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Enzyme-linked immunoassay method for determining content of cell DNA (Deoxyribonucleic Acid) injury marker H2AX

A technology of DNA damage and H2AX, which is applied in the direction of measuring devices, biological testing, material inspection products, etc., can solve the problems of expensive instruments and achieve the effect of improving detection sensitivity and reducing deviation

Active Publication Date: 2014-03-19
CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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AI Technical Summary

Problems solved by technology

The instruments required for these techniques are expensive, and they all use ?H2AX-specific antibodies and fluorescent molecule-labeled secondary antibodies to achieve quantitative determination of the target. The fluorescent molecules used are traditional organic dyes.

Method used

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  • Enzyme-linked immunoassay method for determining content of cell DNA (Deoxyribonucleic Acid) injury marker H2AX
  • Enzyme-linked immunoassay method for determining content of cell DNA (Deoxyribonucleic Acid) injury marker H2AX
  • Enzyme-linked immunoassay method for determining content of cell DNA (Deoxyribonucleic Acid) injury marker H2AX

Examples

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example 1

[0035] In order to investigate the difference in the effect of smoke particulate condensate and gaseous condensate on DNA damage in cigarette sample A, 20 cigarette samples were smoked with a rotary disc type 20H smoking machine (Germany, Borgwaldt company), and the smoking mode was referred to ISO requirements. A total of 178 mg of smoke particulate matter was captured by the Cambridge filter. Add 17.8 mL of DMSO solution and extract on a shaker for 30 minutes. The extract was sterilized through a 0.22 μm filter membrane to obtain a 10 mg / mL extract of total particulate matter in the flue gas. While collecting the total particulate matter in the flue gas, pass the gaseous matter into the absorption bottle containing the PBS solution. After the collection, the PBS absorption solution is made to the same volume as the DMSO in the particulate matter extract, and passed through a 0.22 μm After the filter membrane is sterilized, the corresponding absorption liquid of smoke gas p...

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Abstract

The invention provides an enzyme-linked immunoassay method for determining the content of a cell DNA (Deoxyribonucleic Acid) injury marker H2AX. The enzyme-linked immunoassay method is characterized by comprising the steps that sandwich recognition on the specificity of a target protein H2AX is carried out by using a specific H2AX antibody and an enzyme-linked second antibody, a substrate after enzyme reaction is added, then the substrate is subjected to enzyme catalysis to be changed into a fluorescence product, wherein quantitative determination for the H2AX content can be realized according to a fluorescence value of the substrate. According to the method, the content change of the H2AX in the cells, caused by the fact that cigarette smoke is exposed, is quantitatively determined so that the aim of evaluating the virulence of cigarette smoke genes is realized. Compared with a traditional organic dye, the enzyme-linked antibody has the advantages that the catalyzing frequency of an enzyme is high, an amplification reaction effect is amplified and the detection sensitivity is improved; the enzyme has a stable property at a room temperature, is low in price, and is suitable for a lot of analysis. An enzyme-linked immunosorbent assay has the advantages of rapidness, accuracy, high throughput, high sensitivity and the like.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay technology for detection of cell DNA damage marker phosphorylated histone (?H2AX), which is used for genotoxicity assessment of cigarette smoke, specifically a method for measuring the content of cell DNA damage marker?H2AX Enzyme-linked immunoassay method. Background technique [0002] DNA damage comes in many different forms, of which DNA double-strand breaks are considered the most severe damage to DNA. When a DNA double-strand break occurs in a cell, the serine residue of histone H2AX can be phosphorylated by ataxia capillary mutant gene (ATM) and DNA-dependent protein kinase (DNA-PK) to form ?H2AX. The formation of ?H2AX is directly proportional to the number of double-strand breaks, making ?H2AX an important specific biomarker for DNA double-strand breaks. In recent years, a large number of literatures have carried out studies on DNA double-strand breaks caused by cigarette smoke. Albino et...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/531G01N33/68
Inventor 陈欢侯宏卫付立伟田永峰韩书磊刘彤吴帅宾胡清源张小涛石龙凯
Owner CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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