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Novel ligand assays

一种配体、受体的技术，应用在新型配体测定领域，能够解决昂贵、费用高昂等问题

Pending Publication Date: 2022-03-04
INSTUGEN GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, preparation of nuclear extracts from eukaryotic cells is expensive to manufacture due to the need for expensive cell growth media and the technical expertise required to enrich nuclear material

Method used

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Examples

Experimental program
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Effect test

example 1

[0430] The results presented in Example 1, Table 1 directly compare the effect of manipulating the stoichiometry between steroid hormone receptors and nucleic acid reporter constructs for the same 15bp construct comprising a Cy3 / Cy5 fluorophore combination at the opposite end , when the ratio of steroid hormone receptor to reporter construct / HRE increased from 0.423 to 2.12 in the presence of SHR ligand, ΔE FRET Increased from 15% to 34%. This represents ΔE FRET Significantly and thus assay sensitivity is enhanced.

[0431] Thus, in further examples according to the test kits, assays and methods described herein, the ratio of steroid hormone receptor to nucleic acid reporter construct is between about 1:1 and about 5.0:1 and includes but is not limited to : 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5; 1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2 :1, 2.3:1, 2.4:1, 2.5; 1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1 , 3.5; 1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4.0:...

example

[0645] The following information and data demonstrate the potential for ligands that bind to and activate the androgen receptor, including, for example, testosterone and dihydrotestosterone, or bind to the estrogen receptor, including, for example, estradiol. And various prototype assays for the detection of ligands that activate estrogen receptors. These examples serve to illustrate the activity assay platform described and claimed herein, in which ligands that bind to the androgen receptor or ligands that bind to the estrogen receptor (i.e., ER-α and ER-β) The assay concepts and principles of detection exemplified will be equally applicable to the detection of other receptor ligands of interest, including but not limited to ligands that bind to progesterone receptors including but not limited to progesterone, Ligands that bind to mineralocorticoid receptors including but not limited to aldosterone and ligands that bind to glucocorticoid receptors including but not limited to...

example 2

[0685] Other Fluorometric Prototypes

[0686] 2.1 Fluorescence adjustment via AR / ARE platform

[0687] The main components of fluorescence modulation by the AR / ARE platform include a DNA construct encoding an androgen response element (ARE) and incorporating a fluorescent moiety and a quencher moiety, combined with recombinant androgen receptor (AR), recombinant heat Shock protein 90 (HSP90) and buffer combination.

[0688] When the ARE is bound by the ligand-AR, the DNA construct will exhibit a high level of fluorescence modulation, resulting in a change in the level of fluorescence.

[0689] · DNA constructs include

[0690] o Incorporating ARE SEQ ID No.1 and 2 oligonucleotides

[0691] o A fluorophore dye and a fluorescent quencher matched to the fluorophore dye

[0692] ·Business restructuring AR

[0693] · Commercial recombinant HSP90

[0694] · Androgen ligands (such as testosterone)

[0695] • Necessary buffers.

[0696] 2.2 Fluorescence modulation by AR / ARE

...

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Abstract

The present invention relates to the detection of ligands that bind to and activate a steroid hormone receptor. In particular, the present invention provides test kits and assays for selectively identifying steroid hormone receptor ligands from a test sample. Importantly, the test kits and assays described herein are cell-free and enzyme-free and do not require the production of expensive nuclear extracts to enable the performance of the test kits and assays. In contrast, the test kits and assays described herein employ a reporter construct that includes a hormone response element that, when bound by a ligand-activated steroid hormone receptor, changes physical, mechanical, optical, photochemical, or electrochemical properties of the reporter construct. Thus, the presence of a target ligand in a sample under study can be determined using measured changes in physical, mechanical, optical, photochemical, or electrochemical properties (e.g., fluorescent readings) of the reporter construct.

Description

technical field [0001] The present invention generally relates to assays, methods and test kits for detecting ligands in a sample. In particular, the present invention provides assays, methods and test kits for screening test samples for the presence of a ligand characterized by its ability to form a complex with a steroid hormone receptor and elicit a steroid hormone-specific genomic response. Background technique [0002] The detection of ligands capable of eliciting a genomic response to steroid hormones is important in many fields of biochemistry, molecular biology and medicine. Such ligands include endogenous steroids, exogenous steroids, non-steroidal molecules and synthetic molecules. For example, determination of total hormone bioactivity in serum or plasma is important for monitoring human health-related conditions including aging, perimenopause, menopause, androgen deficiency, androgen hyperactivity, hormone replacement therapy , endocrine cancers including breas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74B82Y15/00
CPCG01N33/743C12Q1/6897H01L31/035218B82Y15/00G01N2333/723C07K14/721G01N21/6428C12Q2565/40C12Q2563/107C12Q2525/205G01N33/94G01N2500/20C07K14/72C12N9/1247C12Y207/07006C12Q1/6804G01N33/542
Inventor A·K·希瑟S·J·索尔比
Owner INSTUGEN GMBH
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