Challenging_Assays

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					  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       HTS                Hit Evaluation     Lead Optimisation



 Bioinformatics  Multiple      'Big pharma'      Potency           Functional effects
                    platforms      systems
 Molecular                                         Selectivity       Target-related
  biology          Multiple     Library choice
                    readouts                                           HERG
 Expression studies
                                                                       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



                                                   Operating system
 80 microplate                                     e.g. OVERLORD™
    On-line
                                                   Data Processing
    Storage
                                                    Activity Base




                             All equipment “off the shelf”
        Schematic




AA #3                    AA #2




            Robot
AA #4                    AA #1
                    bc

          stacker
 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
   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             OVERLORD
Workstation           Scheduler




either
Real Time Instrument Control Options

• Sequential
• Round robin
                                Control system

                                                     Site computer services




                                                      Multiple port switch




Main controller &         AA#1                 AA#2                   AA#3                 AA#4
Data processing     instrument control   instrument control     instrument control   instrument control

  OVERLORD          NetLORD node 1       NetLORD node 2          NetLORD node 3      NetLORD node 4
                Instrument control – Round Robin
                  Cycle

                             AA #1 node   Load, unload     Load plate
                              Ready ?     busy, error ?

                                                           Run AAS
  Start Run
                             AA #2 node
                              Ready ?
                                                          Unload plate


                             AA #3 node
                              Ready ?                        Busy
Data Handling


                             AA #4 node
                              Ready ?
                                                             Error
                  Run
                complete ?
           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    • xMAP technology
• 1 measurement/well   • 100 measurements/well
            Analysis method




• Beads sucked up past fluorescence source
• Fluorescence detected
        Comparison

Q-PCR                     Microarray

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 !

				
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