Tecan Journal Life Sciences ❙ Clinical Diagnostics ❙ Forensics ❙ OEM ❙ Business and Technical News
Edition 1 / 2010
Automated DNA preparation pushes forward evolutionary biology research pages 18–19
TouchTools Suite™ with new enhancements page 4
Efficient vaccine development pages 12 – 13
The missing link for autoimmune disease pages 28 – 29
Developing embryos in eggs of three-spined stickleback (Gasterosteus aculeatus), a model organism for studies in evolutionary biology
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CEO WELCOME TECAN JOURNAL 1/2010
As we begin our 30th anniversary year, it gives me great pleasure to welcome you to the first Tecan Journal of 2010. The 30 year landmark is a good time to look back, take stock, and drive forward to new goals and challenges. We have a strong and well-settled organization, and pride ourselves on maintaining a reputation for high quality and customer loyalty. We look forward to another 30 years of continuing to forge productive relationships with our customers and translating their scientific challenges into automation solutions.
New products featured in this edition of the Journal, like Active Worktable and the updated TouchTools Suite™, demonstrate our dedication to bringing automation closer to more scientists. You can also read about efforts by MedImmune to develop a range of user-friendly automated tools based on the Freedom EVOŽ workstation, and how Inverness Medical has been providing Tecan automation to hospitals and laboratories to process clinical diagnostics kits. Other articles in this issue show the wide range of applications for which Tecan products are being used, including vaccine development, structural analysis, diagnostic testing, and studies of evolutionary biology and electrophysiology. We hope you enjoy this Tecan Journal, and wish you a successful and prosperous 2010.
Welcome
Thomas Bachmann, CEO
CONTENTS TECAN JOURNAL 1/2010
page 5
Automation of the bead washing protocol using the Freedom EVO® 75 is particularly attractive for customers with a high sample throughput page 9
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CEO welcome
4 TouchTools Suite™ 2.0 5
New Active Worktable
6 Next-generation Infinite® F50 6 PowerScanner™ is the new flagship of Tecan’s ‘microarray suite’ range 7 New products and purity levels for MCA 96 disposable tips 8 New partnership with FortéBio for bioprocessing and drug discovery assays 9 A boost for sequencing workflows 10
Tecan hits the road
11 Tecan supports ELRIG sponsorship of ‘TechnoBotts’ 11 Tecan to supply fully automated solution for Hologic Cervista® HPV tests
page 28-29
Developing protein microarrays for serum-based diagnostic testing
Contents
page 16-17
12-13 Automated protein purification for efficient vaccine development 14-15 A long-standing partnership with success 16-17 HydroFlex™ offers a helping hand for automated bead washing 18-19 Automated DNA preparation pushes forward evolutionary biology research 20-21 New tools of the trade 22-23 Plug and play electrophysiology 24-25 Infinite® offers the MIDAS touch 26-27 Automation greatly speeds up protein production for X-ray diffraction experiments 28-29 The missing link for autoimmune disease 30-31 Fully automated ELISA testing in veterinary diagnostics 31
Leading the debate
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Events
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PRODUCT NEWS TECAN JOURNAL 1/2010
TouchTools Suite™ 2.0 Since its launch last year, Tecan’s userfriendly TouchTools Suite has made automated liquid handling more accessible to many scientists, by allowing direct realtime control of automated robotic pipetting systems through a touchscreen interface. It does not require any programming skills or pre-defined scripts to operate, and can virtually turn your robotic system into an automated hand pipette. The popular system has now been further enhanced by a set of added features to provide even more flexibility. In TouchTools Suite 2.0, the unique, patentpending Instant Pipetting™ module is now compatible with any Tecan pipetting arm, including all liquid handling (LiHa) arms, 8 Plus 1 Access™, MultiChannel Arm™ 384 and MultiChannel Arm 96. This makes straightforward procedures of a wider range of applications possible, including fast full plate replication. The new version is also able to control the tip spacing of 8-channel LiHa arms, which allows more complex pipetting patterns, even into 384-well plates.
Hit-Picking mode is another new feature that has been developed in response to many user requests. Hit-Picking mode displays the plate on the graphic user interface and allows the user to select individual wells through the touchscreen, commanding the automated platform to instantly access those positions.
To find out more on Tecan’s TouchTools Suite, visit www.tecan.com/touchtools PowerPoint is a registered trademark of Microsoft Corporation in the United States and other countries. iPod touch is a trademark of Apple Inc., registered in the US and other countries.
