scholarly journals Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology

1999 ◽  
Vol 4 (4) ◽  
pp. 193-204 ◽  
Author(s):  
Sheri Miraglia ◽  
Elana E. Swartzman ◽  
Julia Mellentin-Michelotti ◽  
Lolita Evangelista ◽  
Christopher Smith ◽  
...  
Keyword(s):  
Ligand Binding ◽  
High Throughput ◽  
High Throughput Screening ◽  
Laser Scanner ◽  
Peptide Ligand ◽  
Binding Assays ◽  
Using Data ◽  
Immunological Assays ◽  
The Individual ◽  
Homogeneous Assays

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm2 digitized image of a 100-μm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twister™ (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.

Application of AlphaLISA in bioanalysis

2020 ◽  
pp. 16-37
Author(s):  
Stanislav Cherepushkin
Keyword(s):  
Drug Development ◽  
Ligand Binding ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
High Sensitivity ◽  
Medical Diagnostics ◽  
Protein Protein Interactions ◽  
Binding Assays ◽  
Post Translational Modifications

The use and development of biotherapeutics increases and the need for accurate, sensitive and robust bioanalytical methods is also increasing. ELISA and other ligand-binding assays are the most widely used methods for the quantification of macromolecules in complex biological samples. One of the alternatives to ELISA is AlphaLISA — a versatile chemiluminescent ligand binding assay using a homogeneous no-wash protocol. AlphaLISA assays are suited for automation and exhibit high sensitivity, high throughput and wide analytical range. Since the early 2000s, this method has been used in science, medicine, and drug development for wide variety of applications, including the quantification of analytes, immunogenicity, protein-protein interactions, enzyme activity, post-translational modifications and epigenetics. In this review, we describe the principles of the AlphaLISA assay and its application in bioanalytical studies (pharmacokinetics and immunogenicity) and high-throughput screening in drug development, medical diagnostics and pathogens detection.

scholarly journals High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors

1999 ◽  
Vol 4 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Ilona Kariv ◽  
Michelle E. Stevens ◽  
Davette L. Behrens ◽  
Kevin R. Oldenburg
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Transmembrane Domain ◽  
Receptor Function ◽  
Camp Production ◽  
Ligand Interaction ◽  
G Protein Coupled ◽  
Native Ligand

Impairment of G protein—coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate™ (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within ≤20 μm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein—coupled receptor function as measured by cAMP production and is suitable for high throughput screening.

scholarly journals A Review of Automation Options to Support Plate Preparation, Cherry Picking, and Homogeneous Assays

1998 ◽  
Vol 3 (4) ◽  
pp. 271-275 ◽  
Author(s):  
Jason W. Armstrong ◽  
Rick A. Gerren ◽  
Steven D. Hamilton
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Robotic Systems ◽  
Support Plate ◽  
Improved Stability ◽  
Cherry Picking ◽  
Software And Hardware ◽  
Homogeneous Assays

Developments in high throughput screening (HTS) have led to new needs in automation to enable better handling of applications such as homogeneous assays and cherry picking. Software and hardware integration approaches for screening automation have been changing in concert with these new application needs. The result of this combination has been the production of robotic systems for drug discovery with improved stability and functionality. This review critically assesses some Zymark, Tecan, and Beckman solutions for current HTS requirements.

scholarly journals High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery

2001 ◽  
Vol 6 (6) ◽  
pp. 429-440 ◽  
Author(s):  
Michael W. Pantoliano ◽  
Eugene C. Petrella ◽  
Joseph D. Kwasnoski ◽  
Victor S. Lobanov ◽  
James Myslik ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Ligand Binding ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
General Strategy ◽  
General Applicability ◽  
High Throughput Drug Screening ◽  
Compound Libraries ◽  
Thermal Shift

More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.

scholarly journals A High-Throughput Ligand Competition Binding Assay for the Androgen Receptor and Other Nuclear Receptors

2008 ◽  
Vol 14 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Clémentine Féau ◽  
Leggy A. Arnold ◽  
Aaron Kosinski ◽  
R. Kiplin Guy
Keyword(s):  
Androgen Receptor ◽  
Nuclear Receptors ◽  
High Throughput ◽  
High Throughput Screening ◽  
Low Cost ◽  
Environmental Chemicals ◽  
Binding Assays ◽  
Receptor Ligands ◽  
Competition Binding ◽  
Nuclear Receptor Ligands

