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Increasing the Throughput of Challenging Assays by Automation

Increasing the Throughput of Challenging Assays by Automation. Malcolm Crook Process Analysis & Automation Ltd Farnborough. What is a challenging assay ?. Challenging instrument to automate Physically Software control, no automation interface Multiple PCs required. Why are we doing this.

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Increasing the Throughput of Challenging Assays by Automation

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  1. Increasing the Throughput of Challenging Assays by Automation Malcolm Crook Process Analysis & Automation Ltd Farnborough

  2. What is a challenging assay ? • Challenging instrument to automate • Physically • Software control, no automation interface • Multiple PCs required

  3. Why are we doing this • Assay Development • Low/High Throughput Screening

  4. Target Assay Hit Evaluation HTS Lead Optimisation Drug Discovery Process • Bioinformatics • Molecular biology • Expression studies • Stable cell generation • Multiple platforms • Multiple readouts • 'Big pharma' systems • Library choice • Potency • Selectivity • Functional effects • Target-related • HERG • Cytotoxicity • Apoptosis • Cell proliferation • CYP450

  5. 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

  6. Ion Channels by Atomic Absorption At BioFocus, Cambridge

  7. 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

  8. 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

  9. Non-radiometric ion flux 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

  10. Chosen method • Atomic absorption spectrometry • 85Rb+ • Hollow cathode Rubidium lamp • Air/acetylene flame

  11. 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

  12. Flame Monochromator Hollow cathode lamp source Photomultiplier detector Spray chamber and nebulizer Processing electronics Data processing and instrument control Manual Assay

  13. Detection

  14. Manual Assay Atomic Absorption Burner Autosampler

  15. Automated Assay Ion Channel Screening • Cells processed using appropriate automation • Supernatants analysed for Ion Content • Single burner system (low throughput) • Multi burner system

  16. SOLAAR S AAS #1 SOLAAR S AAS #2 SOLAAR S AAS #3 SOLAAR S AAS #4 AutoSampler 4 Position #1 AutoSampler 4 Position #2 AutoSampler 4 Position #3 AutoSampler 4 Position #4 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”

  17. Robot Schematic AA #3 AA #2 AA #4 AA #1 bc stacker

  18. What is the challenge for this assay ? • Scheduler has to be flexible to time delays • SOLAAR software-no automation interface • Four AAs required to achieve throughput • Autosampler was not robot friendly • Safety • Data handling/data quality

  19. 1. Scheduler needs to be flexible • AA run times not the same • Throughput critical • Error trapping and recovery a necessity

  20. 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 !

  21. 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

  22. 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

  23. real-time OVERLORD Workstation pre-emptive OVERLORD Scheduler either Scheduling - the Choice

  24. Real Time Instrument Control Options • Sequential • Round robin

  25. Site computer services Multiple port switch Main controller & Data processing OVERLORD AA#1 instrument control NetLORD node 1 AA#2 instrument control NetLORD node 2 AA#3 instrument control NetLORD node 3 AA#4 instrument control NetLORD node 4 Control system

  26. Cycle AA #1 node Ready ? Load, unload busy, error ? Load plate Run AAS AA #2 node Ready ? Unload plate AA #3 node Ready ? Data Handling Busy AA #4 node Ready ? Error Run complete ? Instrument control – Round Robin Start Run

  27. 2. SOLAAR software • Requires automation interface • There isn’t one !!!! • Use the OVERLORD keystroke API • Emulates a user

  28. 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

  29. 4. Autosampler was not robot friendly

  30. 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

  31. 6. Data handling/data quality • Barcodes • Data file renaming for integrity • Data transfer to the server • Real time data analysis for quality check

  32. Luminex Instrument

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

  34. Traditional ELISA 1 measurement/well xMAP technology 100 measurements/well Higher throughput

  35. Analysis method • Beads sucked up past fluorescence source • Fluorescence detected

  36. Microarray Q-PCR ELISA Luminex xMAP Technology Multiplex Throughput Comparison

  37. What is the challenge for this assay ? • Luminex software - no automation interface • Luminex instrument – not robot friendly • Caliper Twister I • Lids

  38. 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

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

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