The Laboratory Robotics Interest Group

December 1999 Meeting

High Throughput Screening Using the 1536-Well Format: Distinguishing Fact from Fiction

Date:        Tuesday, December 7, 1999
Place:      Hanover Marriott,1401 Rt 10 E, Whippany, NJ 07981
                  Phone: 973-538-8811, Fax: 973-538-0291
Itinerary:  Social Period & Exhibition - 3:30 to 6:30 PM
                  Presentations  - 6:30 to 8:00 PM
                  Panel Discussion - 8:00 to 9:00 PM
Registration:   Requested, not required.   Pre-registering will allow us to more accurately gauge seating requirements and refreshment needs.  Indicate names of attendees and company affiliation.  Pre-register by email with <mailto:[email protected]> or by phone at (732)302-1038.  In order to speed sign-in at the meeting, please bring a business card to drop into the registration box.  There will be a business card drawing for a PalmPilot IIIx donated by TekCel, www.tekcel.com.

Agenda:  
There will be a variety of vendors available exhibiting the latest developments in laboratory automation hardware and software related to 1536-well and higher density formats during a social period (3:30 P.M. - 6:30 P.M.). Food and refreshments will be available, provided by LJL Biosystems, www.ljlbio.com.   This will be followed by three talks and a panel discussion. Members interested in presenting a poster are encouraged to do so.  Open career positions at your company may be announced or posted.  There is no registration or fee associated with this LRIG function.

Exhibitors:

Presentation:  The Complete Solution to ultra-High Throughput Screening Assay Miniaturization.
David A. Dunn; Senior Research Fellow; Pharmacopeia, Inc.

A complete solution to the challenges of assay development in miniaturized format, including plate miniaturization, liquid handling, optical detection, data management and assay development, is required in order to implement a viable uHTS program. This talk will cover new tools, developed at Pharmacopeia and elsewhere, that address these challenges. Examples of their successful implementation as part of our regular screening operation will be given.

Presentation: An Ultra High-Throughput Approach for an Adenine Transferase Using Fluorescence Polarization
Dr. Julie Li, Hoechst Marion Roussel

We have developed a novel assay for measuring the activity of an enzyme that transfers multiple adenine-containing groups to an acceptor protein. The assay is based on fluorescence polarization (FP) technology in a 1536-well plate format. In the assay, a long wavelength fluorescence tracer, Texas Red (Rhodamine), was covalently conjugated to adenine of the donor substrate through a C6 spacer arm. As a result of the transfer of the adenine-containing moieties to the acceptor protein substrate, the rotation correlation time of the Texas Red conjugate increased, and hence the degree of fluorescence polarization. The pharmacological profile and kinetics of the enzyme measured according to the FP method were consistent with those determined previously by conventional analysis. We have successfully executed a 250,000 compound high throughput screening program based on the FP assay method. The quality and validity of the assay were verified by a variety of statistical analyses.

Presentation: Analysis of Protein-Peptide Interaction by a Miniaturized Fluorescence Polarization Assay Using Cyclin Dependent Kinase 2/Cyclin E as a Model System
Ilona Kariv, Sokhom S. Pin, and Kevin R. Oldenburg
Leads Discovery, DuPont Pharmaceuticals, Wilmington, DE 19880

As a result of the increasing size of chemical libraries, more rapid and highly sensitive strategies are needed to accelerate the process of drug discovery without increasing the cost. One means of accomplishing this is to miniaturize the assays that enter high throughput screening (HTS). Miniaturization requires an assay design that has few steps, has a large degree of separation between the signal and background, and has a low well to well signal variation. Fluorescence polarization (FP) is an assay type that, in many cases, meets all of the above requirements. FP is a homogenous method that allows interactions between molecules to be measured directly in solution. This article demonstrates the application of FP to a miniaturized HTS format, using 1536-well plates, to measure direct binding between cyclin dependent kinase 2/cyclin E complex (CDK2/E) and an 8-mer-peptide kinase inhibitor. The data indicate that low variability and high specificity allow rapid and precise identification of antagonist compounds affecting CDK2/E-peptide interactions.

Panel: Following the talks, there will be a moderated panel discussion with HTS experts who are using 1536-well and higher density formats in their laboratories.
Panel members include:
David A. Dunn, Senior Research Fellow; Pharmacopeia
Dr. Julie Li, Hoechst Marion Roussel
Ilona Kariv, Leads Discovery, DuPont Pharmaceuticals
Daniel Chelsky, BioSignal
Jonathan Burbaum, Associate Director, Technology Business Development, Pharmacopeia
Meng Zhang, M.D., Ph.D.; Senior Applications Scientist, LJL BioSystems, Inc.

Poster: Fluorescence Detection Strategies for Electroseparations in Plastic Micro-fabricated Devices
Shau-Chun Paul Wang, Department of Chemistry, University of Michigan, [email protected]

Fluorescence background interference from the device is inherent in plastic microchips, particularly with blue or UV excitation. Conventionally, microchip background has been reduced with confocal optics or circumvented with specialized long wavelength fluorophores. We show that microchip background can be rejected with analyte velocity modulation. In this scheme the driving voltage is modulated at low frequency. Migration velocities and analyte signals are modulated at the same frequency. Microchip fluorescence is unmodulated, so that lock-in detection (synchronous demodulation) easily separates the analyte signal from background. The technique does not require a laser source. In our implementation a blue (485nm) LED is the light source. Simple optics are used to shape the source and focus it to a spot about 50 microns in diameter inside a microchip. Photomultiplier detection is employed and a lock-in amplifier is used to demodulate the signal. Apertures in the system generate a derivative response, which can be converted to conventional bands by integration. Fluorescence rejection provided by our first generation system lowers detection limits by five to eight fold compared to DC measurements with the same optical train.

Poster: Corning 1536-Well Assay Plate for HTS
Arthur Trombley, Corning Inc. Science Products Division, Portsmouth, NH USA
Jill Veilleux, Corning Inc. Science Products Division, Acton, MA USA
David Dunn, Marc Orlowski, Pharmacopeia, Inc., Princeton, NJ USA
Meng Zhang, Doug Boyd, LJL Biosystems, Inc., Sunnyvale, CA USA

The new Corning 1536–well 2 痞 Plate provides the foundation for a unique solution to assay miniaturization for high throughput screening. The advantages of the 1536-well HTS plate include:

1. Conservation of 96 and 384 well format, which aids in sample transfer, well identification, and data manipulation
2. Low profile, which provides increased sample density over conventional plate height and reduces parallax effects in imaging systems
3. Three point positioning and tight manufacturing tolerances for all dimensions, which enable high-speed automated plate handling functions
4. The lowest volume wells (2 痞) that are commercially available
5. Optimized materials for manufacture, which enhance performance in Fluorescence, luminescence and colorimetric detection formats.

Here, we provide an analysis of evaporative loss and automated dispensing at low volumes. The Corning 1536-well 2 痞 Plate is shown to be an ideal solution to scale up from traditional 96 and 384 well formats in high throughput

Posters will also be presented by BioSignal and Perkin Elmer Tropix.

Take Route 287 to Exit 39 (Route 10 West). Go through one stoplight and take 1st u-turn.

Exhibitors - Click here for Exhibitor Information

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