LRIG New England Chapter
LRIG → 2009 Fall Exhibition → Poster List
2009 Poster Abstracts
1) New HTRF solutions for cell surface
receptors study and screening. Application to GPCR dimerization and
highly selective ligand binding assays.
Cynthia Cormier,Cisbio US Inc.
Cisbio introduces a new technological concepts for the investigation
of cell surface receptors. Based on the combination of SNAP-tagâ„¢
and HTRF® technologies, this new platform can applies for a
comprehensive investigation of receptor mechanistic.
The receptor of interest is fused in its N-terminus to SNAP-tag (20
kDa) and expressed at the cell surface. We describe in this poster
applications to G-protein-coupled receptor (GPCR), and development
of cell-based assays with the new proprietary fluorescent SNAP-tag
substrates. This new technology is streamlined for both highly
selective ligand binding and receptor dimerization assays.
This rapid and non-radioactive platform will meet needs for a
comprehensive and specific investigation of cell-surface-targets,
and preserves the functionality of the receptor and the
intracellular signaling pathway.
The poster describes the simultaneous dynamic localization of both
GPCRs hetero- and homodimers at the cell membrane surface. Based on
this new platform, ligand binding assays were also optimized,
results are presented using COS7 cells expressing the vasopressin
V1A receptor.
2) Experimental Strategies for High
Throughput Experiments
James Cawse, Cawse and Effect
As our ability to generate large numbers of experiments using
robotics and high throughput methods has accelerated, we have become
more conscious of the need to plan these experiments effectively. We
find that the kinds of problems, the desired outcomes, and the
appropriate strategies are significantly different from conventional
experimentation. Classical experimental design strategies grew up in
a period of slow, laborious, error-prone experimentation; massively
parallel experimentation now allows more runs in a day than were
once done in a week, month or year! New experimental strategies and
designs are necessary for success in this exciting new area.
3) An Example of automation and
miniaturization of an ELISA for HTS and Protease drug discovery
Vincent Yu, Novartis Institutes for BioMedical Research
Enzyme-Linked ImmunoSorbent Assays (ELISAs) provide a sensitive and
relatively straightforward method for indirectly measuring enzymatic
activity with minimal signal interference from test compounds.
Typically however, ELISAs require long incubation times and multiple
washing steps, and are thus usually carried out in a low-throughput
manner. In this poster, we describe the adaptation and
miniaturization of a manually performed proteolytic enzyme assay to
an automated fluorescence intensity ELISA suitable for high
throughput screening. The assay was successfully miniaturized to
384-well plate format (6µl total reaction volume per well) and the
full assay time reduced from 2 days to 4.5hr. This was done by a
combination of reduced incubation times and steps and reducing the
multiple washing steps down to 1 extensive wash. Most importantly,
this was done without losing the sensitivity of the assay, as
evidenced by a comparison of IC50 values of identified inhibitors to
the non-automated Disease Area partner results during our pilot
studies. These time and reagent saving measures allowed us to screen
a 250,000 compound collection to identify leads for the Disease
Area. In conclusion, we present here a method for successfully
adapting and miniaturizing a sensitive ELISA for high-throughput
screening.
4) The NA_STAR Influenza Neuraminidase
Inhibitor Resistance Detection Kit: Chemiluminescences Assay for
Detection and Quantification of Influenza Neuraminidase Activity
Alison Sparks, Life Technologies
The NA-Star Influenza Neuraminidase Inhibitor Resistance Detection
Kit provides the NA-Star 1,2-dioxetane chemiluminescent
neuraminidase substrate, together with all necessary assay reagents
and microplates, to measure the resistance level of influenza virus
isolates to neuraminidase inhibitor antiviral therapeutics. The
NA-Star chemiluminescent substrate provides highly sensitive
detection of neuraminidase enzyme activity from influenza virus
types A and B, including human, avian, porcine and equine viruses.
Neuraminidase assays performed with the NA-Star 1,2-dioxetane
chemiluminescent substrate provide approximately 50-fold higher
sensitivity than neuraminidase assays with the MUNANA fluorescent
substrate. The chemiluminescent assay with NA-Star substrate
provides linear results over 3-4 orders of magnitude of
neuraminidase concentration compared to 1-2 orders of magnitude
achieved with the fluorescent assay, providing a greater assay
dynamic range. Virus dilutions are briefly incubated with
neuraminidase inhibitor, and then the two-reagent detection assay is
performed. The entire assay is completed in approximately one hour.
