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Test kits and assays

A technology for kits and test samples, applied in biological testing, microbial determination/inspection, measuring devices, etc., can solve the problems of test difficulties, lower sensitivity and repeatability

Pending Publication Date: 2020-08-18
因斯图根有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, the high level of molecular complexity in living cells makes testing difficult and reduces sensitivity and reproducibility

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0562] Overview of Assay Concepts

[0563] 1.1 IVT platform

[0564] The main components of the IVT platform include a minimal promoter DNA template driven by an androgen response element (ARE) enhancer, a recombinant androgen receptor (AR), cell- or nuclear-extracts containing the transcriptional readiness machinery, and RNA synthesis or RNA Transcription or transcription / translation buffers for both synthesis of proteins. A minimal promoter will drive RNA molecule synthesis and subsequent basal levels of protein. Activation of transcription by AR activated by ligand binding to the ARE / enhancer will increase the amount of RNA molecule synthesis and protein levels.

[0565] androgen response element (ARE)

[0566] The androgen response elements tested in these experiments included:

[0567] 1. Mouse mammary tumor virus (MMTV), a response element strongly transcribed in response to steroid hormones

[0568] 2. Enhancer / ARE - This is more specific for AR due to the enha...

Embodiment 2

[0577] Assay prototype 1: ARE / enhancer-regulated reporter protein synthesis

[0578] 2.1 Overview

[0579] After the AR binds the ARE / enhancer, it prevents RNA polymerase II from interacting with the cell's transcriptional machinery to produce messenger RNA molecules, which in turn will serve as templates for protein synthesis. The end result is the synthesis of new proteins.

[0580] In these experiments, the ARE / enhancer sequence was cloned upstream of the pA-MinProm-green fluorescent protein (GFP) sequence in the plasmid pSF-pA-minPromGFP (OxfordGenetics) such that when introduced into a coupled in vitro transcription / translation reaction, This DNA template will drive ligand-activated AR to produce GFP.

[0581] 2.2 Methods and results

[0582] (1) The ARE / enhancer sequence is designed to have an enhancer sequence followed by a 3×tandem ARE sequence, such as figure 1 shown in (SEQ ID NO: 15).

[0583] Sense and antisense oligonucleotides were synthesized commercial...

Embodiment 3

[0597] Assay prototype 2: ARE / enhancer-regulated RNA synthesis

[0598] A variety of different methods have been investigated for detecting and measuring the amount of RNA molecules produced from in vitro transcription (IVT, no longer coupled to translation) reactions, including:

[0599] (i) Direct detection of RNA molecules (ie) proof of principle

[0600] (ii) Direct detection of RNA molecules by incorporation of fluorophore-labeled NTPs

[0601] (iii) RTqPCR

[0602] (iv) RNA aptamer and fluorophore binding

[0603] Since protein production from mRNA transcripts is no longer required, the mRNA transcripts used in this assay prototype validation were: (a) truncated GFP sequences, as shorter molecules are more resistant to degradation; (b) synthetic mRNA sequence; or (c) RNA aptamer sequence.

[0604] 3.1 Direct detection of prototype 2 RNA

[0605] (i) Visualization of RNA molecules produced by IVT

[0606] To show that the RNA produced by the IVT reaction can be ...

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Abstract

The present invention provides in vitro test kits, assays and methods useful for screening a test sample for the presence of a ligand which is characterized by its ability to form a complex with a steroid hormone receptor and elicit a genomic response when in a cell. Advantageously, the activity-based assays which form the basis of the test kits and methods described herein, are particularly useful in detecting the presence of a ligand of unknown structure, for example, a designer drug used by equine, canine and human athletes in sports doping. Different assay prototypes are disclosed in whichactivation of the hormone receptor by a ligand binding interaction may be detected, for example, though activation of a reporter molecule. In certain examples, the present invention provides test kits, assays and methods involving aptame: fluorophore reporter constructs for detection of a ligand from (e.g.) a sample taken trackside from an athlete.

Description

technical field [0001] The present invention generally relates to assays, methods and test kits for detecting ligands in a test sample. In particular, the invention provides assays, methods and test kits for screening a test sample for the presence of a ligand characterized by its ability to form a complex with a steroid hormone receptor and elicit a genomic response when in a cell. [0002] Background of the invention [0003] Detection of ligands that bind steroid hormone receptor proteins is important in many fields, including, for example, environmental testing for organic pollutants that may affect human health, diagnostic and prognostic testing for human diseases, including (for example) Endocrine- and non-endocrine-cancer detection and / or monitoring, and drug testing, including professional sports organizations responsible for maintaining, for example, a level playing field in human athletes and the horse racing industry. [0004] A common way to detect the presence o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74C12Q1/6897
CPCG01N33/743G01N2333/723C12Q1/6897
Inventor A·K·希瑟S·J·索尔比
Owner 因斯图根有限公司
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