Kit for detecting enzyme activity of thiopurine S-methyltransferase

A technology of mercaptopurine methyl and transferase is applied in the fields of biomedicine, clinical diagnosis and detection, and can solve the problem that the enzyme activity detection method is not easy to popularize and so on.

Active Publication Date: 2016-05-11
山西盈科正欣生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Therefore, based on the defects of the above-mentioned prior art, in order to solve the problem that the enzyme activity detection method of existing thiopurine methyltransferase (TPMT, thiopurineS-methyltransferase, EC2.1.1.67) is no...

Method used

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  • Kit for detecting enzyme activity of thiopurine S-methyltransferase
  • Kit for detecting enzyme activity of thiopurine S-methyltransferase
  • Kit for detecting enzyme activity of thiopurine S-methyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1: Preparation of human red blood cell lysate

[0085] Take 1ml of peripheral venous whole blood from healthy volunteers, centrifuge at 3000rpm for 10min, discard the supernatant, take 100ul of red blood cell pellet at the bottom, add 3ml of normal saline (0.9% NaCl aqueous solution) and shake well, centrifuge at 3000rpm for 10min, discard the supernatant, and then Wash twice with normal saline, centrifuge at 3000rpm for 10min, discard the supernatant, and the collected precipitate is red blood cells. The collected red blood cells can be stored at -40°C for future use. Add 2 volumes of 20mmol / LTris-HCl buffer solution, pH 7.5, to one portion of red blood cell pellet, stir to break up the red blood cells, centrifuge at 10,000g for 10 minutes, and take the supernatant as the red blood cell lysate.

Embodiment 2

[0086] Example 2: Human red blood cell mercaptopurine methyltransferase activity assay (pre-processing)

[0087] The mercaptopurine methyltransferase detection reagent of this embodiment is three reagents, including

[0088] Reagent 1

[0089] Potassium phosphate buffer (pH7.5) 30mmol / L

[0090] SAM0.1mmol / L

[0091] Reagent 2

[0092] Potassium phosphate buffer (pH7.5) 30mmol / L

[0093] 6-TG0.2mmol / L

[0094] Reagent 3

[0095] Potassium phosphate buffer (pH7.5) 30mmol / L

[0096] S-adenosyl-L-homocysteine ​​hydrolase 10KU / L

[0097] The sample of mercaptopurine methyltransferase to be tested is the red blood cells of healthy adults. After crushing, add sodium cholate solution to the sample to make the final volume percentage concentration reach 0.5%, mix gently, and place on ice 30min for use. The total protein dosage in the reaction system is 25ug, and the reaction system is 250ul. A total of 5 parallel samples were made. This detection method uses the end point...

Embodiment 3

[0104] Example 3: Determination of human red blood cell purine methyltransferase activity (comparison of pre-treatment of samples)

[0105] The mercaptopurine methyltransferase detection reagent of this embodiment is three reagents, including

[0106] Reagent 1

[0107] Potassium phosphate buffer (pH7.5) 30mmol / L

[0108] SAM0.1mmol / L

[0109] Reagent 2

[0110] Potassium phosphate buffer (pH7.5) 30mmol / L

[0111] 6-TG0.2mmol / L

[0112] Reagent 3

[0113] Potassium phosphate buffer (pH7.5) 30mmol / L

[0114] S-adenosyl-L-homocysteine ​​hydrolase 10KU / L

[0115] The sample of mercaptopurine methyltransferase to be tested is the mixed red blood cells of healthy adults. After crushing, it is divided into two parts, one of which is added to the sample with sodium cholate solution to make the final concentration reach 0.5%, and mixed gently. After homogenization, place it on ice for 30 minutes; add the same amount of sample suspension to the other part, mix gently and plac...

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Abstract

The invention provides a combination product for detecting enzyme activity of thiopurine S-methyltransferase. The combination product contains S-adenosyl-L-methionine or an enzymatic reaction system producing the S-adenosyl-L-methionine, a methyl receptor and S-adenosyl-L-homocysteine hydrolase, wherein the enzymatic reaction system producing the S-adenosyl-L-methionine comprises adenosine triphosphate, methionine and S-adenosyl methionine synthetase. Preferably, the combination product is in the form of kit, wherein each ingredient contained in the combination product is in a separately self-existent reagent state. The combination product can be used on semi-automatic or fully-automatic analyzing detection equipment, the detection sensitivity is high, the specificity is high, and the operation is simple and convenient, so that the combination product can be practically popularized.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and at the same time belongs to the field of clinical diagnosis and detection. Specifically, the present invention provides a combination product for detecting the enzyme activity of thiopurine methyltransferase (TPMT, thiopurineS-methyltransferase, EC2.1.1.67). Background technique [0002] Mercaptopurine methyltransferase (TPMT) is an intracellular enzyme that specifically catalyzes the sulfhydryl methylation of aromatic and heterocyclic compounds with S-adenosyl-L-methionine as the methyl supplier , to catalyze the sulfur atom methylation of aromatic and heterocyclic compounds. [0003] TPMT was originally found in the kidney and liver of mice, and later found to be distributed in most tissues, such as heart, blood cells, placenta, pancreas, small intestine, etc., and TPMT was also found in these tissues and red blood cells of humans. The structure and biological activity of TPMT are pol...

Claims

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Application Information

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IPC IPC(8): C12Q1/48C12Q1/34
CPCC12Q1/34C12Q1/48G01N2333/91017G01N2333/91171
Inventor 菅晓勇王天泽
Owner 山西盈科正欣生物科技有限公司
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