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Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting beta-carotene pigments

A technology of enzyme-linked immunosorbent reagents and monoclonal antibodies, which is applied in the direction of microorganism-based methods, immunoglobulins, chemical instruments and methods, etc., to achieve the effects of reducing detection costs, strong specificity, and high detection sensitivity

Inactive Publication Date: 2012-07-18
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The applicant retrieved an invention patent with application number 201010140887.6, which provided a method for oxidizing α-ionone and β-ionone, but did not use it as a hapten to synthesize immunogens and apply them to enzymes immunoassay

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting beta-carotene pigments
  • Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting beta-carotene pigments
  • Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting beta-carotene pigments

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of Immunogen and Coating Original

[0036] 1.1 Synthesis of β-ionone acid

[0037] Accurately measure 30 mL of distilled water and add it to a pre-cooled round bottom flask; add 20 g of liquid bromine to the flask, stir for 2 hours, and form A liquid for later use. Accurately 6g of β-ionone was dissolved in 20mL of dioxane to form B liquid. Add solution B to solution A, and react at room temperature for 8 hours. After the reaction was completed, 20% sodium bisulfite was added until the starch potassium iodide test paper no longer turned blue. Concentrated hydrochloric acid was then added until a white solid appeared in solution. After filtering, put the white solid substance of the filter residue into methanol, put it in a 60°C water bath and stir until completely dissolved, then filter. The filtrate was placed at -20°C overnight and filtered, and the filter cake was dried in a vacuum oven at 50°C, and the obtained product was identified as β-i...

Embodiment 2

[0046] Example 2 Preparation of Monoclonal Antibody

[0047] Preparation of hybridoma cells: refer to the method in Xue Qingshan's "Principles and Techniques of In Vitro Culture" (Science Press, 2001 edition): immunize Balb / C mice with the conjugate β-iononic acid-BSA prepared in Example 1 (purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention). The immunization procedure was to emulsify a protein solution containing 125 μg of the conjugate β-iononic acid-BSA with an equal volume of Freund's complete adjuvant (purchased from sigma), and then inject it subcutaneously at multiple points on the back of the mouse. Afterwards, it was strengthened every 2 weeks, and emulsified with incomplete adjuvant (purchased from sigma company). Finally, 3 days before the fusion (preferably more than 4 weeks from the last immunization), intraperitoneal injection, booster immunization, double the amount of antigen, without adjuvant.

[0048]...

Embodiment 3

[0050] The establishment of embodiment 3 ELISA detection method

[0051] 3.1 Preparation of reagents (unless otherwise specified, the reagents used in this example were prepared by the following methods)

[0052] Phosphate buffer: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, add ultrapure water to 1000mL, adjust pH to 7.4;

[0053] Coating solution: Take Na 2 CO 3 1.5g, NaHCO 3 2.9g, add triple distilled water to 1000mL, adjust the pH value to 9.6;

[0054] Washing liquid: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, Tween 200.5mL, add ultrapure water to 1000mL, adjust pH to 7.4;

[0055] Blocking solution: Ovalbumin 0.1g dissolved in 100mL phosphate buffer;

[0056] Substrate solution: provided by Wuhan Feiyuan Technology Co., Ltd.

[0057] Stop solution: 2mol / L sulfuric acid solution.

[0058] 3.2 Preliminary determination of coating source concentration and antibody working concentration

[0059] The combination of wor...

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Abstract

The invention discloses a specific monoclonal antibody capable of identifying beta-carotene pigments and an enzyme-linked immunosorbent assay (ELISA) method and kit for detecting beta-carotene pigments. The monoclonal antibody is secreted by a hybridoma cell DCC / C11 and the hybridoma cell is collected in the China Center for Type Culture Collection, with collection number being CCTCC NO:C201146. The ELISA method comprises the steps of preparation of immunogen, coating antigen and the antibody, treatment and detection of samples, and the like. The ELISA method and the kit can detect the total content of canthaxanthin, beta-carotene, beta-apo-8'-carotenal, xanthophyll, capsorubin and beta-ionone in the samples by one step, thus shortening the detection time and lowering the detection cost; and the ELISA method and the kit have the characteristics of high detection sensitivity, good precision and good accuracy.

Description

technical field [0001] The invention relates to a monoclonal antibody capable of recognizing beta-carotene pigments, an enzyme-linked immunosorbent method and a kit for detecting beta-carotene pigments. Background technique [0002] β-carotene pigments include β-apo-8'-carotene aldehyde, β-apo-8'-carotene acid ethyl ester, lutein, capsanthin, cantharidin, astaxanthin, etc. [0003] At present, the detection methods for β-carotene substances are mainly instrument detection methods, such as paper chromatography, thin layer chromatography, column chromatography, spectrophotometry, high performance liquid chromatography or liquid chromatography-mass chromatography. These detection methods need to be equipped with expensive instruments and professional operators. The pretreatment of samples is complicated and the detection time is long, which is not conducive to the screening of large batches of samples and cannot be quickly detected on site. Enzyme-linked immunoassay (ELISA) is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
Inventor 袁宗辉王玉莲廖峰彭大鹏潘源虎黄玲利陈冬梅陶燕飞戴梦红刘振利闫彩霞
Owner HUAZHONG AGRI UNIV
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