Method for detecting ADAAMTS13 enzyme activity in serum by SELDI-TOF-MS (surface-enhanced laser desorption/ionization-time of flight-mass spectrometry)

A SELDI-TOF-MS, enzyme activity technology, applied in the field of medical molecular biology, can solve problems such as differences in metalloprotease enzyme activity, achieve better treatment, improve survival rate and quality of life, and reduce treatment costs.

Inactive Publication Date: 2017-05-31
杨尚斌
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The clinical manifestations of these two diseases are very similar, but one of the biggest differences

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  • Method for detecting ADAAMTS13 enzyme activity in serum by SELDI-TOF-MS (surface-enhanced laser desorption/ionization-time of flight-mass spectrometry)

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[0024] In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

[0025] The invention discloses a method for detecting ADAMTS13 enzyme activity in serum by using SELDI-TOF-MS, comprising the following steps:

[0026] Include the following steps:

[0027] (1) Expression and purification of ADAMTS13 digestion substrate GST-vWF73-6XHis recombinant protein:

[0028] 1.1. The 73 amino acid fragments D1596-R1668 of vWF and 6 histidines (6XHis) at the C-terminus were inserted into the bacterial expression vector pGEX6p-1 with GST protein at the N-terminus;

[0029] 1.2. Select positive clones and transfect BL21-expressing bacteria;

[0030] The BL21 strain can effic...

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Abstract

The invention discloses a method for detecting the ADAAMTS13 enzyme activity in serum by SELDI-TOF-MS (surface-enhanced laser desorption/ionization-time of flight-mass spectrometry). The method comprises the following steps of (1) performing ADAAMTS13 enzyme digestion substrate GST-vWF73-6XHis recombination protein expression and purification; (2) taking an enzyme digestion reaction; (3) after an ADAAMTS13 enzyme digestion product is enriched by an IMAC chip, performing recognition and detection on the ADAAMTS13 enzyme digestion product; obtaining the enzyme digestion activity of ADAAMTS13 in the serum sample through analysis software. The detection method is fast and effective; the detection is precise; after the detection on all TMA (thrombotic microangiopathy) suspected cases, all cases with the ADAAMTS13 enzyme activity being less than 10 percent are screened out; the patients are treated as TTP (thrombotic thrombocytopenic purpura) patients. For all TTP cases, in the treatment process and the long-time monitoring after the hospital leaving, the regular detection of the ADAAMTS13 enzyme activity needs to be performed, so that the patient can be better and correctly treated; the treatment cost is reduced; the survival rate and the life quality of the patient are effectively improved.

Description

technical field [0001] The invention belongs to the technical field of medical molecular biology and protein polypeptide detection, and particularly relates to a method capable of detecting metalloprotease ADAMTS13 enzyme activity by using SELDI-TOF-MS, which is applied to TTP disease screening in TMA disease and TTP disease Treatment monitoring. Background technique [0002] SELDI-TOF-MS (Surface Enhanced Laser Desorption Ionization Time-of-Flight Mass Spectrometry) is a new type of mass spectrometry instrument developed by Ciphergen Corporation of the United States. It mainly consists of three parts: the first part is the protein chip (Protein Chip), which is its core technology; The second part is the time-of-flight mass spectrometer (TOF MS), which can also be called the chip reader (protein chip reader); the third part is the system-specific analysis software. [0003] The protein chip is the core technology of SELDI-TOF-MS. This detection method will use the metal aff...

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6851G01N33/6893
Inventor 杨尚斌
Owner 杨尚斌
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