Detection method for medicine with protease inhibition action

A detection method and protease inhibitor technology are applied in the field of drug detection, which can solve the problems of low detection sensitivity and complicated detection technology operation, and achieve the effects of high sensitivity, reduced cost and high purity

Inactive Publication Date: 2006-12-13
爱华生物科技(香港)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In order to achieve high yield, ensure the high purity and high activity of the product, simplify the process and reduce the cost at the same time, the present invention provides a preparation method of a drug capable of inhibiting protease. Situation, the present invention provides a kind of detection method that is simple and easy, the detection method of the drug that has the function of inhibiting protease with high detection sensitivity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] After Cohn's Component IV was dissolved with 20 times the weight of purified water, 37% sucrose and 0.38M sodium citrate were added as stabilizers for virus inactivation treatment. Then tributyl phosphate with a final concentration of 0.3% and Triton X-100 with a final concentration of 1.0% were added and incubated at 30° C. for 4 hours. After virus inactivation, adjust the pH to 6.5, reduce the ionic strength to 2.5mS / cm with water, and add it to a DEAE Sepharose Fast Flow chromatography column pre-balanced at pH 6.5. The target protein α1-PI was selectively eluted with 20mM sodium dihydrogen phosphate and 100mM sodium chloride solution. Increasing the concentration of sodium chloride can elute strongly adsorbed foreign proteins, including ceruloplasmin, from the ion-exchange chromatography column. DEAE Sepharose Fast Flow resin can be reused after being sterilized with 0.5M sodium hydroxide.

[0031] The eluate containing α1-PI was passed through a 10 kD ultrafiltra...

Embodiment 2

[0036] The dissolved Cohn's fraction IV was adjusted to pH 5.4 with 1M hydrochloric acid and ionic strength to 1.5mS / cm with water, and then added to a CM Sepharose Fast Flow column pre-equilibrated with 5mM sodium citrate (pH 5.4). Collect the effluent from the ion exchange resin, adjust the pH to 6.5, adjust the ionic strength to 2.5mS / cm, and add it to a DEAE Sepharose Fast Flow chromatography column pre-balanced with 20mM sodium dihydrogen phosphate solution (pH 6.5). Active α1-PI was selectively eluted using 20 mM sodium dihydrogen phosphate and 100 mM sodium chloride solution. In this example, 58.9 mg of α1-PI with a purity of 92.5% was purified from 5.34 g of fraction IV. The yield of active α1-PI was as high as 81.2%. Table 3 is the experimental data of this example.

[0037] Total protein (mg)

[0038] All the other are identical with embodiment 1.

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Abstract

The invention relates to a method for making and detecting protease inhibitor. The method comprises steps of: deactivating the water solution that contains alpha1-protease inhibitor with virus; ion exchanging resin absorbing and eluting; making serine proteinases react with alpha1-protease inhibitor to change wave length absorbed by light; measuring activity of thealpha1-protease inhibitor quantitatively. The invention has the advantages of being able to ensure product high purity and high activity.

Description

[0001] The application of the present invention is a divisional application of the application document "a preparation method and detection method of a drug capable of inhibiting protease". The application number of the original application is 2004100310574, and the application date is April 12, 2004. 【Technical field】 [0002] The invention belongs to the field of protein detection, and in particular relates to a detection method of a drug capable of inhibiting protease. 【Background technique】 [0003] α1-Protease Inhibitor (also known as α1-antitrypsin, alpha-1 Antitrypsin, α1-PI for short) is a glycoprotein with a molecular weight of about 53,000 Daltons, which contains an average of 1.5 grams per liter of plasma. The protein mainly inhibits the decomposition of elastase and other serine proteases in the body. When the concentration of active α1-PI in the body is significantly lower than the concentration of serine proteases, as occurs in people with a genetic defect of α...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31C12Q1/37
Inventor 黄炳镠谢亦武司徒子杰
Owner 爱华生物科技(香港)有限公司
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