Kit (Developing substrate method) for testing protein C (PC)

A kit and reagent technology, which is applied in the field of kits for detection of protein C activity by chromogenic substrate method, can solve the problems that activators cannot be mass-produced and applied, restrictions on the routine development of detection items, and difficult promotion and use of PC detection, etc. Achieve the effect of promoting routine development, low production cost, and not easy to interfere

Active Publication Date: 2012-09-26
SHANGHAI SUNBIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the activators used in such kits currently on the market are isolated and purified from the venom of Agkistrodon contortrix, which is mostly distributed in the southeastern United States and northeastern Mexico. It is relatively difficult to obtain this snake venom in China, so this activator cannot be mass-produced and applied in China
Recently, it has been reported in the literature that the PC activator isolated and purified from Agkistrodon halys venom has t

Method used

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  • Kit (Developing substrate method) for testing protein C (PC)
  • Kit (Developing substrate method) for testing protein C (PC)
  • Kit (Developing substrate method) for testing protein C (PC)

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0040] Example 1 Preparation of protein C activator

[0041] (1) Take 1g of Qinling Agkistrodon crude snake venom dry powder (purchased from the snake farm of Ningdong Forestry Bureau, Shaanxi Province), dissolve it in 10ml 20mM PBS buffer (pH7.5), let it stand, take the supernatant and add it to the same buffer In the liquid-equilibrated DEAE-Sepharose FF anion exchange column, after loading the sample, rinse the column again to equilibrium with the above-mentioned buffer, so as to remove the remaining contaminant proteins that are not bound to the column. Then use the same buffer containing 0-0.5mol / L NaCl for linear gradient elution, with a flow rate of 1ml / min and a detection wavelength of 280nm. Take 0.1ml eluate, add 0.1ml normal mixed plasma and 0.1ml APTT reagent (cephalin-kaolin) pre-warmed at 37°C, mix well and incubate at 37°C for 5min, then add 0.025mol / L pre-warmed at 37°C CaCl 2 After 0.1ml of the solution, determine the coagulation time. If prolonged, it means tha...

Example Embodiment

[0044] Example 2 Preparation of the kit of the invention

[0045] The protein C activity assay kit in this example is a solid reagent, including R1 reagent and R2 reagent, which are prepared according to the following ingredients and dosages:

[0046] A) R1 reagent

[0047]

[0048] After all the above reagents are completely dissolved, 1ml / bottle is filled into bottles and used as freeze-dried powder.

[0049] B) R2 reagent

[0050]

[0051]

[0052] After all the above reagents are completely dissolved, 1ml / bottle is filled into bottles and used as freeze-dried powder.

Example Embodiment

[0053] Example 3 Method for detecting protein C activity by the kit of the present invention

[0054] Take protein C calibrator (purchased from American ANIARA company, article number: A222101), and prepare 6 calibrator solutions with different activities with normal saline, the activities are 150%, 100%, 75%, 50%, 25%, 0 %. The solid R1 reagent in the kit prepared in Example 2 was dissolved with 1 ml of distilled water, and the R2 reagent was dissolved with 1 ml of distilled water. Take the operation of the Japan East Asia CA530 automatic blood coagulometer as an example: set the reaction temperature to 37°C, the measurement wavelength is 405nm, and take 20μl of calibrator solutions of different concentrations. After preheating for 60s, add 125μl of R1 reagent, and add R2 after 300s of reaction. Reagent 30μl, measure the absorbance difference (△OD) between the 11th and 100th s of the reaction. Repeat the measurement 3 times for each tube. The average value of the absorbance △OD...

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Abstract

The invention discloses a kit for detecting the activity of a protein C. The kit for detecting the activity of the protein C provided by the invention comprises a protein C activating agent obtained by separating and purifying venom of vipers in Qinling Mountains of China, and a developing substrate reagent, and belongs to detection by a developing substrate method. The kit has good detection specificity and is not easy to be interfered; and raw materials are easy to obtain, the preparation is simple, the operation is simple and fast in a detection process, and an instrument applicable range is wide, so that the kit is convenient to popularize and use in each level of hospitals, hygiene departments and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting protein C activity by a chromogenic substrate method. Background technique [0002] Protein C (referred to as PC) is a vitamin K-dependent protein, in Ca 2+ In the presence of conditions, it can be activated by thrombin-thrombin regulatory protein and converted into active protein C (abbreviated as APC). APC can inactivate activated blood coagulation factors Va (referred to as FVa) and Ⅷa (referred to as FⅧa) through proteolysis, thus showing strong anticoagulant activity; at the same time, it can promote fibrinolysis by releasing vascular plasminogen activator . The lack of PC will affect the balance of blood coagulation and fibrinolysis in the human body, and cause hypercoagulation, resulting in thrombotic diseases. [0003] Decreased plasma PC levels predispose to disseminated intravascular coagulation (DIC) and liver diseases such as cirrhosis and chronic h...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 谢永华
Owner SHANGHAI SUNBIO TECH
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