Data analysis, using non-linear curve fitting dose response analysis
software (not provided), is performed to determine the IC50 value of
the neuraminidase inhibitor with each viral isolate. The NA-Star
chemiluminescence assay has been compared to fluorescence assays
performed with MUNANA with isolates of the major influenza types,
H1N1, H3N2 and influenza B strains, including NI-sensitive and
resistant strains. Results obtained with both oseltamivir and
zanamivir inhibitors will be presented.
The NA-Star Influenza Neuraminidase Inhibitor Resistance Detection
Kit combines highly sensitive and rapid chemiluminescent
quantitation of neuraminidase activity from flu virus isolates using
supplied reagents and a simple assay protocol to provide a
convenient method for use in research laboratories to monitor the
resistance levels of both human and animal influenza virus isolates
to neuraminidase inhibitors.
5) Chemiluminescent Substrates and
Chemiluminescent Assays for Monitoring Cytochrome P450 Enzyme
Activity
Zhixian Wang, Life Technolodies, Inc
We have developed chemiluminescent CYP450 1,2-dioxetane substrates
and assays to address CYP450 activity surveillance in a simple,
microplate-based assay format amenable to high or medium throughput
screening. Preliminary CYP450 inhibition assays based on these
substrates have been developed for the five isozymes responsible for
the majority of drug metabolism: CYP1A2, CYP2C9, CYP2C19, CYP2D6,
and CYP3A4. These end-point assays have a robust signal to
background, are simple to perform, and provide IC50 values for known
inhibitors in the expected ranges. Most substrates have an apparent
Km below 10mM for the matched CYP isozyme. These assays compare
favorably with commercially available fluorescent and indirect
chemiluminescent assays. We have recently developed additional
chemiluminescent CYP450 substrates that show adequate isozyme
specificity to support isozyme-specific enzyme monitoring in human
liver microsomes. These include sensitive and specific CYP3A4
substrates and substrates that allow for monitoring 2C19-specific
activity in as little as 10 min assay time.
6) Simple and Fast Performance
Verification for Ultra-Low-Volume Liquid Handlers:
Nanoliter Aqueous Fluid Transfers Assessed with Dual-Dye Technology
Keith Albert, Artel
Poor liquid handling performance can lead to poor assay results.
When the volume verification methodology used to assess liquid
handler performance is not properly executed, errors will enter into
the process and the results could lead to incorrect conclusions
regarding instrument performance. This presentation discusses proper
techniques and best practices for assessing aqueous liquid transfer
accuracy and precision for low-volume liquid handlers. These
practices were established using both acoustic droplet ejection (Echo®
555 liquid handler, Labcyte Inc.) and a feedback-controlled,
tip-based dispenser (Deeracâ„¢ GX reagent dispenser, Labcyte Inc.).
Volumetric accuracy and precision were measured using a standardized
volume verification platform based on a dual-dye absorbance
technology (MVS®, Artel) and compared to fluorescence-based tests.
For absorbance testing, manufactured aqueous-based dye solutions
were employed for all target volumes and diluents. For fluorescence
testing, sodium fluorescein (150 mM) in water was used for target
volumes and sodium hydroxide (10 mM) was used as diluent. In both
cases, the dye solution was transferred into a 384-well test plate
with the Echo (30 – 200 nL) or the Deerac GX system (200 – 800
nL) followed by the addition of buffer. Using the MVS, the measured
accuracy and precision for both the Echo and Deerac GX Series were
below 5% inaccuracy and 5% CV for the volume transfers indicated.
Some of the best practices developed and discussed herein include
source plate preparation, assay plate preparation and assay plate
reagent mixing. By following these recommended practices, optimal
conditions for measuring liquid handler performance can be achieved.
7) Identification of Chemical Probes for the
Study of Platelet Granule Secretion
Lynn VerPlank, Broad Institute
The NIH Molecular Libraries Probe Centers Network (MLPCN) initiative
supports the testing of a publicly available 300K compound library
to identify small molecule probes effective at modulating a given
biological process or disease state. As a network member, The Broad
Institute Probe Development Center (BIPDeC) participates in a
comprehensive program that supports projects through assay
development, execution of primary HTS, confirmation in secondary
assays, and lead optimization with medicinal chemistry to identify
biological probes. All data from completed probe development
campaigns are made available to the wider community through PubChem.