Other improvements to the TouchTools Suite allow customized input screens to tailor the user interface to the laboratory’s needs. A custom-loading guide is now able to display customized instructions saved as PowerPoint®, PDF or other formats. In the QuickStart module, the settings can be personalized, so that users can only access the methods that they are authorized to use. Users of Tecan’s robotic instruments can also look forward to a new version of the Common Notification System (CNS), due to launch in the near future, which will make the remote access and monitoring of the laboratory’s automated systems from mobile devices even easier.
TouchTools Suite allows direct real-time control of automated robotic pipetting systems through a touchscreen interface
PRODUCT NEWS TECAN JOURNAL 1/2010
New Active Worktable
Active Worktable has been designed for simplicity in operation, bringing extra process security, control and monitoring to research scientists.
The new Active Worktable option for the Freedom EVO® 100, 150 and 200 platforms, launching at Lab Automation 2010, offers benefits of extra process security, control and monitoring to research scientists. It brings the functionality available in Freedom EVOlyzer® and Freedom EVO Clinical workstations into the researchers’ domain, with added flexibility to run their own scripts for various applications. Active Worktable has been designed for simplicity in operation, keeping the user informed of what is on the deck through a graphical user interface and, with red and green lights to signal carrier status. All automated applications can benefit from the quick operational status update that is available at a glance.
Worktable monitoring ensures safety The Active Worktable closely monitors all user interactions with the worktable, and confirms all loading and unloading actions using a magnetic sensor on each individual grid on the worktable. Active Worktable notifies the user what needs to be done, and where carriers need to be positioned with multicolored LEDs on each grid. It also monitors the worktable during a run and, if it detects that a required carrier has been removed, it will stop the system to avoid false results. Optional PosID™ adds automatic barcode scanning and evaluation of the samples, loaded plates and reagent containers to ensure further process security.
Simple interaction with the operator The Active Worktable’s integrated system of green and red lights simplifies the placement of sample and reagent racks on the instrument. The lights, together with an acoustic signal, alert the user whenever interaction with the system is needed, making it easy for the operator to know the status of the workstation. Active Worktable has been tightly integrated into Freedom EVOware® Sample Oriented, so customers are offered the flexibility in the control of added modules as well as wizardbased sample pipetting. To find out more on Tecan’s Freedom EVO, visit www.tecan.com/freedomevo
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PRODUCT NEWS TECAN JOURNAL 1/2010
Next-generation Infinite® F50 New state-of-the-art ELISA microplate reader with latest LED technology The Infinite F50 is Tecan’s next-generation, 8-channel absorbance reader that provides accurate, reproducible and fast measurements out of a compact, innovative design, and is ideal for a variety of ELISA applications. The Infinite F50 uses the latest LED technology to replace the traditional halogen lamp light source, providing longer service-free life, lower energy consumption and compact dimensions. LEDs typically last 10 times longer than halogen lamps, making inconvenient lamp changes a thing of the past.
The Infinite F50 comes complete with Magellan™ – Tecan’s established reader control and data analysis software – to allow quick and easy measurements. The wizardbased software’s interface in eight languages (Chinese, English, French, German, Italian, Portuguese, Russian and Spanish) provides full qualitative and quantitative EIA analysis, including endpoint, kinetic and multi-label measurements and all major curve fittings. A comprehensive QC function and a range of data import and export options are also available. Both the Infinite F50 absorbance reader and Magellan software have been designed, tested, verified and validated according to ISO 13485:2003 quality system standards,
ensuring technical compliance and integrity of data. Furthermore, with the optional Magellan Tracker software, the Infinite F50 has been validated to meet the 98/79/EC IVD directive for in vitro diagnostic products and, by providing electronic signatures, records and multilevel user management, meets FDA’s Title 21 CFR Part 11 to allow its use in diagnostic applications. To find out more on Tecan’s Infinite F50, visit www.tecan.com/infiniteF50
PowerScanner™ is the new flagship of Tecan’s ‘microarray suite’ range The PowerScanner is Tecan’s open-format microarray scanner, designed for imaging of regular and high density microarrays of tissues, proteins and nucleic acids. This state-of-the-art system features: • High resolution – from 2 to 40 μm, with two configurations available (4 μm system is upgradable to 2 μm) • Excellent sensitivity – offering outstanding signal-to-noise ratio for reproducible detection • Large dynamic range – plus multiple scans at varying gain • Unrivaled versatility – allowing the widest possible range of applications • Flexible analysis – including Array-Pro® Analyzer software
The highly customizable PowerScanner allows fully automated processing of up to 48 slides in one run, and features an open platform design to ensure compatibility with virtually all major brands of microarrays using transparent, opaque or reflective substrates. The system is configurable and upgradable according to individual resolution requirements, providing excellent resolution for imaging of both high- and low-density microarrays on a single platform. To find out more on Tecan’s PowerScanner, visit www.tecan.com/powerscanner
Array Pro Analyzer is a registered trademark of Media Cybernetics, Inc.
