate buffer solution (PBS): 8.00g NaCl, 0.20g KCl, 0.20...

Embodiment 2

[0086] Example 2: Double-antibody sandwich ELISA detection method for glycinin

[0087] The detection of glycinin with the monoclonal antibody produced by the anti-glycinin monoclonal antibody cell line 4B2 CGMCC No.2791 may include the following reagents:

[0088] 1) The monoclonal antibody produced by the anti-glycinin monoclonal antibody cell line 4B2 CGMCC No.2791 was purified according to Step 5 of Example 1;

[0089] 2) Polyclonal antibody against glycinin. The preparation and purification methods of the polyclonal antibody are as follows:

[0090] a. Preparation of polyclonal antibodies

[0091] The glycinin prepared in Example 1 was mixed with physiological saline to make 5 mg / mL, mixed with an equal amount of Freund's adjuvant, and emulsified completely with a vortex mixer. Select healthy female New Zealand white rabbits, the first immunization is the medial epithelium of the foot, inject complete adjuvant emulsified antigen, select two sites, each site 0.5mL; boos...

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Abstract

The invention discloses a monoclonal antibody of glycinin and the application thereof. Monoclonal antibody of glycinin provided by the invention is made of anti-glycinin monoclonal antibody cell line 4B2 with preserving number being CGMCC No.2791. When being used for detecting the glycinin, the glycerin monoclonal antibody of the invention has the advantages that the defects, such as no reference conducted between different laboratories, low sensitivity and accuracy, and the repeated preparation for polyvalent antibody in an immune manner, existing in the traditional methods, such as SDS-PAGE and polyvalent antibody indirect ELISA; the detection accuracy is close to that obtained by adopting reversed-phase hplc method (RP-HPLC); the cost is very low; the requirement for detecting the content of glycinin in soybeans, bean pulp, and bean products in a high throughput manner by the feed industry, the food industry, and relevant enterprises; and the broad application prospect of quantitatively detecting antigenic substances of bean products and evaluating the hypersensitivity-inducing ability is possessed.

Description

technical field [0001] The invention relates to a monoclonal antibody of glycinin and its application. Background technique [0002] As a high-quality vegetable protein and oil source for humans and animals, soybean has a wide range of applications. Soybeans and soy products are widely used in the food industry, and corn-soybean meal-based rations are widely used in livestock and poultry production as the main animal feed. However, glycinin, as a soybean antigen protein with rich content, strong thermal stability and strong immunogenicity, often causes allergic reactions, often manifested as infantile diarrhea in the medical field, and 5-6% of children and 2-4 % of adults show clinical symptoms to soybeans and soybean products, often manifested as diarrhea, severe symptoms such as trembling, throat edema and acute asthma; in animal husbandry, soybean antigen protein can cause weaned piglets and pregnant sows, Allergic reactions in young animals such as calves and rats ofte...

Claims

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Application Information

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IPC IPC(8): C07K16/16C07K1/22C12N5/20G01N33/68
Inventor 马曦李德发贺平丽尹靖东王凤来王军军于贵平
Owner CHINA AGRI UNIV
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