As part of this program, we undertook a screen to identify novel
probes that inhibit platelet activation. Such probes will provide
additional insights into the mechanisms regulating platelet granule
secretion, and may also result in a regulator(s) of
platelet-mediated arterial thrombosis. The screen was conducted
entirely in platelet-rich plasma (PRP) obtained from normal, healthy
adult donors. The screen assessed the ability of compounds to
inhibit the activation of platelets when stimulated with the
recombinant thrombin peptide fragment SFLLRN. To assess platelet
activation, a luciferin/luciferase reaction was used to detect ATP
that is released upon granule secretion. In the primary screen, the
criterion for a hit was set at 50% or greater inhibition relative to
the positive control Cilostazol, a known inhibitor of platelet
activation. The screen showed a hit-rate of 0.2%. A ‘cherry-pick’
list of 1684 compounds was selected including all hits from the
primary screen plus non-active compounds of related chemical
structures. These compounds were retested in an 8-point dose
response. Further secondary screens were conducted to identify false
positives and select for specificity toward platelet response
pathways.
8) A LIMS integrated dashboard tool for
automation asset management and protocol reliability
Joshua Bittker, Broad Institute
The high throughput screening and compound management groups in the
Broad Institute Chemical Biology Platform execute automated compound
formatting and assay protocols using a set of integrated modular
robotic systems (High Resolution Biosolutions.) A common concern in
HTS labs that is a particular issue with modular systems is the
tracking of physical assets and their integration with the
automation informatics environment.
We have developed and implemented an equipment tracking system that
is tightly integrated with our existing LIMS system, CBIP (Chemical
Biology Informatics Platform.) This system provides a centrally
located inventory and status report of all automation-related
instruments and accessories that can be accessed through any web
browser. The most basic function tracks the physical locations and
the current functional status of each piece of equipment, providing
coordination for all personnel in the screening and compound
management groups.
Thanks to the integration of the Broad LIMS, automation, and record
keeping databases, the system is also able to validate protocols
prior to execution to avoid runtime failures due to nonfunctional
equipment or invalid automation parameters. The High Resolution
Biosolutions scheduling software, Cellario, assigns physical
instruments to virtual resources required for protocols. By using
this mapping through the equipment tracker, the system can prevent
the execution of any protocol that would require an instrument
marked as nonfunctional or not robotically accessible. Protocols can
also be automatically checked prior to runtime to ensure that the
expected accessories, such as pin transfer tools or reader filters,
are assigned to each instrument. Finally, by accessing the screening
group's record keeping database, Cambridgesoft E-Notebook,
information on the most recent QC status of all instruments can be
tracked and flagged at appropriate intervals. By integrating in a
central location information from all the databases necessary to
design and execute automated HTS and compound formatting protocls,
we are able to avoid unnecessary errors and improve overall
automation efficiency.
9) Flexible, Customized Liquid
Dispensing Systems for Use in High-Throughput Experimentation
Joe Marchionna, TransForm Pharmaceuticals, Inc
As liquid handling technology has continued to advance, experience
in running high-throughput experiments with these systems has
increased as well, as have the ideas of engineers and scientists on
how to do even more with these tools. Blending off-the-shelf and
in-house developed hardware and software to deliver more flexible
solutions has been a goal at TransForm Pharmaceuticals from the
company’s founding. This poster will outline the hardware and
software features of two automated flow-through dispensing systems
recently developed at TransForm to perform liquid dispensing
operations in various work-flows.
The first system is based on a 30-channel IVEK liquid dispenser with
custom motion control, environmental control, fluid-line temperature
control, and fluid handling components, controlled by a custom
software application developed based on input from TransForm’s
high-throughput experimentation scientists. This system has been
successfully used to dispense dozens of screening experiments for
vaccine re-formulation efforts. These types of screens usually
contain in the range of 768 to 1728 unique, sterile samples,
containing quinary combinations of 800 ul per well, and resulting in
dispense times of approximately 35 min per 96 well plate.
The second system is based on a 32-channel BioDot liquid dispenser
outfitted with custom environmental control and other hardware
improvements, as well as a similar custom software interface. It has
been used to dispense similar-sized screening batches of aqueous
combinations for biological experiments with a dispense time of
approximately 15 min per 96 well plate.