PRODUCT NEWS TECAN JOURNAL 1/2010
New products and purity levels for MCA 96 disposable tips Tecan has introduced new purity levels for the MCA 96 disposable tips, to satisfy a broader range of applications. All Tecan Pure MCA 96 disposable tips are free of human DNA, RNase, DNase and PCR inhibitors, making them suitable for genomics research and forensics use. Tecan Sterile disposable tips offer the same high purity standards, with the additional benefits of being sterile and free from pyrogens/endotoxins, and are ideally suited to cell biology, microbiology and cell-based screening applications. Nested tips for the MCA 96 are now also available in Tecan Sterile purity, extending the benefits of Tecan nested tips into these fields. Tecan Pure and Tecan Sterile disposable tips are manufactured in a controlled environment, and subject to periodic purity testing according to USP31 and ISO 11137 guidelines. Tecan’s new Certificate of Conformity provides access to detailed information on purity level specifications, lot number and expiry date, offering full product traceability.
Tecan is also introducing wide bore tips for the MultiChannel Arm™ 96. The wider orifice of these tips minimizes shearing and fragmentation, allowing effective pipetting of fragile cell suspensions or genomic DNA. Handling of viscous fluids is also improved, because of the decreased flow resistance. The narrower aperture of conventional disposable tips can get clogged, making wide bore tips a superior alternative in areas such as crop research, where suspensions of plant material – such as leaves and seeds – are handled. They are available as non-filtered Tecan Pure or Tecan Sterile with filters, and have already completed successful external testing at Bayer CropScience in Belgium, where the filtered version is now in routine laboratory use. To find out more, visit www.tecan.com/purity and www.tecan.com/mca96diti All products: Not for use in human clinical or diagnostic procedures.
Sterile nested tips
Tecan Pure and Tecan Sterile disposable tips are manufactured in a controlled environment, and subject to periodic purity testing according to USP31 and ISO 11137 guidelines.
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PARTNERSHIPS TECAN JOURNAL 1/2010
New partnership with FortéBio for bioprocessing and drug discovery assays Tecan and FortéBio have worked together to integrate the new FortéBio Octet® 384 platform on the Freedom EVO® workstation for automated real-time, label-free assays to support bioprocessing and drug discovery workflows. Tecan has collaborated with FortéBio to deliver a robust, off-the-shelf integration that allows the user to get the most out of the Octet 384, allowing increased walkaway time as well as optional liquid handling for assay plate set-up.
Label-free, real-time methods avoid artifacts caused by label interference. FortéBio’s Octet instruments provide reliable high throughput measurements for proteinprotein, protein-peptide and protein-small molecule interactions using the innovative Dip and Read™ biosensor, that has been designed to analyze specific biomolecular interactions. The Octet RED384 performs label-free quantitation, kinetics and affinity measurements from protein-protein to protein-small molecule interaction analysis, including fragment or small molecule screening.
Tecan has designed a Freedom EVO deck extension plate to mount the Octet securely on the right hand side of the workstation. A robotic manipulator (RoMa) arm performs automated transport and handling of plates and sensor trays. An 8-channel liquid handling (LiHa) arm with fixed or disposable tips, or a MultiChannel Arm™ (MCA) with a 384- or 96-channel pipetting head can also be specified for liquid handling tasks. Octet is a registered trademark and Dip and Read is a trademark of FortéBio Inc.
The integrated FortéBio Octet 384 platform on the Freedom EVO workstation for automated real-time, label-free assays
PARTNERSHIPS TECAN JOURNAL 1/2010
A boost for sequencing workflows
The Applied Biosystems SOLiD System genetic sequencing platform performs next-generation sequencing
Tecan has been working with Applied Biosystems, part of Life Technologies Corporation to develop an off-the-shelf system for automated bead washing, based on a Freedom EVO® 75 workstation equipped with a Te-MagS™ separation module.
