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

Inactive Publication Date: 2013-07-04
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

[0027]As explained hereinbefore, the detection method for environmental hormone of the present invention has an improvement from the conventional one that requires the step of purification of dioxins from the serum before measurement and has an advantage of saving time and effort because of using serum as a whole, which thereby enables the analysis with increased accuracy and easiness even with multiple numbers of samples. This method is advantageous in bi

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 hexa

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

Example 1

Construction of a Cell Line Expressing a Recombinant Reporter Gene Stably

Construction of a Recombinant Reporter Gene Vector

[0087]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 HindIII, which was cloned in HindIII site of pGL3-basic vector digested with HindIII. 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. 1).

Construction of a Transformed Cell Line Expressing the Recombinant Reporter Gene

[0088]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 pcDNA...

Example

Example 2

Response Test with the Transformed Cell Line Expressing the Recombinant Reporter Gene

Response Test Using Indole-3-Carbinol

[0089]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.

[0090]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, signifi...

Example

Example 3

Detection of Dioxins in Human Serum

Preparation of Human Serum Samples

[0093]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

[0094]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 ...

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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

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]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.[0003]2. Description of the Related Art[0004]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 the representatives. Once POPs are come into human body, they are not easily decomposed or excreted, resulting in the accumulation in human body. Recently, the public is more interested in the situation of these chemicals in our environment, including industrial byproducts, pesticides, or drugs cause potential endocrine disruption in human and many other wild species. Accordingly, many reports have been made saying that blood levels of various PO...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/66G01N2800/04G01N33/5308C12Q1/6897C12N2015/8572G01N33/5067
Inventor LEE, HONG KYUKIM, YOUNGMI
Owner UNIV IND COOP GRP OF KYUNG HEE UNIV
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