Transcript Document
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 • Assay Development • Low/High Throughput Screening Drug Discovery Process Target Assay Bioinformatics Multiple platforms Molecular biology Multiple readouts Expression studies HTS 'Big pharma' systems Hit Evaluation Lead Optimisation Potency Functional effects Selectivity Target-related Library choice HERG Cytotoxicity Stable cell generation Apoptosis Cell proliferation CYP450 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 Ion Channels by Atomic Absorption At BioFocus, Cambridge 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 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 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 Chosen method • Atomic absorption spectrometry • 85Rb+ • Hollow cathode Rubidium lamp • Air/acetylene flame 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 Manual Assay Flame Monochromator Hollow cathode lamp source Photomultiplier detector Spray chamber and nebulizer Processing electronics Data processing and instrument control Detection Manual Assay Atomic Absorption Burner Autosampler Automated Assay Ion Channel Screening • Cells processed using appropriate automation • Supernatants analysed for Ion Content – Single burner system (low throughput) – Multi burner system Automated Platform: Reader platform initial design SOLAAR S SOLAAR S SOLAAR S SOLAAR S AAS #1 AAS #2 AAS #3 AAS #4 AutoSampler AutoSampler AutoSampler AutoSampler 4 Position #1 4 Position #2 4 Position #3 4 Position #4 Linear Track Robotic arm 80 microplate On-line Storage Operating system e.g. OVERLORD™ Data Processing Activity Base All equipment “off the shelf” Schematic AA #4 AA #2 Robot AA #3 stacker AA #1 bc What is the challenge for this assay ? 1. 2. 3. 4. 5. 6. 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 1. Scheduler needs to be flexible • AA run times not the same • Throughput critical • Error trapping and recovery a necessity 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 ! 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 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 Scheduling - the Choice real-time pre-emptive OVERLORD Workstation OVERLORD Scheduler either Real Time Instrument Control Options • Sequential • Round robin Control system Site computer services Multiple port switch Main controller & Data processing AA#1 instrument control AA#2 instrument control AA#3 instrument control AA#4 instrument control OVERLORD NetLORD node 1 NetLORD node 2 NetLORD node 3 NetLORD node 4 Instrument control – Round Robin Cycle AA #1 node Ready ? Load, unload busy, error ? Load plate Run AAS Start Run AA #2 node Ready ? AA #3 node Ready ? Data Handling AA #4 node Ready ? Run complete ? Unload plate Busy Error 2. SOLAAR software • • • • Requires automation interface There isn’t one !!!! Use the OVERLORD keystroke API Emulates a user 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 4. Autosampler was not robot friendly 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 6. Data handling/data quality • • • • Barcodes Data file renaming for integrity Data transfer to the server Real time data analysis for quality check Luminex Instrument Luminex 100 • • • • Bead based with 100 assays per well Small sample volume 1000 samples a day reproducible Higher throughput • Traditional ELISA • 1 measurement/well • xMAP technology • 100 measurements/well Analysis method • Beads sucked up past fluorescence source • Fluorescence detected Comparison Microarray Q-PCR ELISA Luminex xMAP Technology Multiplex Throughput What is the challenge for this assay ? • • • • Luminex software - no automation interface Luminex instrument – not robot friendly Caliper Twister I Lids 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 Caliper Twister I • Limited control system – OVERLORD has own Twister I control • Lids – Can be a problem – Relidding especially Luminex Twister I Delidding Shaker 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 > 220 instruments And remember ….. Real high throughput screening !