The power of next-generation sequencing methods comes from simultaneous, massively parallel analysis of small DNA fragments. Because of this, meticulous sample preparation is essential to get the maximum information from an analysis run. Preparation of templated beads for the Applied Biosystems’ SOLiD™ System genetic sequencing platform is a multi-step process, requiring several washes of magnetic beads for ePCR product purification. In a close collaboration between Tecan and Life Technologies, a new system based on the Freedom EVO 75 workstation has been carefully developed to eliminate the variation commonly associated with manual techniques, minimizing loss of beads and ensuring complete removal of chemicals which could interfere with results. Automation of the bead washing protocol using the Freedom EVO 75 is particularly attractive for customers with a high sample throughput, as Karen Poulter, a staff scientist at Life Technologies, explained: “Automation of ePCR purification for the SOLiD System eliminates a lot of the timeconsuming and laborious work associated with manual techniques. This not only reduces hands-on time, allowing users to
concentrate on more challenging tasks, but also improves consistency.” The Freedom EVO 75 bead washing station is equipped with a two-channel liquid handling (LiHa) arm, a TeMagS magnetic separation module and all the racks and carriers required for parallel preparation of 24 templated bead samples. In addition, the system’s Freedom EVOware® software features washing protocols specifically developed by Life Technologies’ Genetic Systems Division for optimal sequencing performance of the SOLiD System. “We chose the Freedom EVO 75 platform to automate templated bead washing for a combination of reasons,” said Kathy Perry, science director at Life Technologies. “Tecan is a very easy company to work with, and the instrument represents a compact, costeffective solution for automation of bead washing protocols. This allows us to offer our customers a validated, off-the-shelf solution for genetic sample preparation, helping to streamline laboratory workflows and improve consistency of results.” To find out more on Tecan’s Freedom EVO 75, visit www.tecan.com/freedomevo75 SOLiD is a trademark of Life Technologies.
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CORPORATE NEWS TECAN JOURNAL 1/2010
Tecan hits the road Over the last few months, Tecan application specialists around the world have been organizing roadshows and seminars to discuss the benefits of laboratory automation with customers from the biotechnology, pharmaceutical and academic sectors. In the US, Tecan has jointly hosted a series of bioprocessing seminars. Working with applications partners Atoll and FortéBio, these meetings focused on use of Tecan’s Freedom EVO® liquid handling workstations in the bioprocess sector. Tecan representatives Kevin Moore and Daniel Leach presented a summary of Tecan’s automation options for the bioprocessing laboratory, covering topics such as cell transfection, high throughput process development and sample preparation for HPLC, MALDI-TOF and ELISA. They were joined by Atoll’s Tim Schroeder, who illustrated how MediaScout® RoboColumns can be used with the Freedom EVO platform for both process development screening and process analytical technology (PAT). Islay Campbell and Will Riches from FortéBio continued this theme, explaining the principles of the FortéBio Octet® 384 platform’s BioLayer Interferometry technology, and its integration with Tecan systems.
For the Scandinavian market, Tecan’s Didrik Rosen and Roland Durner hosted seminars on cell culturing in Copenhagen and Stockholm. Invited speakers from leading companies in the field shared their experiences of combining experimental theory with practical applications in the laboratory, introducing attendees to the latest developments in cell culturing technology. There were also workshops to demonstrate the practical advantages of automation for a range of applications, illustrating how easy it can be to implement laboratory automation, even without previous experience, using Tecan’s innovative TouchTools Suite™ touchscreen control system. Finally, representatives from Tecan’s UK offices have been touring England and Scotland in a series of one day exhibitions. This roadshow introduced the latest laboratory automation technologies and multifunctional microplate reader designs to a broad scientific audience, visiting universities, research institutes and science parks. Visitors were able to discuss the huge wealth of applications which can now be automated, and the potential benefits of Tecan’s technology, including increased throughput, improved reproducibility and greater confidence in results. To find out which events Tecan will be attending in the coming months, see the back cover or visit www.tecan.com/events MediaScout is a registered trademark of Atoll GmbH, Weingarten, Germany. Octet is a registered trademark of FortéBio Inc.
CORPORATE NEWS TECAN JOURNAL 1/2010
Tecan supports ELRIG sponsorship of ‘TechnoBotts’ Tecan continues to encourage the growth of innovative technology through a donation to help European Laboratory Robotics Interest Group (ELRIG), at the ELRIG Drug Discovery conference, gold-sponsored by Tecan, in Liverpool, UK in September 2009. Tecan contributed to the sponsorship of a promising group of young automation specialists named TechnoBotts, a UK-based team of teenage robotics developers. With funding from Tecan and other vendor companies, a check for £5,000 was donated to support the team in its venture to enter RoboCup 2010, an international robotics competition in Singapore in June 2010.