This poster will describe the benefits of custom hardware solutions
implemented and the flexibility the custom control software has
provided in operating the instrument, as well as dispensing results
achieved.
10) Label-free BIND Technology Automation of
SRU Biosystems Label-free BIND® Technology Using Beckmans Biomek®
Platform: A Universal Solution for 384- and 1536-well GPCR Screening
and Profiling.
Amy Mitchell, SRU Biosystems
Today’s drug discovery efforts require technologies that produce
physically relevant data in a high throughput, cost effective
manner. SRU Biosystems™ label-free BIND® technology provides the
application flexibility, robust performance, and automation
simplicity to meet those needs in a high density, miniaturized
format. SRU had created a custom, plug-and-play platform in which
the BIND ultra-high throughput reader, the SCREENER, is fully
integrated within Beckmans Biomek FX robot for streamlined screening
and profiling. This poster highlights the BIND SCREENER-Biomek FX
integration platform and discusses platform components, assay
performance and throughput capabilities using a cell-based GPCR
assay.
Key Assay Performance Results:
Screen over 130,000 samples in an 8 hour day
1536-well screening with z-factors as high as 0.87
384-well assays with z-factors as high as 0.93
High level well-to-well, plate-to-plate and day-to-day
reproducibility
11) Tag-lite, the new HTRF
solutions for cell surface receptor studies and screening.
Application to GPCR dimerization and highly selective ligand binding
assays
Anna Sinsigalli, Cisbio US Inc.
Cisbio introduces Tag-lite, a new technological concept for the
investigation of cell surface receptors. Tag-lite combines HTRF®
and SNAP-tag technology, a unique method to accurately label a
protein of interest with a fluorescent dye. Tag-lite can be used in
a comprehensive range of applications such as receptor mechanistics
and dimerization, ligand binding assays, and second messenger
assessment.
SNAP-tag is a suicide enzyme which can be fused to the N-terminal
position of a GPCR’s 7TM fragment. A plasmid construction can be
engineered and once transfected into cells, leads to the expression
of the GPCR of interest tagged with the SNAP at the cell membrane
surface.
For this platform, Cisbio has developed highly selective derivatized
SNAP substrates. These substrates are labeled with HTRF fluorophores
such as terbium cryptate (Lumi4-Tb) and green or red HTRF acceptors.
As the Tag-lite SNAP substrates are non-permeating, only the SNAP
tagged GPCR expressed at the cell surface can be labeled with the
appropriate HTRF-compatible fluorophores.
12) Reducing the Cost of Poor Quality
Screening Using Vision Technology
Significant time and effort is expended in screening sample
libraries; however, no matter how advanced the screening system, the
end results are only as good as the quality of the sample in the
plate wells.
Sue Jones, RTS Life Science
Survey data gathered by RTS suggests that as many as 5% of plate
wells may be empty, and 3 to 5% of wells may have samples at the
wrong concentration; whilst the cost of this poor quality can
readily be calculated, the implications are perhaps not fully
appreciated by screening and compound management groups.
In order to improve the quality of the delivered plates, and reduce
the cost of wasted screening (time, effort, reagents, missed hits
etc) a major pharma company partnered with RTS to incorporate a
vision system that routinely and accurately audits sample tubes to
calculate volumes and also identify any particulate matter; key in
understanding the quality of the final delivered plate wells.
This system has now been in operation for over 12 months, and this
paper highlights the benefits achieved, and outlines how this
established technology might be more widely utilised to reduce the
cost of poor quality screening.
13) Dual Resolution Syringe lets syringe
drives go 10-20X lower with high precision and contact-free
delivery.
Donald Schwartz, DRSLongstroke/DRD Diluter
Dual Resolution Syringe LONGSTROKE's very long Differential Mode
stroke minimizes variance to give unsurpassed precision and accuracy
for minute samples. Built-in high flow Single Mode handles large
volumes and eliminates bubbles and priming problems for robustness
and low maintenance. Smooth controllable flow power gives optimal
Tip Escape Velocity (TEVIA) for contact-free delivery from plopoff
through splatoff morphology of even sub-microliter (nanoliter)
volumes, including viscous and biological materials, with disposable
tips. LONGSTROKE can be swapped for a conventional syringe in any
syringe drive, typically empowering automated liquid-handling
systems to go 10-20X lower with excellent P & A while keeping high
volume capability. Mechanical design, data, automated robot use and
6 powerful applications are shown.