Details for Patent: 8,097,431

Title:	Methods and compositions for detecting steroids
Abstract:	The present invention provides for methods and systems for detecting steroids. Examples of such steroids include estrogen, progesterone, androgen, testosterone, and derivatives and analogs thereof. Systems useful for carrying out the method include tripartite constructs including a DNA-binding domain, a ligand binding domain, and an activation domain. The present invention provides numerous improvements over previous diagnostic systems for detection of steroids, such advantages include that the method allows for detection of steroid analogs and derivatives, whose structures may not yet be known, the method is generally applicable to a wide variety of organisms, and numerous ligand binding domains may be used in conjunction with the present system.
Inventor(s):	Meikle; A. Wayne (Salt Lake City, UT), Stillman; David J. (Salt Lake City, UT), Orrock; Jared M. (Salt Lake City, UT), Terry; Alan H. (Bountiful, UT), Sandrock; Tanya M. (Salt Lake City, UT)
Assignee:	University of Utah Research Foundation (Salt Lake City, UT)
Filing Date:	Aug 04, 2005
Application Number:	11/631,725
Claims:	1. A method for detecting steroids comprising the steps of: (a) providing a host cell transformed with a reporter gene and a tripartite construct, said tripartite construct encoding a ligand binding domain (LBD) wherein said LBD is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, operably linked to both a DNA binding domain (DBD) comprising SEQ ID NO:1 and an activation domain (AD) comprising SEQ ID NO:9, wherein said host cell expresses said reporter gene upon binding of the LBD to generate a detectable signal; (b) contacting said host cell with a mammalian sample to be screened for a compound that binds to said LBD, wherein the contacting is in vitro; and (c) detecting the presence of the signal generated by expression of said reporter gene in order to detect the amount of steroids present in the sample. 2. The method of claim 1, wherein said host cell is a yeast cell or a modified yeast cell. 3. The method of claim 2, wherein said yeast cell or modified yeast cell is Saccharomyces cerevisiae. 4. The method of claim 1, wherein said reporter gene is selected from the group consisting of .beta.-galactosidase, green fluorescent protein, luciferase, .beta.-glucuronidase, chloramphenicol acetyltransferase, and alkaline phosphatase. 5. A method for determining the steroid activity of a mammalian sample, said method comprising the steps of: (a) contacting the mammalian sample with a cell comprising: (i) a reporter plasmid; and (ii) a fusion protein comprising a ligand-binding domain (LBD), wherein the LBD is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, operably linked to both a DNA binding domain (DBD) comprising SEQ ID NO:2 and an activation domain (AD) comprising SEQ ID NO:10, where said fusion protein binds to binding sites in said reporter plasmid, wherein the contacting is in vitro; (b) allowing the sample to incubate with said cell; (c) measuring the reporter activity of said cell; (d) comparing the measured reporter activity to an amount of reporter activity, observed when known quantities of a steroid are contacted with said cell, to give a relative reporter activity of the sample, and (e) using the relative reporter activity to detect or quantify an active steroid in the sample. 6. The method according to claim 5, wherein the mammalian sample is selected from the group consisting of a serum sample, a plasma sample and a urine sample. 7. The method of claim 5, wherein said reporter gene is selected from the group consisting of .beta.-galactosidase, green fluorescent protein, luciferase, 13-glucuronidase, chloramphenicol acetyltransferase, and alkalinephosphatase. 8. A tripartite construct comprising: (a) a DNA binding domain comprising SEQ ID NO:1; (b) a ligand binding domain selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and (c) an activation domain comprising SEQ ID NO:9. 9. A plasmid comprising a tripartite construct comprising: (a) a DNA binding domain comprising SEQ ID NO:1; (b) a ligand binding domain selected from the group consisting of SEQ ID NO:3 SEQ ID NO:5, and SEQ ID NO:7; and (c) an activation domain comprising SEQ ID NO:9. 10. An isolated host cell comprising a plasmid comprising: (a) a DNA binding domain comprising SEQ ID NO: 1; (b) a ligand binding domain selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and (c) an activation domain comprising SEQ ID NO:9 wherein said host cell further comprises DNA that transcribes a reporter gene when associated with an activated form of said tripartite construct. 11. The host cell of claim 10, wherein said reporter gene is selected from the group consisting of .beta.-galactosidase, green fluorescent protein, luciferase, .beta.-glucuronidase, chloramphenicol acetyltransferase, and alkaline phosphatase. 12. A kit for detecting steroidal activity of a sample, said kit comprising: (a) host cells comprising a plasmid comprising (i) a DNA binding domain comprising SEQ ID NO:1; (ii) a ligand binding domain selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; and (iii) an activation domain comprising SEQ ID NO:9; and (b) instructions for detecting said steroidal activity.