Method for detecting activity of O6-methylguanine-DNA (Deoxyribose Necleic Acid) methyltransferase

A technology of methyltransferase and methylguanine, applied in measuring devices, instruments, scientific instruments, etc.

Inactive Publication Date: 2014-11-26
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite this, so far there is no report on the application of BRET to measure MGMT activity

Method used

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  • Method for detecting activity of O6-methylguanine-DNA (Deoxyribose Necleic Acid) methyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 O 6 -Methylguanine-DNA methyltransferase (MGMT) biotin labeling

[0031] In 4ml TBS buffer solution (pH 6.4) containing 4mg MGMT to be tested (taken from tumor cells) was added 1ml containing 2mg biotin-labeled O 6 -TBS buffer solution of benzylguanine (pH 6.4), mix well and place in a water bath at 37°C for 4 hours. After the reaction is over, add the reaction solution to a 14KD dialysis bag, and use a TBS buffer solution of pH 6.4 as the dialysate Dialysis was performed at 4°C, and the dialysate was changed every 3 hours, for a total of three changes, then the retentate was filtered with cross-linked agarose gel CL-6B, the filtrate was taken, and freeze-dried at 4°C for 10 hours to obtain biotin-labeled O 6 -Methylguanine-DNA methyltransferase.

Embodiment 2

[0032] Example 2 Detection of MGMT activity

[0033] (1) Coating: The streptavidin-firefly luciferase fusion protein was prepared with a 0.1mol / L TBS buffer solution with a pH of 6.4 to make 50ml of a 1mg / mL coating solution. Add the coating solution to the wells of a 96-well white-bottomed polystyrene microtiter plate, 200μl per well, place at 37°C for 60 minutes, discard the coating solution, and add 200μl of 10% bovine serum albumin (BSA) to each well. The TBS buffer solution of) was used as the blocking solution, sealed at 37°C for 1 hour, and the plate was washed with the above TBS buffer solution, soaked for 3 minutes each time, washed three times, dried under vacuum at 30°C, and stored in a closed bag under vacuum to prepare Microplate coated with streptavidin-firefly luciferase fusion protein;

[0034] (2) Add samples: add the streptavidin-firefly luciferase fusion protein-coated microtiter plate prepared in step (1) to the sample holes of the microtiter plate prepared in ...

Embodiment 3

[0039] Example 3 Detection of MGMT activity

[0040] (1) Coating: The streptavidin-alkaline phosphatase fusion protein was used to prepare 50 ml of a 3 mg / mL coating solution with a 0.5 mol / L TBS buffer of pH 6.4. Add the coating solution to the loading wells of a 96-well white-bottomed polystyrene microtiter plate, 200μl per well, and place at 25°C for 1h. Discard the coating solution, and add another 200μl mass concentration of 5% bovine serum albumin to each well ( BSA) the above TBS buffer as a blocking solution, blocked at 37°C for 1 hour, washed with the above TBS buffer, soaked for 3 minutes each time, washed the plate 3 times, dried under vacuum at 25°C, and stored in a closed bag under vacuum. Streptavidin-alkaline phosphatase fusion protein;

[0041] (2) Sample addition: Add the concentration of 1000, 500, 250, 125, 62.5, 62.5, 1000, 500, 250, 125, 62.5, to the sample well of the microtiter plate coated with streptavidin-alkaline phosphatase fusion protein prepared in st...

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Abstract

The invention discloses a method for detecting the activity of O6-methylguanine-DNA (Deoxyribose Necleic Acid) methyltransferase. The method is that the activity of O6-methylguanine-DNA (Deoxyribose Necleic Acid) methyltransferase is detected by adopting a bioluminescence resonance energy transfer technology and a chemiluminescence technology for the first time, and comprises the steps of incubating streptavidin-luciferase fusion protein and O6-methylguanine-DNA methyltransferase labeled by biotin for 30-60 minutes at the temperature of 0-40 DEG C, then adding O6-methylguanine-DNA methyltransferase antibody labeled by fluorochrome, incubating for 30-60 minutes at the temperature of 0-40 DEG C, then adding a fluorescein substrate, standing for 5-30 minutes at the temperature of 4-30 DEG C, detecting the lighting intensity at the position of 670nm, and further calculating to obtain the activity of O6-methylguanine-DNA methyltransferase. According to the method disclosed by the invention, the operation is simple and convenient, the applicable range is wide, the scattering caused by exogenous exciting light and the interference of backgrounds such as sample matrix fluorescence can be avoided, and the sensitivity and the accuracy are high.

Description

(1) Technical field [0001] The present invention relates to an O 6 -Methylguanine-DNA methyltransferase activity detection method, especially involving the use of bioluminescence resonance energy transfer technology and chemiluminescence technology immunoassay O 6 -Methylguanine-DNA methyltransferase activity method. (2) Background technology [0002] Alkylating agents are the largest class of anti-tumor drugs. Commonly used clinical examples include cyclophosphamide, nimustine, carbachol, nitrocaine, nitrogen mustard and temozolomide. They are widely used in the treatment of brain, head and neck, liver, and esophagus. , Lung, urinary, reproductive, blood and other malignant tumors. Alkylating agent drugs kill tumor cells mainly through the DNA molecule guanine O 6 Is achieved by alkylation at the position, and O 6 -Methylguanine-DNA methyltransferase (O 6 -Methylguanine DNA-Methyltransferase, MGMT) can remove this alkyl adduct, so that tumor cells and normal cells can survive an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/573
Inventor 陈灿玉梁媛媛吕常庆张现侠黄志坚章鹏飞
Owner HANGZHOU NORMAL UNIVERSITY
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