Synthesis process of horseradish peroxidase enzymelabeled zearalenone

A technology of horseradish peroxidase and zearalenone is applied in the field of synthesis technology of enzyme-labeled reagents to achieve the effects of avoiding cross-linking reaction, increasing enzyme activity and improving effectiveness

Active Publication Date: 2013-04-03
CHEMCLIN DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because the preparation technology of horseradish peroxidase-labeled zearalenone has been protected by the kit manufacturer as a technical secret, there are no relevant reports at home and abroad.

Method used

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  • Synthesis process of horseradish peroxidase enzymelabeled zearalenone
  • Synthesis process of horseradish peroxidase enzymelabeled zearalenone
  • Synthesis process of horseradish peroxidase enzymelabeled zearalenone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of horseradish peroxidase-labeled zearalenone

[0028] Weigh 0.5mg ZEN and 4.0mg carboxymethylhydroxylamine hydrochloride, dissolve in 0.2ml pyridine, and stir at room temperature for 24h. Add 0.2ml of distilled water to the reaction solution, adjust the pH to 8.5 with 0.2M NaOH, and shake to clarify the solution. Extracted 3 times with benzene (0.2ml), adjusted the pH of the aqueous layer to 3 with 2M HCl, extracted 3 times with ethyl acetate (0.5ml), and dried in vacuo, the obtained carboxylated product was carboxylated zearalenone .

[0029] Weigh 0.2 mg carboxylated zearalenone, 0.4 mg succinimide and 1 mg carbodiimide, dissolve in 0.7 ml DMF, and stir at room temperature for 1 h. Slowly add this reaction solution dropwise to 0.1M NaHCO containing 13.6mg HRP 3 The solution was stirred at room temperature for 2 hours. Dialyzed against 0.02M PBS overnight, the resulting product (ZEN-HRP) was protected with an equal volume of glycerol and store...

Embodiment 2

[0030] Example 2 Preparation of horseradish peroxidase-labeled zearalenone

[0031] Weigh 0.5mg ZEN and 4.3mg carboxymethylhydroxylamine hydrochloride, dissolve in 0.3ml dimethyl sulfoxide, and stir at room temperature for 24h. Add 0.3ml of distilled water to the reaction solution, adjust the pH to 9 with 0.2M NaOH, and shake to clarify the solution. Extracted 3 times with benzene (0.3ml), adjusted the pH of the aqueous layer to 4 with 2M HCl, extracted 3 times with ethyl acetate (0.6ml), and dried in vacuo, the obtained carboxylated product was carboxylated zearalenone .

[0032] Weigh 0.2 mg of carboxylated zearalenone, 0.4 mg of succinimide and 1 mg of carbodiimide, dissolve in 0.7 ml of ethanol, and stir at room temperature for 1 h. Slowly add this reaction solution dropwise to 0.1M NaHCO containing 6.8mg HRP 3 The solution was stirred at room temperature for 2 hours. Dialyzed against 0.02M PBS overnight, the resulting product (ZEN-HRP) was protected with an equal volum...

Embodiment 3

[0033] Example 3 Preparation of horseradish peroxidase-labeled zearalenone

[0034] Weigh 0.4mg ZEN and 3.2mg carboxymethylhydroxylamine hydrochloride, dissolve in 0.2ml pyridine, and stir at room temperature for 24h. Add 0.2ml of distilled water to the reaction solution, adjust the pH to 8 with 0.2M NaOH, and shake to clarify the solution. Extracted 3 times with benzene (0.2ml), adjusted the pH of the aqueous layer to 3 with 2M HCl, extracted 3 times with ethyl acetate (0.5ml), and dried in vacuo, the obtained carboxylated product was carboxylated zearalenone .

[0035]Weigh 0.2 mg carboxylated zearalenone, 0.4 mg succinimide and 1 mg carbodiimide, dissolve in 0.7 ml DMF, and stir at room temperature for 1 h. The reaction solution was slowly added dropwise to 0.1M NaHCO3 solution containing 10.2 mg HRP, and stirred at room temperature for 2 h. After dialyzing overnight with 0.02M PBS, the resulting product (ZEN-HRP) was protected with an equal volume of glycerol and stored...

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Abstract

The invention discloses a synthesis process of horseradish peroxidase enzymelabeled zearalenone. In the synthesis process, a carboxylated product is obtained by carboxylating zearalenone, and the horseradish peroxidase enzymelabeled zearalenone is obtained by a condensation reaction of the carboxylated product and horseradish peroxidase enzyme. The preparation process of detecting the key component horseradish peroxidase enzymelabeled product in the CLIA (Chemiluminescence Immunoassay) field is a formidable difficult all along. Accordingly, the synthesis process of the horseradish peroxidase enzymelabeled zearalenone, which is provided by the invention, is simple and practical and has low cost and favorable market application prospects.

Description

technical field [0001] The invention relates to the field of synthesis technology of enzyme-labeled reagents, in particular to a synthesis technology of horseradish peroxidase-labeled zearalenone. Background technique [0002] Zearalenone (ZEN), also known as F2 toxin, is a fungal secondary metabolite with estrogen-like effects produced by Fusarium fungus, and belongs to 2,4-dihydroxybenzoic acid lactone compounds. The chemical name is 6-(10-hydroxy-6oxy-undecenyl) β-rapenolide, which is widely found in moldy corn, sorghum, wheat and other cereal crops and milk. [0003] Zearalenone has a strong estrogen effect. If feed contaminated with zearalenone is ingested, it can cause hyperestrogenism and severe reproductive tract symptoms in pigs, rats, mice, poultry and other animals and infertility. At the same time, zearalenone has been proved to have immunotoxicity, genotoxicity, cytotoxicity, etc., and it is suspected that the estrogen effect of zearalenone leads to the cause ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/62
Inventor 胡国茂唐宝军柴淑芳
Owner CHEMCLIN DIAGNOSTICS CO LTD
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