High-Throughput Screening Applications of a Novel
Scanning Laser Imaging Technology

Presented at the October 7th, 1999 meeting of the LRIG Mid

The increasing number of compounds available for screening in drug
development has driven the requirement for higher throughput screening technologies, as
well as unique technologies that address a broader application portfolio. Our laboratory
has been involved in the development of multiplexed mix-and-read assays using Fluorometric
Microvolume Assay Technology (FMAT). FMAT is a fluorescence based assay system that
incorporates a laser scanner and optical detection system that provides a direct
measurement of cellular or bead-based fluorescence on a well-to-well and on an individual
cell/bead basis. This design is ideal for the homogenous identification of hits in primary
screening, as well as for lead optimization in the form of IC50 determinations, and for
assessment of lead compound cytotoxicity. Fluorescent beads of various sizes can be
distinguished from one another, allowing the multiplexing of two or more targets present
on different sized beads in the same well. In addition, the digitized image data is
compiled from two PMTs permitting the development of multiplexed assays based on dye
color. A variety of different mix and read applications for FMAT will be described,
including peptide-receptor ligand interactions, and multiplexed bead based immunocapture
assays. The results of a novel high-throughput screen performed in collaboration with a
major Pharma oncology group will also be discussed. Employing a simple mix-and-read
Annexin V binding assay, a variety of purified natural products were identified that are
potent inducers of apoptosis in tumor cells. Taken together, the data to be presented will
demonstrate the versatility and feasibility of fluorescence-based homogeneous and
multiplexed assays for a variety of cell-based and molecular targeted screens used in drug

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Table of Contents

PPT Slide


FMAT Instrumentation

FMAT Optics – Excitation

Scan Area is 1mm X 1mm

FMAT Optics – Emission

Image Processing

Capillary Scan

There is significant variety in the sizes and shapes
of cells:

FMAT User Interface

FMAT Analysis

FMAT – 384 Well Plate Reproducibility

Competitive Inhibition of anti-HLA-Cy5 Binding
to HL-60 Cells with Unlabeled anti-HLA

Whole Cell Peptide Ligand Binding

IC-50 Curves Utilizing Cells Expressing Endogenous

HTS Detection of a Y1 Antagonist

FMAT images of peptide binding to adherent cells

IC50 Determination using Adherent Cells

Summary of Ligand Binding Data Relevant to FMAT

Effects of Colored Compounds

PPT Slide

Multiplex Assay to Detect Apoptosis

Dose Response Curves for Chemotherapeutics using the
FMAT Apoptosis Assay – Novartis

Novartis Pure Natural Products Screen – Sample Plate

ELISA Enzyme Linked Immunosorbent Assay

Bead Based ELISA Formatted for FMAT

FLISA Fluorescence Linked Immunosorbent Assay

Comparison Between FLISA and ELISA

PPT Slide

PPT Slide

PPT Slide

PPT Slide

Multiplexed FLISA

Multiplexing with Color

Multiplexing with Size

Multiplexing with Size

Multiplexing with Size

In Summary:

Author: Sheri Miraglia, Ph.D., Senior
Scientist, PE Biosystems


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