Sepsis diagnosis method and reagent

A technology of sepsis and reagents, which is applied in the field of judgment and prognosis assessment of multiple organ dysfunction, and can solve problems such as poor prognosis and great difference

Inactive Publication Date: 2015-07-15
AFFILIATED CHILDRENS HOSPITAL OF CAPITAL INST OF PEDIATRICS +1
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Mitochondrial damage found in patients with severe sepsis prompts the search for new reliable biomarkers at the mitochondrial subcellular level that, combined with current biomarkers, may contribute to multiple organ dys

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment one Extraction of Mitochondria from Human Peripheral Blood Leukocytes

[0071] Take 2ml of human whole blood, add 8ml of lysate (0.1mM EDTA), process for 15min to break the red blood cells, centrifuge at 3000rpm for 10min, discard the supernatant, collect the precipitate that is the white blood cells, wash the white blood cells twice with normal saline, centrifuge at 3500rpm for 5min, discard the supernatant Clear, collect white blood cell pellet. Add 5ml of cell suspension (0.25M Sucrose, 5mM HEPES, 0.5mM EGTA, pH7.4) to the pellet to suspend the cells, homogenize 20 times with a glass homogenizer, centrifuge the homogenate at 1000g for 10 minutes, discard the pellet, and collect the supernatant Centrifuge the supernatant at 10,000 g for 10 minutes, discard the supernatant, and collect the precipitate as mitochondria, which can be stored at -80°C.

Embodiment 2

[0072] Embodiment two Preparation of Crude Enzyme Solution of Mitochondrial Respiratory Chain Supercomplex NCR

[0073] Suspend the mitochondria prepared in Example 1 with a suspension medium (containing 0.25M Sucrose, 5mM HEPES, 0.5mM EGTA, pH7.4), and then sonicate for 10s, with an interval of 10s, for a total of 10 sonications, and then use BCA The legal protein concentration is 0.5ug / ul, and the suspension is the crude enzyme solution containing the respiratory chain supercomplex NCR. After the concentration is determined, it is placed in an environment at 4°C for use.

Embodiment 3

[0074] Embodiment Three Enzyme Activity Determination of Mitochondrial NCR in Human Peripheral Blood Leukocytes

[0075] The mitochondrial NCR detection reagent of this embodiment is a two-dose reagent, including

[0076] Reagent 1

[0077] Phosphate buffer (pH7.2) 50mmol / L

[0078] Tween 20 0.1%

[0079] NADH0.1mmol / L

[0080] Oxidized Cytochrome C 0.1mmol / L

[0081] Reagent 2

[0082] Rotenone 0.02mmol / L

[0083] Samples of mitochondrial NCR to be tested For the crude enzyme solution containing mitochondrial respiratory chain supercomplex NCR prepared in Example 2, the dosage of the crude enzyme solution is 10 ug, and the reaction system is 200 ul. This detection method uses enzyme kinetics to detect mitochondrial NCR.

[0084] Determination of total activity: Place reagent 1 in a warm bath at 30°C for 3 minutes, then add reagent 1 into the microplate reader, perform a baseline scan at 550nm for 1 minute at 30°C, add protein samples to start the reaction...

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Abstract

The invention provides a method for sepsis diagnosis, severity degree monitoring, and prognosis evaluation. The enzymatic activity level of mitochondrial respiratory chain supramolecular complex: NADH-cytosome coxidoreductase, NCR in the peripheral blood leucocyte namely mitochondrial NCR enzymatic activity level is closely related with the organ dysfunction and prognosis of sepsis. The invention also provides a detection method and detection reagent for detecting the peripheral blood leucocyte mitochondrial respiratory chain supramolecular complex NCR enzymatic activity. The provided method can rapidly, specifically, and sensitively measure the mitochondrial NCR enzymatic activity of a sample, and has an important meaning on the sepsis diagnosis, severity degree monitoring, and prognosis evaluation.

Description

technical field [0001] The invention belongs to the technical field of criticality judgment and prognosis evaluation of infectious and inflammatory diseases, that is, the judgment and prognosis evaluation of multiple organ dysfunction in sepsis (Sepsis). Specifically, the present invention provides a detection method for sepsis diagnosis, criticality monitoring and prognosis assessment from the mitochondrial subcellular level, the detection method is mainly to measure the mitochondrial respiratory chain supercomplex NADH-cytochrome C redox Enzyme activity of enzyme (NADH: cytosome C oxidoreductase, NCR), at the same time, the invention also provides a method and reagent for analyzing and detecting the enzyme activity of mitochondrial NCR. Background technique [0002] Sepsis refers to the systemic inflammatory response syndrome (SIRS) caused by infection, which is a severe systemic inflammatory response induced by microorganisms (such as bacteria, fungi, viruses, parasites...

Claims

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Application Information

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IPC IPC(8): G01N21/31
Inventor 张琪朱伟文李宁郝淑静胡洁郭琳瑛李伟卢秀秀王志龙
Owner AFFILIATED CHILDRENS HOSPITAL OF CAPITAL INST OF PEDIATRICS
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