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

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.

1 . A detection method for dioxin compounds in serum comprising the following steps:
 1) preparing a transformed cell line by introducing a recombinant vector comprising a gene construct, in which at least one of dioxin-responsive elements (DRE) represented by SEQ. ID. NO: 1, a promoter, and a reporter gene are operably linked, into a host cell;   2) preparing a sample by heat-inactivating the serum obtained from a test subject;   3) culturing the transformed cell line prepared in step 1) with the sample obtained in step 2) in a serum-free medium; and   4) detecting the expression of the protein expressed by the reporter gene in the transformed cell line cultured in step 3).   
     
     
         2 . The detection method according to  claim 1 , wherein the dioxin compound is selected from the group consisting of polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzo-furans (PCDFs), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarcons (PAHs), flavonoids, and pesticides. 
     
     
         3 . The detection method according to  claim 1 , wherein 3˜4 of the dioxin-responsive elements of step 1) are preferably included in the gene construct. 
     
     
         4 . The detection method according to  claim 1 , wherein the promoter of step 1) is selected from the group consisting of MMTV (Mouse Mammary Tumor Virus) promoter, SV40 promoter, and CMV (cytomegalovirus) promoter. 
     
     
         5 . The detection method according to  claim 1 , wherein the reporter gene of step 1) is selected from the group consisting of luciferase, alkaline phosphatase, chloramphenicol acetyl transferase, and β-galactosidase. 
     
     
         6 . The detection method according to  claim 1 , wherein the host cell of step 1) is the mammalian tumor cell line. 
     
     
         7 . The detection method according to  claim 6 , wherein the host cell is the mouse hepatocarcinoma cell line. 
     
     
         8 . The detection method according to  claim 1 , wherein the serum of step 2) is total serum. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . The detection method according to  claim 1 , wherein the heat-inactivation of step 2) is performed at 60˜70° C. for 20˜40 minutes. 
     
     
         15 . A method for diagnosing diabetes or metabolic syndrome and predicting the likelihood thereof comprising the following steps:
 1) preparing a transformed cell line by introducing a recombinant vector comprising a gene construct, in which at least one of dioxin-responsive elements (DRE) represented by SEQ. ID. NO: 1, a promoter, and a reporter gene are operably linked, into a host cell;   2) preparing a sample by heat-inactivating the serum obtained from a test subject;   3) culturing the transformed cell line prepared in step 1) with the sample obtained in step 2) in a serum-free medium; and   4) detecting the expression of the protein expressed by the reporter gene in the transformed cell line cultured in step 3).   
     
     
         16 . The method according to  claim 15 , wherein the serum of step 2) is total serum. 
     
     
         17 . The method according to  claim 15 , wherein the heat-inactivation of step 2) is performed at 60˜70° C. for 20˜40 minutes. 
     
     
         18 . A method for monitoring the prognosis of diabetes or metabolic syndrome comprising the following steps:
 1) preparing a transformed cell line by introducing a recombinant vector comprising a gene construct, in which at least one of dioxin-responsive elements (DRE) represented by SEQ. ID. NO: 1, a promoter, and a reporter gene are operably linked, into a host cell;   2) preparing a sample by heat-inactivating the serum obtained from a test subject;   3) culturing the transformed cell line prepared in step 1) with the sample obtained in step 2) in a serum-free medium; and   4) detecting the expression of the protein expressed by the reporter gene in the transformed cell line cultured in step 3).   
     
     
         19 . The method according to  claim 18 , wherein the serum of step 2) is total serum. 
     
     
         20 . The method according to  claim 18 , wherein the heat-inactivation of step 2) is performed at 60˜70° C. for 20˜40 minutes.