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Method for determining activity of IMPDH in human peripheral blood

A single nucleotide dehydrogenase and hypoxanthine technology, applied in the field of medical testing, can solve the problems of large variation and lack among individuals, and achieve the effect of high sensitivity and convenient operation

Inactive Publication Date: 2020-01-07
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Clinical practice shows that there is a certain correlation between the plasma concentration of IMPDH inhibitors and clinical efficacy, but the inter-individual variability of this correlation is relatively large. Determination of IMPDH activity can be used as an effective means to monitor the drug effect, but the current clinical In the detection, there is still a lack of accurate, sensitive and simple methods for the determination of IMPDH enzyme activity in peripheral blood

Method used

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  • Method for determining activity of IMPDH in human peripheral blood
  • Method for determining activity of IMPDH in human peripheral blood
  • Method for determining activity of IMPDH in human peripheral blood

Examples

Experimental program
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Embodiment 1

[0037] Embodiment 1: Determination of IMPDH enzyme activity in the peripheral blood of healthy volunteers

[0038] Blood sample pretreatment: Take 2.0mL heparin anticoagulated venous blood and add 2.0mL PBS buffer to mix, add to the upper layer of 3.0mL lymphocyte separation medium, centrifuge at 400g for 40min at 20°C, remove the mononuclear cell layer, add 6mL PBS, Pipette to suspend the cells, centrifuge at 500g for 10min at 20°C, discard the supernatant, add 3mL of PBS to wash again, add 200μL of ultra-filtered water to the obtained cell pellet, pipette and mix well, then dispense into 1.5mL EP tubes, each The volume of the tube is 50 μL, and it is frozen at -80°C for 30 minutes;

[0039] Take 50 μL of PBMC cryopreservation solution, after thawing at room temperature, add 150 μL of incubation solution, the concentration of substrate IMP is 1.0 mmol / L, and the concentration of coenzyme NAD is 1 mmol / L; take another 6 parts of 50 μL 1mg / mL BSA and add 150 μL of BSA without s...

Embodiment 2

[0052] Example 2: Determination of IMPDH enzyme activity in peripheral blood of kidney transplant patients

[0053] Blood sample pretreatment:

[0054] Take 2.0 mL of heparin anticoagulated venous blood and add 2.0 mL of PBS buffer to mix well, add to the upper layer of 3.0 mL of lymphocyte separation medium, centrifuge at 400 g for 40 min at 20 °C, and remove the mononuclear cell layer. Add 6mL of PBS, pipette to suspend the cells, centrifuge at 500g for 10min at 20°C, discard the supernatant, add 3mL of PBS to wash again, add 200μL of ultra-filtered water to the obtained cell pellet, pipette and mix well, then dispense to 1.5mL EP tubes, each with a volume of 50 μL, and frozen at -80°C for 30 minutes;

[0055] Take 50 μL of PBMC cryopreservation solution, after thawing at room temperature, add 150 μL of incubation solution, the concentration of substrate IMP is 0.5 mmol / L, and the concentration of coenzyme NAD is 0.5 mmol / L; take another 6 parts of 50 μL 1mg / mL BSA and add ...

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Abstract

The invention belongs to the field of medical detection and relates to an analytical determination method for in vivo enzyme activity, in particular to a method for determining the activity of IMPDH in human peripheral blood. The method comprises the step of detecting human blood samples by using an ultraviolet detector after peripheral blood mononuclear cell extraction and in vitro enzymatic reaction and liquid chromatographic column separation. The method is fewer in sampled samples, high in determination speed, high in specificity, accurate in analysis result and high in precision, is applicable to clinical pharmacodynamics study on an IMPDH inhibitor and contributes to promotion of individualized administration of related drugs.

Description

technical field [0001] The invention belongs to the technical field of medical examination, and relates to an analysis and determination method for enzyme activity in vivo, in particular to a method for determining the activity of hypoxanthine mononucleotide dehydrogenase in human peripheral blood. Background technique [0002] The prior art discloses that inosine 5'-monophosphatedehydrogenase (IMPDH) is a nicotinamide adenine dinucleotide (nicotinamide adeninedinucleotide, NAD)-dependent enzyme, which is widely present in all differentiations of the body. cell. Studies have shown that IMPDH is the rate-limiting enzyme in the process of purine metabolism, which can catalyze the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), and then synthesize guanine nucleotides. It plays an important role in cell growth, differentiation and apoptosis; when IMPDH is inhibited, it can lead to the consumption of intracellular guanosine triphosphate storage, slow...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/74G01N30/06
CPCG01N30/02G01N30/06G01N30/74G01N2030/027G01N2030/8822
Inventor 焦正刘飞燕盛长城徐璐扬
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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