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Double-cross-linking chemical synthesis method of citrinin-protein-coupled antigen and preparation method of anti-citrinin monoclonal antibody

A chemical synthesis method and monoclonal antibody technology, applied in the biological field, can solve the problem of inability to prepare high-affinity and high-specificity anti-citrinin antibodies, and achieve reduced steric hindrance effect, strong immunogenicity, and high specificity. Effect

Inactive Publication Date: 2010-09-29
FUZHOU UNIVERSITY
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Problems solved by technology

[0006] At present, there is no commercialized citrinin ELISA detection kit in China, only the citrinin detection test paper (Xu Yang CN1603823A) that can be used for qualitative detection. Therefore, it is of great significance to synthesize strong immunogenic antigens and prepare high affinity and high specificity anti citrinin antibodies

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  • Double-cross-linking chemical synthesis method of citrinin-protein-coupled antigen and preparation method of anti-citrinin monoclonal antibody

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Effect test

Embodiment 1

[0024] Preparation of Citrinin-Bovine Serum Albumin (CIT-BSA) Immunization Antigen

[0025] Dissolve 0.5 mg of citrinin in 0.5 mL of sodium hydroxide (0.6 mol / L), add 0.5 mL of sodium borohydride solution (2 mg / mL), and then add 0.5 μL of 1,4-butanediol diglycidyl ether For activation, shake and react at room temperature for 4 hours; add 31.8 mg of sodium carbonate, adjust the pH to 9.0, add 5 mg of bovine serum albumin, and conduct a coupling reaction at 37°C for 24 hours; The reaction was shaken at 37°C for 48 hours. The reaction product solution was purified by G-25 chromatography column and eluted with phosphate buffer (0.01 mol / L, pH 7.4). The first elution peak collected was the target product CIT-BSA. The coupling product CIT-BSA was analyzed by a UV full-wavelength scanner, and the UV spectrum of the coupled antigen CIT-BSA showed two characteristic absorption peaks (278nm and 326nm) at the same time, and the absorption peak at 278nm was similar to the characteristic ...

Embodiment 2

[0027] Preparation of citrinin ovalbumin (CIT-OVA) detection antigen

[0028]10 mg of citrinin was dissolved in 10 mL of sodium hydroxide (0.6 mol / L), and 10 mL of sodium borohydride (2 mg / mL) was added, followed by 10 μL of 1,4-butanediol diglycidyl ether. Activation, shaking reaction at room temperature for 10 hours; adding sodium carbonate to adjust the pH to 9.0, adding 43 mg ovalbumin, and coupling reaction at 37°C for 24 hours; after the reaction, adding 200 mg glycine for blocking, and shaking at 37°C React for 48 hours. The reaction product solution was dialyzed in 0.01mol / L PBS (pH 7.4) buffer solution at 4°C for 2 days, and the dialysate was changed 3 times in the middle. The collected reaction liquid in the dialysis bag is the target product CIT-OVA. The coupling product CIT-OVA is analyzed by an ultraviolet full-wavelength scanner. The coupling product CIT-OVA has an obvious characteristic absorption peak at 279nm, which corresponds to the absorption peak of OVA ...

Embodiment 3

[0030] Preparation of citrinin-keyhole limpet hemocyanin (CIT-KLH) detection antigen

[0031] Dissolve 5 mg of citrinin in 5 mL of sodium hydroxide (0.6 mol / L), add 5 mL of sodium borohydride solution (2 mg / mL), and then add 5 μL of 1,4-butanediol diglycidyl ether For activation, shake and react at room temperature for 8 hours; add sodium carbonate to adjust the pH to 10.0, add 30 mg keyhole limpet hemocyanin, and perform coupling reaction at 37°C for 24 hours; The reaction was shaken at 37°C for 48 hours. The reaction product solution was purified by G-25 chromatography column and eluted with phosphate buffer (0.01 mol / L, pH 7.4). The first elution peak collected was the target product CIT-KLH. The coupling product CIT-KLH was analyzed by a UV full-wavelength scanner, and the UV spectrum of the coupled antigen CIT-BSA showed two characteristic absorption peaks (278nm and 326nm) at the same time, and the absorption peak at 278nm was similar to that of KLH. Consistent, there ...

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Abstract

The invention relates to a double-cross-linking chemical synthesis method of a citrinin-protein-coupled antigen and a preparation method of an anti-citrinin monoclonal antibody, belongs to the field of biotechnology. The synthesis method of the citrinin-protein-coupled antigen comprises the following steps that: the citrinin and a double-cross-linking agent (C2H3O)-(CH2)n-(C2H3O) react at first and is then coupled with a carrier, glycine is used for occlusion after reaction, and separation and purification are carried out finally, thereby obtaining the citrinin-carrier-coupled antigen. The preparation method of the anti-citrinin monoclonal antibody comprises the following steps: carrying out cell fusion and screening with the hybridoma technique to obtain a hybridoma cell line of the anti-citrinin monoclonal antibody with stable secretion, preparing ascitic fluid, and purifying, thereby obtaining the anti-citrinin monoclonal antibody. The invention has the advantages of mild preparation conditions, simple operation and controllable process. The obtained anti-citrinin monoclonal antibody has high specificity and high affinity, and can be further used for preparation of the enzyme linked immunosorbent assay kit or test paper card for citrinin detection.

Description

technical field [0001] The invention relates to a double-crosslinking chemical synthesis method of a citrinin-protein coupled antigen and a preparation method of an anti-citrinin monoclonal antibody, belonging to the field of biotechnology. Background technique [0002] Citrinin or citrinin is a toxin produced by filamentous fungi such as Penicillium, Aspergillus, and Monascus. It has severe liver and kidney toxicity, as well as teratogenic, carcinogenic, and mutagenic effects. Investigations and studies in recent years have found that citrinin can be detected in many foods and agricultural products, mainly because it is related to the mildew of corn, rice, bread, cheese, apples, pears and other agricultural products or food. Therefore, the food safety problem caused by citrinin has attracted more and more attention. [0003] Monascus has a long history of application in my country, and it can be fermented with Monascus to produce a variety of traditional Chinese foods, suc...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/77C07K14/795C07K1/14C07K1/10C07K16/44
Inventor 李泳宁郭养浩汪媛媛
Owner FUZHOU UNIVERSITY
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