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Vitamin B2 conjugate and preparation method and application thereof

A vitamin and conjugate technology, applied in the field of vitamin B2 conjugate and its preparation, can solve problems such as complicated steps, save detection time, and make up for long time-consuming effects

Inactive Publication Date: 2015-11-25
TIANJIN SUNGENE BIOTECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Third, the steps are cumbersome. Even commercial microbiological kits require strict sample pretreatment, medium configuration, result processing, etc.
After retrieval, there is no vitamin B in the world 2 Reports on the synthesis of immunogens and antibody preparation, so research on vitamin B 2 It is very necessary to synthesize immunogens and prepare antibodies

Method used

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  • Vitamin B2 conjugate and preparation method and application thereof
  • Vitamin B2 conjugate and preparation method and application thereof
  • Vitamin B2 conjugate and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] (1) Preparation of solution A: vitamin B 2 19.4mg, N,N'-carbonyldiimidazole (CDI) 25.0mg, 4-dimethylaminopyridine (DMAP) 0.6mg dissolved in 4ml dimethylformamide, the reaction produces vitamin B 2 Active intermediate with N,N'-carbonyldiimidazole, spare;

[0042] (2) Preparation of cBSA: Under the condition of 0~4°C, first dissolve 18.0mg of ethylenediamine in 20ml of phosphate buffer solution with a pH of 7.40 and a concentration of 0.01M, adjust the pH to 7.40 with concentrated hydrochloric acid; weigh 1000.0 mgBSA (molecular weight 68,000) and 57.51mg EDC, then added to the ethylenediamine solution, stirred and reacted at 20°C for 2 hours; the reaction solution of ethylenediamine and BSA was buffered with the above-mentioned phosphate buffer at 0~4°C The dialyzed solution was stirred and dialyzed for 70 hours, and then dialyzed with distilled water for 24 hours, and the dialysate was replaced every 6 hours; the dialyzed solution was centrifuged at 13,000 rpm for 15 ...

Embodiment 2

[0048] (1) Preparation of solution A: vitamin B 2 19.4mg, N,N'-carbonyldiimidazole (CDI) 30.0mg, 4-dimethylaminopyridine (DMAP) 1.0mg dissolved in 5ml dimethylformamide, the reaction produces vitamin B 2 Active intermediate with N,N'-carbonyldiimidazole, spare;

[0049] (2) Preparation of cBSA: Under the condition of 0~4°C, first dissolve 18.0mg of ethylenediamine in 20ml of phosphate buffer solution with a pH of 7.40 and a concentration of 0.01M, adjust the pH to 7.40 with concentrated hydrochloric acid; weigh 1000.0 mgBSA (molecular weight 68,000) and 57.51mg EDC, then added to the ethylenediamine solution, stirred and reacted at 20°C for 2 hours; the reaction solution of ethylenediamine and BSA was buffered with the above-mentioned phosphate buffer at 0~4°C The dialyzed solution was stirred and dialyzed for 70 hours, and then dialyzed with distilled water for 30 hours, and the dialysate was replaced every 6 hours; the dialyzed solution was centrifuged at 13,000 rpm for 15 ...

Embodiment 3

[0055] (1) Preparation of solution A: vitamin B 2 19.4mg, N,N'-carbonyldiimidazole (CDI) 35.0mg, 4-dimethylaminopyridine (DMAP) 1.3mg dissolved in 7ml dimethylformamide, the reaction produces vitamin B 2 Active intermediate with N,N'-carbonyldiimidazole, spare.

[0056] (2) Preparation of cBSA: Under the condition of 0~4°C, first dissolve 18.52mg of ethylenediamine in 20ml of phosphate buffer solution with a pH of 7.56 and a concentration of 0.02M, adjust the pH to 7.56 with concentrated hydrochloric acid; weigh 1000.00 mgBSA (molecular weight 68,000) and 57.51mg EDC, then added to the ethylenediamine solution, stirred and reacted at 22°C for 3 hours; the reaction solution of ethylenediamine and BSA was buffered with the above-mentioned phosphate buffer at 0~4°C The dialyzed solution was stirred and dialyzed for 80 hours, and then dialyzed with distilled water for 20 hours, and the dialysate was replaced every 6 hours; the dialyzed solution was centrifuged at 13,000 rpm for 1...

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Abstract

The invention discloses a vitamin B2 conjugate. The vitamin B2 conjugate is formed by coupling vitamin B2 hapten and bovine serum albumin or ovalbumin which generates immunogenicity and is a carrier material, wherein n refers to number of molecules, of vitamin B2, combined with one bovine serum albumin molecule and is an integer from 1-20, BSA refers to bovine serum albumin, and molecular weight ranges from 6.6KDa to 6.9KDa. The invention further discloses a preparation method of the vitamin B2 conjugate. The preparation method includes: connecting vitamin B2 with the carrier material generating immunogenicity to form the coupling material capable of inducing an animal immune system to generate antibodies. Antiserum with titer reaching 1:256000 is prepared through immune New Zealand rabbits, and limit of detection is 1.07ng / ml. The preparation method has the advantages of simplicity, convenience, quickness, specificity and accuracy. A foundation is provided for manufacturing an enzyme-linked immunosorbent assay reagent box of vitamin B2.

Description

technical field [0001] The invention relates to a conjugate of vitamin B group vitamins and its preparation method and application, in particular to a vitamin B 2 Conjugates and their preparation methods and applications. The invention belongs to the field of nutrient immune detection. Background technique [0002] The following titles involved in the present invention apply to the entire specification and claims: [0003] BSA: serum albumin (BovineSerumAlbumin), Sigma company product [0004] PBS: Phosphate Buffered Saline (0.01M, pH=7.40) [0005] Sephadex-G75: dextran gel, product of Sigma [0006] cBSA: bovine serum albumin modified with ethylenediamine [0007] Dialysis Membrane: Solaibao Technology Co., Ltd. [0008] Vitamin B 2 : Sigma product [0009] CDI: N,N'-carbonyldiimidazole, product of Sigma [0010] DMAP: 4-dimethylaminopyridine, product of Sigma Company. [0011] Vitamin B 2 Riboflavin, also known as riboflavin, was discovered in milk by the Briti...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/77C07K1/107C07K16/44
CPCC07K14/765C07K1/1077C07K14/77C07K16/44C07K19/00
Inventor 郗日沫李小刚孟萌尹永梅张丽沙董亚庆宋兆瑞王玉芬许坤龙浩
Owner TIANJIN SUNGENE BIOTECH
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