As team TechnoBotts3, Andrew Wightwick (14), Oliver Wightwick (12), Eric Wieser (15) and Alex Gilday (14) enjoyed great success in 2008 – 2009, becoming the UK and Ireland FIRST LEGO® League (FLL) champions. They also represented the UK in the FLL World Festival in Atlanta, Georgia in April 2009,
coming 4th in Robot Performance and winning the first place trophy for Innovative Design. For 2009 – 2010, Andrew, Oliver and Eric have re-formed as TechnoBotts4 and are looking for further success in RoboCup Junior, in both the Rescue and Soccer categories. LEGO is a registered trademark of the LEGO Group.
(l to r) Kevin Moore (Tecan), the TechnoBotts team, Adrian Kinkaid (ELRIG), Robin Grimwood (FluidX)
Tecan to supply fully automated solution for Hologic Cervista® HPV tests Tecan has made a global OEM agreement with Hologic, Inc., a leading developer, manufacturer and supplier of premium diagnostics and surgical products for the healthcare needs of women, to supply a fully automated solution for Hologic’s Cervista molecular diagnostics human papillomavirus (HPV) tests. The system is based on Tecan’s Freedom EVO® liquid handling platform with an integrated Tecan detection device. Prototypes of the automated solution have been tested successfully with select Hologic customers. Validation for regulatory approval of the automated system has started before the end of 2009, and Tecan expects to start shipping the Cervista system in the US in
2010. Tecan will also supply Hologic with plastic consumables as an integral part of the validated system. HPV is the most common sexually transmitted disease in the US and is recognized as the cause of most cervical cancers. To help prevent the onset of disease, the American College of Obstetrics and Gynecology (ACOG) suggests routine Pap and HPV testing for women over the age of 30 to identify individuals most likely to develop cervical cancer. Hologic and Cervista are trademarks and/or registered trademarks of Hologic, Inc. and/or its subsidiaries in the US and/or other countries.
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PROTEOMICS TECAN JOURNAL 1/2010
Automated protein purification for efficient vaccine development Biochemists at the Novartis Vaccines and Diagnostics Research Center in Siena, Italy, are processing vaccine candidates by automated purification of recombinant proteins from huge numbers of bacterial lysates on a Freedom EVO® platform, saving a lot of their time without compromising reproducibility, consistency or quality.
(l to r) Sara Marchi, Francesca Scala and Massimo Mariani
The Protein Purification Group at the multi-disciplinary Novartis Vaccines and Diagnostics Research Center purifies proteins cloned from a range of bacteria. The research center is using ‘reverse vaccinology’, an approach that starts with in silico analysis of entire bacterial genomes, to identify hundreds of possible vaccine candidates, followed by cloning, expressing the proteins in E. coli and purifying the recombinant proteins, which are tested in screening studies using appropriate animal models. Dr Massimo Mariani, biochemist at the Protein Purification Group, explained: “We have about 10 different projects in vaccine development covering a diverse group of pathogenic bacteria, mainly meningococci, both human and animal streptococci, staphylococci, Clostridium, pneumococcus and pathogenic mutants of E. coli. All these projects generate a large number of recombinant proteins that require purification from bacterial lysates. Originally, we did this manually using affinity chromatography. This involved up to three operators who performed bacterial lysis, loading and washing columns, eluting and characterizing the eluted proteins, and
determining concentrations as well as the degree of purity. This was tedious and timeconsuming, taking almost a whole day, so we decided to look for an automated system for protein purification.” “We started to collaborate with Tecan in 2005 and, in just two months, were able to optimize our new Freedom EVO platform with tailored software to reproduce many of the steps that were previously performed manually. We still prepare the bacterial lysates manually, but all subsequent steps are automated, starting with loading of samples onto affinity chromatography columns, followed by washing and elution of each sample into two fractions, colorimetric quantification of each fraction using a Bradford assay, and preparation of samples for SDS-PAGE. On the next day, samples are analyzed by SDS-PAGE, giving information on molecular weights and protein profiles. The software script has the capability to enter our selection of fractions after gel electrophoresis for further analysis, and these fractions can be pooled if required. We run eight to 24 samples at a time, and the system can handle a variety of sample
PROTEOMICS TECAN JOURNAL 1/2010
The Freedom EVO platform at the Novartis Vaccines and Diagnostics Research Center