Standardized, automated ligand-binding assays facilitate evaluation of endocrine activities of environmental chemicals and identification of antagonists of nuclear receptor ligands. Many current assays rely on fluorescently labeled ligands that are significantly different from the native ligands. The authors describe a radiolabeled ligand competition scintillation proximity assay (SPA) for the androgen receptor (AR) using Ni-coated 384-well FlashPlates® and liganded AR-LBD protein. This highly reproducible, low-cost assay is well suited for automated high-throughput screening. In addition, the authors show that this assay can be adapted to measure ligand affinities for other nuclear receptors (peroxisome proliferation-activated receptor γ, thyroid receptors α and β). ( Journal of Biomolecular Screening 2009:43-48)

scholarly journals Fluorescence Polarization and Anisotropy in High Throughput Screening: Perspectives and Primer

2000 ◽  
Vol 5 (5) ◽  
pp. 297-306 ◽  
Author(s):  
John C. Owicki
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
The Past ◽  
Assay Performance ◽  
The Subject ◽  
Homogeneous Assays

Fluorescence polarization and anisotropy are two nearly equivalent techniques that have together, over the past 5 years, achieved wide use in high throughput screening in drug discovery. These are single-label methods that can be used to construct homogeneous assays that are fast, sensitive, and resistant to some significant interferences. Moreover, the assays are relatively inexpensive. This review surveys the peer-reviewed literature on the subject and explores some of the fundamental issues that bear on assay performance.

scholarly journals An Effective Tool for Homogeneous Assays Using a Simple Robot Arm with Stackers—TRISTAN

2002 ◽  
Vol 7 (1) ◽  
pp. 89-94
Author(s):  
Kazuyoshi Yajima ◽  
Takanori Ohgaru ◽  
Yoshifumi Hashimoto ◽  
Naoki Suto ◽  
Toru Okuda
Keyword(s):  
Cost Effectiveness ◽  
High Throughput ◽  
High Throughput Screening ◽  
Integrated System ◽  
Robotic Systems ◽  
Robot Arm ◽  
Parameter Setting ◽  
Plate Reader ◽  
Homogeneous Assay ◽  
Homogeneous Assays

The Twister &! RapidPlate Integrated System by TANabe, or TRISTAN, consists of a 96-channel dispenser (Rapid-Plate 96), a plate reader (V-MAX), and a simple robot arm (Twister). We developed TRISTAN for effectively conducting a homogeneous assay. Although this system accommodates fewer than 20 microplates, it has several advantages over conventional robotic systems for high-throughput screening in the following aspects: parameter setting, running time, hardware errors, manpower, and cost-effectiveness. The system proved to be effective and efficient for homogeneous assays.

scholarly journals Oncology Drug Discovery Applications Using the FMAT™ 8100 HTS System

2003 ◽  
Vol 8 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Jennifer Y. Lee ◽  
Sheri Miraglia ◽  
Xiongwei Yan ◽  
Elana Swartzman ◽  
Susan Cornell-Kennon ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
High Throughput Screening ◽  
Enzyme Assay ◽  
Annexin V ◽  
Enzyme Assays ◽  
Binding Assays ◽  
Src Tyrosine Kinase ◽  
Apoptosis Assay ◽  
Peptide Interaction ◽  
Homogeneous Assays

High-throughput screening (HTS) for potential anticancer agents requires a broad portfolio of assay platforms that may include kinase enzyme assays, protein-protein binding assays, and functional cell-based apoptosis assays. The authors have explored the use of fluorometric microvolume assay technology (the FMAT™ 8100 HTS System) in three distinct homogeneous HTS assays: (1) a Src tyrosine kinase enzyme assay, (2) a Grb2-SH2 protein-peptide interaction assay, and (3) an annexin V binding apoptosis assay. Data obtained from all three assays suggest that the FMAT system should facilitate the implementation of homogeneous assays for a wide variety of molecular targeted and cell-based screens. ( Journal of Biomolecular Screening 2003:81-88)

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