Novel method for analyzing human thymidine kinase fluorescence immune based on magnetic nanometer particular

A fluorescent immunoassay and magnetic nanoparticle technology, which is applied in the field of clinical medical determination methods and detection analysis, can solve the problems of complicated process and long time, and achieve the effect of simple operation, simple method and simple detection method

Inactive Publication Date: 2011-08-10
丁克祥
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the effect of this washing and separation process is better, the process is more complicated and the time is longer.

Method used

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  • Novel method for analyzing human thymidine kinase fluorescence immune based on magnetic nanometer particular
  • Novel method for analyzing human thymidine kinase fluorescence immune based on magnetic nanometer particular
  • Novel method for analyzing human thymidine kinase fluorescence immune based on magnetic nanometer particular

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Effect test

Embodiment 1

[0054] Preparation of hTK1-coated core-shell magnetic particles

[0055] ① Preparation of Magnetic Nanoparticles (MNPs): Accurately weigh 0.59g FeCl 3 ·H 2 O and 0.40FeCl 2 ·4H 2 O was dissolved in 4 ml of 2M HCl to give concentrations of 0.82M and 0.43M, respectively. Quickly mixed in a 250ml three-necked flask, then rapidly added dropwise 40ml of 1M ammonia water, added 0.0202g ascorbic acid (analytical grade) after 5min, stirred for 30min, statically layered and removed the supernatant, added 1.5ml of 25% tetramethyl hydrogen Ammonium oxide (analytical grade) solution was stirred for 10min, distilled water was added to make the total volume 40ml, ultrasonically oscillated for 15min, filtered with a sand funnel to remove large particles to obtain magnetic nanoparticles, and a gray-brown magnetic nanoparticle solution was obtained.

[0056] ②Aminosilanization of magnetic nanoparticles: Mix 1ml of magnetic nanoparticles with 100ml of isopropanol and sonicate for 30min, add...

Embodiment 2

[0062] Detection of hTK1

[0063] Take 20 μl of hTK1 solutions of different concentrations (0-2.0ng / ml) and 10 μl of 50ng / ml horseradish peroxidase-labeled hTK1 monoclonal antibody (by immunizing Balb / C with human thymidine kinase expressed and purified in E. coli). Mice, immunized mice spleen cells were fused with myeloma cells, positive clones were selected and cultured by HAT and ELISA, and the hybridomas of positive clones were injected into the peritoneal cavity of mice to prepare ascites, and then purified and prepared) for 40 minutes, and then 10 μl of magnetic nanoparticles were added to label hTK (hTK1-MNPs), reacted at 37°C for 40 min, washed three times with phosphate buffer solution (pH=7.4), separated under the action of a magnetic field, dispersed in 10 μl pH=7.4 phosphate buffer solution, and added sequentially 150μl pH5.5 Phosphate Buffer, 20μl 4.4×10 -4 M HPPA and 20 μl 8.8 x 10 -4 M H 2 O 2 , react for 30min, under the action of a magnetic field, use a mi...

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Abstract

The invention relates to a new fluoroimmunoassay method for human thymidine kinase fluoroimmunoassay based on magnetic nanometer grains. The invention sets up a quick and simple immunological detecting technology for the high flux human thymidine kinase (hTK1), and the technology is used for the hTK1 clinical examination. The hTK1 is fixed with covalence on the surface of an amido silanization superparamagnetism nanometer grain immobilized carrier through glutaric dialdehyde, a competitive immunoassay method is adopted, horse radish peroxidase is used as an enzyme labeling, ethyl-para-hydroxyphenyl acid is used as a fluorogenic substrate, thereby realizing the detecting for the hTK1. The amido silanization nucleocapsid-shaped magnetic nanometer grains are evenly dispersed in liquidoid, thereby having the advantages of fixation and uniformity, high specificity, good repeatability, quick reaction velocity, etc., under the action of an adscititious magnetic field, the invention can effectively realize the separation and enrichment of the immune complex and the reaction liquid of the nucleocapsid-shaped magnetic nanometer grains, the operation of the analytical method is simple, accurate, sensitive and fast, automatic detection is easy to be realized, and large batch of test task can be completed.

Description

technical field [0001] The present invention relates to clinical medical measurement methods and detection analysis, in particular to a new method for human thymidine kinase fluorescent immunoassay based on magnetic nanoparticles, that is, using the combination of magnetic nanotechnology and modern fluorescent immunoassay technology to detect human type I thymidine kinase (hTK1) Novel method for accurate detection of blood and interstitial fluids. Background technique [0002] Technical background about human thymidine kinase type I (hTK1): [0003] Human type I thymidine kinase, abbreviated as human type I thymidine kinase in Chinese; its English name is Human thymidine kinase, abbreviated as hTK1. Before DNA is synthesized, thymidine (TdR) is taken into the cell and must be catalyzed by phosphorylation kinase when converted into TMP, and the phosphorylation reaction process must be catalyzed by TK enzyme. [0004] Under normal physiological conditions, TK in human cells ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/532G01N33/577
Inventor 丁克祥吴朝阳刘卫国费定宇刘莉丁宇
Owner 丁克祥
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