Increasing the Throughput of Challenging Assays by Automation

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Increasing the Throughput of Challenging Assays
by Automation

• Malcolm Crook
• Process Analysis Automation Ltd
• Farnborough

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What is a challenging assay ?

• Challenging instrument to automate
• Physically
• Software control, no automation interface
• Multiple PCs required

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Why are we doing this

• Assay Development
• Low/High Throughput Screening

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Drug Discovery Process

• Bioinformatics
• Molecular
• biology
• Expression studies
• Stable cell generation

• Potency
• Selectivity

• Multiple
• platforms
• Multiple
• readouts

• 'Big pharma'
• systems
• Library choice

• Functional effects
• Target-related
• HERG
• Cytotoxicity
• Apoptosis
• Cell proliferation
• CYP450

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Requirements for an Assay

• High-throughput
• Low false-positive rate
• Low false-negative rate
• Direct measure of function
• Good correlation with electrophysiology
• Reliability
• Reproducibility
• Amenable to miniaturization
• Low cost

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Ion Channels by Atomic Absorption

• At BioFocus, Cambridge

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What is an ion channel ?

• assembly of proteins
• circular arrangement of proteins
• closely packed around water-filled pore
• large-pore channels permit
• passage of ions
• more or less indiscriminate

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What is an ion channel ?

• archetypal channel pore
• one or two atoms wide
• conducts a specific species of ion
• sodium or potassium
• conveyed through membrane single file
• fast
• access to the pore is governed by a "gate
• opened or closed by
• chemical/electrical signals
• Temperature
• mechanical force

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Measuring Ion Channels

• Redistribution voltage-dependent dyes
• FRET-based technology
• Radioligand binding
• Automated two-electrode voltage clamp
• Automated whole-cell patch clamp
• Planar patch clamp
• Radiometric ion flux
• Non-radiometric ion flux

• Radioactive
• Cost
• Low throughput

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Chosen method

• Atomic absorption spectrometry
• 85Rb
• Hollow cathode Rubidium lamp
• Air/acetylene flame

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Advantages of AAS

• Health and Safety
• Ease of handling
• Cost of components
• Cost of disposal
• Environmental Impact
• Sensitivity
• No time limits to read samples
• Decay or Licence constraints

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Manual Assay
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Detection
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Manual Assay
Atomic Absorption Burner
Autosampler
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Automated Assay Ion Channel Screening

• Cells processed using appropriate automation
• Supernatants analysed for Ion Content
• Single burner system (low throughput)
• Multi burner system

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Automated Platform Reader platform initial design
Linear Track Robotic arm
Operating system e.g. OVERLORD Data Processing
Activity Base
80 microplate On-line Storage
All equipment off the shelf
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Schematic
AA 3
AA 2
AA 4
AA 1
bc
stacker
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What is the challenge for this assay ?

1. Scheduler has to be flexible to time delays
2. SOLAAR software-no automation interface
3. Four AAs required to achieve throughput
4. Autosampler was not robot friendly
5. Safety
6. Data handling/data quality

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1. Scheduler needs to be flexible

• AA run times not the same
• Throughput critical
• Error trapping and recovery a necessity

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Scheduler control options

• Time resolved
• Static
• Pre-emptive
• All decisions are taken before the run starts.
  i.e. before the GO button is pressed
• Event driven
• Real time
• Dynamic
• All decisions are taken during the time frame of
  the run, i.e. once the GO button is pressed
• Process Analysis Automation have both !

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Time resolved

• Advantages
• one or more accurate timings are guaranteed
• easy to set up
• Disadvantages
• inflexible
• decisions can not be taken during the run
• error recovery more difficult

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Event Driven

• Advantages
• flexible
• decisions can be taken during the run
• error recovery easy
• Disadvantages
• accurate timings are more difficult to set up
• two accurate timings in a run are impossible

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Scheduling - the Choice
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Real Time Instrument Control Options

• Sequential
• Round robin

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Control system
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Instrument control Round Robin
Start Run
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2. SOLAAR software

• Requires automation interface
• There isnt one !!!!
• Use the OVERLORD keystroke API
• Emulates a user

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3. Four AAs required for throughput

• Use NetLORD
• Remote OVERLORD nodes
• Makes control structure easier
• Node in 4 states
• Ready for load
• Ready for unload
• Busy
• Error state
• NetLORD nodes addressed by IP address
• Common folders for data transfer
• Redundancy

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4. Autosampler was not robot friendly
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5. Safety

• Hazards
• AAS instrument
• Locked room
• Air conditioning
• Acetylene gas
• Only run during the day
• Gas detectors
• Time out on AAS flames
• Robot
• Hamilton SWAP stops when touched
• Hamilton SWAP can be recovered

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6. Data handling/data quality

• Barcodes
• Data file renaming for integrity
• Data transfer to the server
• Real time data analysis for quality check

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Luminex Instrument
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Luminex 100

• Bead based with 100 assays per well
• Small sample volume
• 1000 samples a day
• reproducible

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Higher throughput

• Traditional ELISA
• 1 measurement/well

• xMAP technology
• 100 measurements/well

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Analysis method

• Beads sucked up past fluorescence source
• Fluorescence detected

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Comparison
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What is the challenge for this assay ?

• Luminex software - no automation interface
• Luminex instrument not robot friendly
• Caliper Twister I
• Lids

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Luminex instrument not robot friendly

• Plate tray only suitable for fingers
• Modify the tray with a hacksaw
• Software
• Version 1.7 no automation interface
• Keyboard control possible

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Caliper Twister I

• Limited control system
• OVERLORD has own Twister I control
• Lids
• Can be a problem
• Relidding especially

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Conclusions

• Instruments can be automated
• Keyboard control
• Plate access
• Unusual assays
• Real time scheduler
• Round robin method
• Twister I robots good tools
• Need more than basic software
• OVERLORD can control gt 220 instruments
• And remember ..

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Real high throughput screening !