Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for detecting 6-methylmercaptopurine

A detection method, the technology of methylmercapto, applied in the field of 6-purine detection, can solve the problems of complex 6-MMP method, and achieve the effects of high sensitivity, accurate results and convenient operation

Active Publication Date: 2012-08-01
石家庄利德康医药科技有限公司
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention is to overcome the complex defects of the detection method of 6-MMP existing in the prior art, and uses the immunoassay reagent developed by the 6-MMP specific antibody to make up for it.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detecting 6-methylmercaptopurine
  • Method for detecting 6-methylmercaptopurine
  • Method for detecting 6-methylmercaptopurine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Preparation of anti-6-MMP specific antibody

[0069] 1. Synthesis of 6-MMP derivatives, the chemical structure of which is shown in formula (II).

[0070]

[0071] The synthetic approach and method of this 6-MMP derivative are as follows:

[0072]

[0073] (1) Dissolve 5.0g 6-MMP and 4.45g K with 30ml DMF 2 CO 3 , add 10ml of DMF solution containing 4.06g of ethyl 6-bromohexanoate, and stir the reaction at 40°C for 30min.

[0074] (2) The reacted solution was then neutralized with 1N HCl solution, and extracted with ethyl acetate. The organic phase was dried, filtered, dried in vacuo and purified on normal phase silica gel. 1.5 g of the purified product was dissolved in 25 ml of methanol, then an aqueous solution containing 600 mg of LiOH.H2O was added, and the reaction was stirred at room temperature for 4 hours. After concentration, the product was diluted with water and washed with ether, and the pH of the aqueous phase solution was adjusted to 5...

Embodiment 2

[0083] Example 2 Preparation of homogeneous enzyme immunoassay reagent for 6-MMP

[0084] (1) Preparation of R1 reagent: Dilute the prepared antibody into R1 buffer, the homogeneous R1 buffer contains 50 mM Tris, 0.25% BSA, 50 mM G-6-P and 50 mM NAD. The volume ratio of antibody to R1 buffer was 1:1000.

[0085] (2) Preparation of R2 reagent

[0086] 1) Preparation of G6PDH-6MMP

[0087] a) Weigh 15mg G6PDH, dissolve it in 12ml, 0.05M Tris buffer, add 100mg NADH, 0.5ml carbitol and 1ml DMF in sequence and mix well;

[0088] b) Dissolve 10 mg of 6-MMP derivative in 420 μl dimethyl sulfoxide and 180 μl DMF, add 6 μl tributylamine and 3 μl isobutylchloroformate, and stir the reaction at 2-8°C 30min;

[0089]c) Stir overnight at 2-8° C., and purify the obtained G6PDH-6MMP.

[0090] 2) Dilute the prepared G6PDH-6MMP into R2 buffer. R2 buffer was 100 mM Tris, 0.25% BSA. The volume ratio of antibody to R2 buffer was 1:1000.

Embodiment 3

[0091] Example 3 Homogeneous enzyme immunoassay method and calibration results of 6-MMP

[0092] Table 1 Hitachi 7180 analyzer 6-MMP homogeneous enzyme immunoassay parameter list

[0093]

[0094] Through the automatic biochemical analyzer, set the parameters according to the data in Table 1. First add the sample, then add the R1 reagent (the mixture of the antibody and the R1 buffer) in Example 2, and finally add the R2 reagent (the mixture of the G6PDH-6MMP conjugate and the R2 buffer), and measure the OD340 at different time points Absorbance value, calculate the absorbance change rate of different concentrations of standard substances, and obtain a more ideal reaction standard curve, the results are as follows figure 1 shown.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
absorbanceaaaaaaaaaa
Login to View More

Abstract

The invention discloses a homogeneous phase enzyme immuno-detection method for 6-methylmercaptopurine (6-MMP), a method for detecting an enzyme linked immuno-adsorbent and a detection reagent used by the two methods. According to the two detection methods, whether the 6-MMP exists in a sample to be detected can be determined by using the detection reagent developed from an antibody for resisting 6-MMP specificity, and the content of the 6-MMP can be quantitatively determined. Compared with the conventional genotyping and high performance liquid chromatography (HPLC) method, the immuno-detection method has the advantages of simplicity and convenience and quickness in operation, accurate detection result, low cost and the like, and has a very good prospect for large-scale clinical popularization and application in the future, particularly in middle-small hospitals lack of expensive instruments.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a detection method for 6-(methylmercapto)purine. Background technique [0002] 6-(methylmercapto) purine (6-Methylmercaptopurine, 6-MMP), its structural formula is shown in formula (I). [0003] [0004] 6-MMP is a metabolite of 6-(mercapto)purine (6-Mercaptopurine, 6-MP). Clinically, 6-MP is widely used in the treatment of many important diseases, including: acute leukemia, organ transplantation and some autoimmune diseases, etc., but if this drug is used improperly, it can cause serious or even life-threatening blood toxicity, according to Mardini [1] It is recommended to determine the dosage of 6-MP by measuring the concentration of its metabolite 6-MMP in the patient's blood when using 6-MP. The results of the literature show that when the concentration of 6-MMP in the blood is less than 0.6 μg / mL, The dosage cannot reach the relevant curative effect; and when the concentration...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/53G01N33/535C07K16/06C07K14/765C07K14/795C07K14/435
Inventor 虞留明田军袁红霞蔡江丽
Owner 石家庄利德康医药科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com