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Novel biological detection method for dioxins in serum, and a diagnostic use therefor in metabolic syndrome and related conditions

a biological detection and dioxin like technology, applied in the field of new biological detection methods for dioxin like activity in serum, can solve the problems of high price, compound is not easily decomposed or excreted in general, and accumulates in human body, so as to improve accuracy and ease, the effect of saving time and effor

Inactive Publication Date: 2017-04-20
UNIV IND COOP GRP OF KYUNG HEE UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides an improved detection method for environmental hormones which does not require purification of dioxins from serum before measurement. This method uses serum as a whole, leading to increased accuracy and ease of analysis with multiple samples. The method is efficient in analyzing small amounts of samples without pre-treatment with hexane. It can also research correlations between specific POPs and patients' disease factors by accurately detecting the presence of dioxins in serum. This method can effectively predict disease occurrence and determine treatability.

Problems solved by technology

Once POPs are come into human body, they are not easily decomposed or excreted, resulting in the accumulation in human body.
Some of the compound taken in human body is excreted through urine and bile juice, but the compound is not easily decomposed or excreted in general.
However, for high-throughput assay, high price has to be paid.
In addition, a large amount of serum sample (25˜200 ml) is required for the analysis as well.
However, with the recent technology, analysis with the said method takes long and asks a large amount of serum for the extraction or purification process using hexane from human serum samples (at least, 1 me of serum is necessary, and generally 10˜20 ml of sample is required).
When the transient transfection cell line is used, inter-assay variation is very big, leaving a question in reliability.
As the number of samples increases, handling speed and reliability decrease.
The conventional methods require high priced equipments for the analysis and the complicated pre-treatment step for sample purification, for which a large volume of organic solvents and other toxic reagents such as radio-labeled materials are used and longer analyzing time is taken.

Method used

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  • Novel biological detection method for dioxins in serum, and a diagnostic use therefor in metabolic syndrome and related conditions
  • Novel biological detection method for dioxins in serum, and a diagnostic use therefor in metabolic syndrome and related conditions
  • Novel biological detection method for dioxins in serum, and a diagnostic use therefor in metabolic syndrome and related conditions

Examples

Experimental program
Comparison scheme
Effect test

example 1

ion of a Cell Line Expressing a Recombinant Reporter Gene Stably

Construction of a Recombinant Reporter Gene Vector

[0089]1.8 kb virus promoter fragment comprising mouse CYP1A1 promoter (482 bp) having 4 DRE binding sites (AhR binding sites) (5′-TNGCGTG-3′) and long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) excluding glucocorticoid response element was cut out from pGudLuc1.1 vector by using Hind III, which was cloned in Hind III site of pGL3-basic vector digested with Hind III. As a result, pCYP1A1-luc vector was constructed (Han et al., BioFactors, 20:11-22, 2004). The direction of the promoter was confirmed by sequencing (FIG. 1B).

Construction of a Transformed Cell Line Expressing the Recombinant Reporter Gene

[0090]Hepa1c1c7, the mouse hepatocarcinoma cell line, was distributed in a 6 well plate (1×105 cells / well), followed by culture for 24 hours until the confluency reached 50%. 2 μg of pCYP1A1-luc vector constructed in Example and 0.5 μg of pcDNA3.1 vector wer...

example 2

Test with the Transformed Cell Line Expressing the Recombinant Reporter Gene

Response Test Using Indole-3-Carbinol

[0091]The cell line stably expressing pCYP1A1-luc constructed in Example was distributed in 60 mm culture dish at the density of 1×105 cells, followed by culture for 48 hours. Upon completion of the culture, the medium was replaced with DMEM supplemented with 0.5% charcoal-stipped FBS but not supplemented with phenol red, and indole-3-carbinol was treated thereto at the concentrations of 0, 0.01, 0.1, 1, 10, and 100 μM for 4, 8, or 24 hours. Then, the cells were collected, followed by luciferase assay.

[0092]As a result, as shown in FIG. 3, luciferase activity of the group treated with 0.001˜10 μM of indol-3-carbinol was increased dose-dependently, compared with that of the non-treated control group. In particular, when indol-3-carbionol was treated at the concentration of 100 μM, luciferase activity was increased at least 3 times. At this time, significant difference ov...

example 3

of Dioxins in Human Serum

Preparation of Human Serum Samples

[0096]Serum samples obtained from 97 people were heat-inactivated at 65° C. for 30 minutes. This heat-inactivation process secures the safety of cell culture.

Detection of Dioxins in Human Serum and Analysis of the Correlation Between Dioxins and Physical Variables

[0097]The cell line stably expressing pCYP1A1-luc constructed in Example was distributed in a 96 well plate at the density of 5×104 cells / well, followed by culture for 24 hours. The medium was replaced with DMEM not containing phenol red (90 μl / well), to which 10% human serum sample was treated (10 μl / well) for 24 hours. Then, the cells of each well were lysed by using luciferase lysis buffer (Promega). The lysate of each well was transferred into a 96 well plate (1 μl / well), followed by protein quantification by BCA method. The remaining cell lysates proceeded to luciferase assay using Promega luciferase assay kit. The luciferase activity was modified with prote...

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Abstract

The present invention relates to a novel biological detection method for dioxins in serum and a diagnostic use thereof in metabolic syndrome and related conditions. More particularly, according to the present invention, a significant correlation between dioxin content in serum and physical variables has been confirmed by measuring luciferase activity through the use of serum as a whole and cells transformed with a recombinant vector comprising a gene construct in which a dioxin responsive element, a promoter and a reporter gene are operably linked. And there is an improvement in this method of the present invention over the conventional methods which require a preprocessing step in order to purify dioxins from serum, by which plural samples can be analyzed easily and accurately even with a very small amount of each because of the use of serum as a whole. Therefore, the detection system of the present invention which involves total serum processing in a genetically transformed cell line comprising a recombinant reporter gene whose expression is modified by dioxin compounds can be effectively used for the biological detection of dioxins in serum. This method can also be used to research the correlation between specific POPs and patient's disease factors by accurate detection of POPs such as dioxins in the serum, and further this method can be effectively used to predict the occurrence of disease and to determine treatability.

Description

RELATED APPLICATIONS[0001]This application is to a divisional of U.S. patent application Ser. No. 13 / 821,779, filed on Mar. 8, 2013, which is a national stage application under 35 U.S.C. §371 of International Patent Application No. PCT / KR2011 / 006583, filed on Sep. 6, 2011, which claims priority to Korean Patent Application No. 10-2010-0088905, filed on Sep. 10, 2010. Each of these applications is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to a novel biological detection method for dioxin like activity in serum and a diagnostic use thereof in metabolic syndrome and related conditions.[0004]2. Description of the Related Art[0005]Persistent organic pollutants(pops) do not exist in the nature or only very small amounts of them if they exist. They are mostly organic chemicals generated artificially for human needs or byproducts of industrialization. Dioxin and dioxin like substances are th...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6897C12Q1/66G01N33/5308G01N2800/04C12N2015/8572G01N33/5067
Inventor LEEKIM, YOUNGMI
Owner UNIV IND COOP GRP OF KYUNG HEE UNIV
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