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 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.

scholarly journals Inhibitors of Difficult Protein–Protein Interactions Identified by High-Throughput Screening of Multiprotein Complexes

2013 ◽  
Vol 8 (9) ◽  
pp. 1988-1997 ◽  
Author(s):  
Laura C. Cesa ◽  
Srikanth Patury ◽  
Tomoko Komiyama ◽  
Atta Ahmad ◽  
Erik R. P. Zuiderweg ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Protein Protein Interactions ◽  
Multiprotein Complexes

scholarly journals Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

2019 ◽  
Author(s):  
Christina K. Kim ◽  
Kelvin F. Cho ◽  
Min Woo Kim ◽  
Alice Y. Ting
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Cellular Protein ◽  
Protein Protein Interactions ◽  
Lov Domain ◽  
Omics Technologies ◽  
Lower Background

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested “AND” gate design of SPARK2—in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest—yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.

Development and Application of Activity-based Fluorescent Probes for High-Throughput Screening

2021 ◽  
Vol 28 ◽  
Author(s):  
Juan Cheng ◽  
Xin Li
Keyword(s):  
Drug Development ◽  
High Throughput ◽  
Fluorescent Probes ◽  
Analytical Methods ◽  
High Throughput Screening ◽  
High Sensitivity ◽  
Rapid Identification ◽  
Screening Assays ◽  
Hit Identification ◽  
Working Mechanisms

: High-throughput screening facilitates the rapid identification of novel hit compounds; however, it remains challenging to design effective high-throughput assays, partially due to the difficulty of achieving sensitivity in the assay techniques. Among the various analytical methods that are used, fluorescence-based assays dominate owing to their high sensitivity and ease of operation. Recent advances in activity-based sensing/imaging have further expanded the availability of fluorescent probes as monitors for high-throughput screening of result outputs. In this study, we have reviewed various activity-based fluorescent probes used in high-throughput screening assays, emphasizing their structure-related working mechanisms. Moreover, we have explored the possibility of the development of additional and better probes to boost hit identification and drug development against various targets.

scholarly journals A High-Throughput Screening Strategy to Identify Inhibitors of SSB Protein–Protein Interactions in an Academic Screening Facility

2017 ◽  
Vol 23 (1) ◽  
pp. 94-101 ◽  
Author(s):  
Andrew F. Voter ◽  
Michael P. Killoran ◽  
Gene E. Ananiev ◽  
Scott A. Wildman ◽  
F. Michael Hoffmann ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Bacterial Infections ◽  
Small Molecule Inhibitors ◽  
Klebsiella Pneumonia ◽  
Protein Protein Interactions ◽  
Response Curves ◽  
Single Stranded Dna

Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein–protein interactions (PPIs) to recruit more than a dozen important genome maintenance proteins to single-stranded DNA. Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal, suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial agents. As a first step toward implementing this strategy, we have developed orthogonal high-throughput screening assays to identify small-molecule inhibitors of the Klebsiella pneumonia SSB-PriA interaction. Hits were identified from an initial screen of 72,474 compounds using an AlphaScreen (AS) primary screen, and their activity was subsequently confirmed in an orthogonal fluorescence polarization (FP) assay. As an additional control, an FP assay targeted against an unrelated eukaryotic PPI was used to confirm specificity for the SSB-PriA interaction. Nine potent and selective inhibitors produced concentration–response curves with IC50 values of <40 μM, and two compounds were observed to directly bind to PriA, demonstrating the success of this screen strategy.

scholarly journals Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Christina K Kim ◽  
Kelvin F Cho ◽  
Min Woo Kim ◽  
Alice Y Ting
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Cellular Protein ◽  
Protein Protein Interactions ◽  
Lov Domain ◽  
Omics Technologies ◽  
Lower Background

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested ‘AND’ gate design of SPARK2—in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest—yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.

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