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

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

Inactive Publication Date: 2012-11-28
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

[0031] Example 1 O 6 -Biotin labeling of methylguanine-DNA methyltransferase (MGMT)

[0032] Add 1 ml of 2 mg of biotin-labeled O 6 - TBS buffer solution of methylguanine (pH 6.4), mix well and place in water bath at 37°C for 4 hours. After the reaction, add the reaction solution into a 14KD dialysis bag, and use TBS buffer solution with pH 6.4 as the dialysate Dialyze at 4°C, change the dialysate every 3 hours, and change three times in total, then filter the retentate with cross-linked agarose gel CL-6B, take the filtrate, freeze-dry at 4°C for 10 hours, and obtain biotin-labeled O 6 -Methylguanine-DNA methyltransferase.

Embodiment 2

[0033] Embodiment 2 detects MGMT activity

[0034] (1) Coating: The streptavidin-firefly luciferase fusion protein was prepared into 50ml of 1mg / mL coating solution with 0.1mol / L TBS buffer solution with pH 6.4. Add the coating solution to the sample wells of a 96-well white-bottomed polystyrene microwell plate, 200 μl per well, place at 37°C for 60 minutes, discard the coating solution, and add 200 μl of 10% bovine serum albumin (BSA) to each well ) above TBS buffer solution as blocking solution, blocked at 37°C for 1 hour, washed the plate with the above TBS buffer solution, soaked for 3 minutes each time, washed the plate three times, dried in vacuum at 30°C, put it into a sealed bag for vacuum storage, and prepared Microwell plates coated with streptavidin-firefly luciferase fusion protein;

[0035] (2) Adding samples: add the concentration of 1000, 500, 250 , 125, 62.5, 31.2, 15.6, 7.8, 3.4, 0ng / mL and unknown concentration (sample to be tested, taken from a tumor tissu...

Embodiment 3

[0040] Embodiment 3 detects MGMT activity

[0041] (1) Coating: The streptavidin-alkaline phosphatase fusion protein was prepared with 0.5mol / LTBS buffer solution with pH 6.4 to prepare 50ml of 3mg / mL coating solution. Add the coating solution to the sample wells of a 96-well white-bottomed polystyrene microwell plate, 200 μl per well, place at 25°C for 1 hour, discard the coating solution, and add 200 μl of 5% bovine serum albumin ( The above-mentioned TBS buffer solution of BSA) was used as the blocking solution, blocked at 37°C for 1 hour, washed with the above-mentioned TBS buffer solution, soaked for 3 minutes each time, washed the plate three times, dried in vacuum at 25°C, and stored in a sealed bag in vacuum to obtain streptavidin-alkaline phosphatase fusion protein;

[0042] (2) Adding samples: Add concentrations of 1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.4, 0 ng / mL and unknown concentration (sample to be tested, taken from a tumor tissue sample) 50 μl of TBS ...

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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 -A detection method for methylguanine-DNA methyltransferase activity, particularly involving the use of bioluminescence resonance energy transfer technology and chemiluminescence technology for immunodetection O 6 - Method for methylguanine-DNA methyltransferase activity. (2) Background technology [0002] Alkylating agents are the largest class of antineoplastic drugs. Commonly used clinical examples include cyclophosphamide, nimustine, carbazine, nitromustine, 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. The killing of tumor cells by alkylating agent drugs is mainly through the guanine O of DNA molecules. 6 achieved by alkylation on the position, while the O 6 -Methylguanine-DNA methyltransferase (O 6 -Methylguanine DNA-Methyltransferase, MGMT) can remove this alk...

Claims

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Application Information

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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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