LabAutomation 2006 - SLAS

TP39
Robert Newman
Merck Sharp And Dohme
Harlow Essex, United Kingdom
robert_newman@merck.com
Where Laboratory Technologies Emerge and Merge
Co-Author(s)
Samentha Ellis
Julie Kerby
Mathew Leveridge
Peter Simpson
Carmel Nanthakumar
Kevin Moore
Merck, Sharp and Dohme
Development of an Automated High Throughput Assay to Measure Amyloid’s Peptides
Using Meso Scale Discovery Electrochemiluminescence Detection
Alzheimer’s disease is pathologically characterised by extracellular deposits of amyloid-â (Aâ) in the cerebral parenchyma or surrounding
blood vessels and intracellular neurofibrillary tangles. Robust and reliable assay methodologies for identifying small molecules that inhibit Aâ
formation could provide useful tools in drug discovery. The aim of the present study was to develop an automated high throughput assay to
quantify Aâ peptides secreted from SH-SY5Y cells over-expressing human amyloid precursor protein (APP). The Meso Scale Discovery
(MSD) platform is based on the electrochemical properties of the ruthenium cation and carbon electrode arrays inserted into microtitre
plates. Cells were seeded into 96-well plates at a range of densities and incubated for 20 h at 37°C/5% CO2. Aâ peptides were detected
by transferring cell plate supernatant to 96 well streptavidin coated MULTI-ARRAY plates with 4µg/ml biotinylated 4G8 and 1µg/ml
ruthenylated anti-amyloid beta antibodies, and incubating for times ranging between 1h to 24h. Electrochemiluminescence was detected
using a MSD SECTORTM 6000 plate reader.
Secretion of Aâ peptides was detected at all cell densities with the greatest signal from cells seeded at 3.5 x104. DMSO concentrations of
up to 2% were well tolerated. Generation of a stable antibody-peptide complex was detected at 1h incubation, and reached equilibrium
at 12h to 24h, yielding a Z’ between 0.6 and 0.7. Using synthetic Aâ peptides, the limit of detection of the assay was determined to be
between 10-100pM. This sensitive and robust assay to quantify Aâ peptides has been automated on a Beckman-Sagian robotic system.
TP40
Peter Nollert
deCODE biostructures
Bainbridge Island , Washington
pnollert@decode.com
Co-Author(s)
Mark Mixon, Stuart Bowers, Brendan Gan
deCODE biostructures
Geetha Rao, Norgren Systems
Larry Nickell, Appalachian Electronic Instruments
Lance Stewart, deCODE biostructures
DETECT-X: an Automated Microscope for Bulk Crystal Contrast Enhancement
A mission-critical event in X-ray crystallographic protein structure determination is the accurate detection of protein crystals in a
crystallization chamber. While protein crystallization trials are usually prepared using high-throughput liquid dispensation technology, the
assessment of such experiments is usually carried out by visual inspection of each individual crystallization chamber by an experienced
individual. This poses a substantial burden on operator-based resources especially when large numbers of crystallization experiments (i.e.
more than 10,000/day) need to be evaluated.
The presence of crystals is generally established by the observation of crystal edges in 2D images taken with a microscope. For a number
of reasons such edges are frequently disguised and it is desirable to detect crystals by virtue of bulk contrast. Polarization microscopy, a
method that can provide bulk color for non-cubic space group crystals, would be formidable tool for bulk contrast generation. However,
its application is severely hampered due to the ubiquitous use of polymer-based birefringent crystallization trays that provide a substantial
background in which it is difficult to spot crystals.
We show how bulk contrast of micro crystals grown in birefringent plastic trays can be enhanced dramatically by digital processing of
images that were captured with an integrated Extinction Polarization Microscope with Birefringence Difference Contrast.
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