LabAutomation 2006 - SLAS

TP77
Susan Yan
Pierce Biotechnology
Woburn, Massachusetts
susan.yan@perbio.com
LabAutomation2006
Co-Author(s)
Scott Van Arsdell
Christine Burns
High-Throughput Detection of Human Cytokines Using Fluorescent Multiplex
SearchLight Assay
We have developed a fluorescent multiplex protein array on the SearchLight platform for the quantification of 12 human cytokines that
are involved in Th1/Th2 and inflammatory responses. Twelve different human cytokines (IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10,
IL-12p70, IL-13, IFNg, and TNFa) were printed in each 96-well plate. The assay was carried out as typical ELISA assay until the last step:
instead of adding HRP conjugated to streptavidin for chemiluminescent detection, a fluorescent dye-streptavidin conjugate was introduced
followed by laser scanning. The results show that this multiplexed assay can be used to measure 12 or more analytes at once in a single
50 ul patient sample. We demonstrate the fluorescent multiplexed protein array as a powerful tool for screening patient samples or profiling
cytokines in different disease stages.
TP78
Kate Yu
Waters Corporation
Milford, Massachusetts
kate_yu@waters.com
Co-Author(s)
Peter Alden, Rob Plumb
Waters Corporation
Li Di, Susan Li, Edward Kerns
Wyeth Research
Paul Chilvers
Waters Micromass UK Limited
Automated ESCi LC/MS/MS Quantification Protocol Applied for Microsome Stability
Test in Drug Discovery
Physical chemical information plays an important role in drug discovery. For example, the stability test screens compound stability in
microsomes etc. providing important information about potential liabilities of drug candidates. The application of LC/MS/MS in drug
discovery demands throughput, sensitivity and wide coverage of diverse structures. A combined ESI and APCI ionization source (ESCi)
increases the throughput and sample coverage by eliminating the need to physically change the ionization source and to repeat injections
for failed samples. We have developed an automated analytical protocol using the multi-mode ionization cusing an UPLC-Tandem
Mass Spectrometer; this protocol was applied to a microsome stability test. A 2.1x50 mm UPLC column (1.7 ƒÝm) was used for the
separation at 0.6 ml/minute flow rate. Four ionization modes (ESI+/ESI-/APCI-/APCI+) were used simultaneously during the analysis.
The quantitative analysis was automatically accomplished from MS and MSMS scan optimizations, to MRM method creation, to data
acquisition and processing as well as report generation. For the 96 well plate microsome stability test, standards with known metabolism
were incorporated as QC checks. Samples at Time 0 and Time 20 were measured. % remaining is calculated by divided the area at Time
15 over the area at Time 0 and times 100. From that, half-life is derived. We will present the MS optimization results as well as the MRM
analysis results from the microsome stability test samples.
190