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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 the prospect of clinical PC activity detection, but experiments have proved that the activator partially activates other blood coagulation factors in plasma, and is susceptible to interference from various factors. The specificity is relatively low, and the activator has not been able to be applied to the PC assay kit at present
[0009] The above drawbacks make it difficult to promote the use of PC testing in hospitals, thus limiting the routine development of this testing project

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

Embodiment 1

[0040] The preparation of embodiment 1 protein C activator

[0041] (1) Take 1g of Qinling Agkistrodon halys venom dry powder (purchased from Shechang, 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 that was used in advance. In the DEAE-Sepharose FF anion exchange column equilibrated with liquid, after loading the sample, wash the column again with the above buffer solution to balance, so as to remove the remaining impurity proteins that are not bound to the column. Then use the same buffer solution containing 0-0.5mol / L NaCl for linear gradient elution, the flow rate is 1ml / min, and the detection wavelength is 280nm. Take 0.1ml of the eluate, add 0.1ml of normal mixed plasma and 0.1ml of 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, measure the coagu...

Embodiment 2

[0044] Embodiment 2 Preparation of kit of the present invention

[0045] The protein C activity assay kit in this example is a solid reagent, including R1 reagent and R2 reagent, prepared according to the following components and dosage respectively:

[0046] A) R1 reagent

[0047]

[0048] After the above reagents are completely dissolved, put them into bottles at 1ml / bottle, and make them into freeze-dried powders for use.

[0049] B) R2 reagent

[0050]

[0051]

[0052] After the above reagents are completely dissolved, put them into bottles at 1ml / bottle, and make them into freeze-dried powders for use.

Embodiment 3

[0053] Embodiment 3 The method that kit of the present invention detects protein C activity

[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%, respectively. %. Dissolve the solid R1 reagent in the kit prepared in Example 2 with 1 ml of distilled water, and dissolve the R2 reagent with 1 ml of distilled water. Take the operation of CA530 automatic hemagglutination analyzer in East Asia, Japan as an example: set the reaction temperature to 37°C and the measurement wavelength to 405nm, take 20μl of calibrator solutions with different concentrations, preheat 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 100s of the reaction, repeat the measurement 3 times for each tube, take the average of the absorbance △...

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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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