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Gamma-H2AX based biomarker genetic toxicity detection method

A biomarker, -H2AX technology, applied in biological testing, measurement devices, material inspection products, etc., can solve the problems of high false positive rate, low sensitivity, and few detection indicators, and achieve low false positive rate, high sensitivity, The effect of detecting many indicators

Pending Publication Date: 2020-02-25
SHANGHAI INNOSTAR BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a genotoxicity detection method based on gamma-H2AX biomarkers for the defects of high false positive rate, low sensitivity and few detection indicators in the prior art for detecting genotoxicity of in vitro cells. Detection method

Method used

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  • Gamma-H2AX based biomarker genetic toxicity detection method
  • Gamma-H2AX based biomarker genetic toxicity detection method
  • Gamma-H2AX based biomarker genetic toxicity detection method

Examples

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Embodiment 1

[0040] Take the TK6 growing in the logarithmic phase in culture, after counting, adjust the cell density to 3×10 5 / mL. When adding samples, in a U-bottom 96-well plate, add 2 μL of test substance or solvent control to each well, and then add 198 μL of cell suspension, and the number of cells inoculated in each well is about 6×10 4 / hole. For the 24h long-term treatment group (24h-S9 group) without adding in vitro metabolic activation system, four concentrations of ETO (etoposide) of 0.2, 0.4, 0.8 and 1.6 μg / mL and 0.0063, 0.0125, 0.025 and 0.05 μg / mL were selected. 4 concentrations of COL (colchicine); add in vitro metabolic activation system 4h short-term treatment group (4h+S9 group) choose 3.75, 7.5, 15 and 30μg / mL 4 concentrations of CP (cyclophosphamide). The 4h+S9 treatment group treated TK6 cells for 4h, washed the cells with serum-free medium, removed the medium and added complete medium to continue culturing. Three replicate wells were set up for each concentratio...

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Abstract

The invention discloses a Gamma-H2AX based biomarker genetic toxicity detection method. The detection method comprises the following steps of (1) employing a flow cytometry to perform fluorescent detection after mixing and incubation of to-be-detected cells, after being mixed with counting microspheres, an antibody, a transparent liquid and a confining liquid, wherein the antibody comprises a Gamma-H2AX antibody, a p53 protein antibody, a phosphorylation protein H3 antibody and an anti-Cleaved PARP antibody; and (2) analyzing a result, particularly eliminating dying cells, and analyzing cell toxicity information according to expression of Gamma-H2AX of living cells after the apoptotic cells are positively eliminated by Cleaved-PARP. The detection method is a method for detecting in-vitro cell genetic toxicity and is simple, rapid and accurate, and various mechanism information in-vitro cell genetic toxicity detection methods can be provided.

Description

technical field [0001] The invention belongs to the field of genotoxicity detection, in particular to a genotoxicity detection method based on gamma-H2AX biomarkers. Background technique [0002] Among the different DNA damages, DNA double-strand breaks are the most serious one, and DNA damage is closely related to the genotoxicity of chemicals. Chemicals with spindle toxicity can affect the synthesis of cellular spindles or the formation of spindles, resulting in cell cycle arrest, apoptosis, and in severe cases, aneuploid changes in the cell chromosome group. The aneuploidy of cell chromosomes can lead to various diseases, such as trisomy 21, and the formation of cancer is also closely related to the aneuploidy of cells. Detection of compound-induced DNA damage and aneuploidy changes has become an important marker for evaluating the genotoxicity of compounds. [0003] Commonly used in vitro tests for genotoxicity detection at this stage mainly include bacterial reverse m...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N15/14
CPCG01N33/68G01N15/14
Inventor 黄鹏程李若婉周长慧常艳
Owner SHANGHAI INNOSTAR BIO TECH
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