formats – 12 ml tubes, 1 ml columns and 24and 96-well microtiter plates; the 96-well plates are used for the Bradford assay.” “Working closely with Tecan, we tailored the software so the procedure automatically stops at three points, during the loading, washing and elution steps. At these points, the system temporarily stops operating, and a warning on the screen prompts the operator to manually check that the columns are empty prior to loading samples, avoiding the risk of process failure due to occlusion of columns by overloading. When the samples are eluted from the columns and the Bradford assay begins, we can leave the Freedom EVO completely unattended because the samples are stored on a refrigerated plate on the Freedom EVO
platform deck, to be collected during the next day. The automated procedure takes longer than the manual one – 11 hours compared to eight hours – for purification of 24 samples, but it requires far less operator time, as no human intervention is needed after elution of samples (Table). Yield and purity of proteins are similar between manual and automated procedures for both low and higher expression proteins.” Dr Vittoria Pinto, who has been involved in setting up the procedures on the Freedom EVO platform, said: “The system is fully reliable, we are very happy with its performance and our results, and they are consistent with those from our manual procedures. The local Tecan engineer comes regularly to give assistance
Table: Comparison of manual and automated protein purification procedures. Although the automated procedure takes longer, it saves considerable time for the operator. Purification procedure step
Manual processing
Manual processing
Robotic processing
Automated processing
Duration
No. of operators
Duration
No. of operators
Loading
1h
1
1h
0 (1)
Washing
1h
1
1h
0
Elution and fraction collection
1h
1
1h
0
Bradford assay
3h
1
6h
0 (1)
Sample preparation for SDS PAGE
2h
1
2h
0
Total for 24 samples
8h
3
11 h
1
Occasional attention for control of loading procedure and analytical results.
(1)
when needed and we have developed a good working relationship with Tecan.” Massimo concluded: “The Tecan system has freed us from the tedium of manual protein purification, so we can do other experiments while the screening purification procedure is running on the platform. We are now investigating, in collaboration with Tecan, changing from the Bradford assay to the BCA protein assay, an alternative colorimetric method for protein quantitation that is faster and less affected by protein composition compared to the Bradford assay, with a greater linear range; this will further advance the automated technique, speeding up the procedure by a few hours.”
Figure: SDS PAGE of samples processed by manual and automated purification procedures. The protein profiles and yields are comparable between the two methods.
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CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2010
A long-standing partnership with success Inverness Medical has been using Tecan liquid handling systems to automate ELISA testing in Spain for over 15 years. The Company supplies Tecan instruments free of charge to hospitals and laboratories using its diagnostic kits and reagents, an agreement which has been benefiting its customers since 1990.
Inverness Medical Innovations, Inc., is a global company at the forefront of medical diagnostics and health management. The Company’s Spanish subsidiary, Inverness Medical Ibérica, is a major supplier of diagnostic testing kits to hospitals and medical laboratories, providing a range of over 500 distinct ELISA kits, including tests for respiratory, immunodeficiency, autoimmunity and infectious diseases. In addition, Inverness Medical Ibérica also provides reagent sets for immunofluorescence assays, ToRCH tests and RT-PCR protocols. For many years, Inverness Medical Ibérica has supplied Tecan liquid handling workstations to customers buying its ELISA testing kits, allowing straightforward
automation on validated platforms. This ensures the reliability and reproducibility of its diagnostic tests, and offers greater throughput for busy diagnostic laboratories. Sergi Gordillo is responsible for the Tecan instruments supplied to Inverness Medical Ibérica customers, and explained the origins of the Tecan association: “We have a long, well established working relationship with Tecan, that started in the early 1990s. At that time we were looking for a system to automate all the steps of ELISA processing, and Tecan was the only manufacturer able to offer a platform capable of automating the entire assay. This original instrument was very successful, and greatly appreciated by our customers, and we have been working with Tecan ever since. We are continuously updating our liquid handling workstations
to reflect the latest developments from Tecan, and we validate our kits for each new system in our own development laboratory in Barcelona. We are offering the latest Freedom EVOlyzer® platforms to our high throughput customers, and the only change we make to the systems is the addition of specially adapted carriers to suit our reagent bottles.” “Our customers find the Tecan instruments very easy to use, and give us good feedback in terms of suggestions and improvements for diagnostic use. We have a very good relationship with our Tecan representatives, and the customer feedback we share helps to drive the development of new systems. The Freedom EVOlution™ software that controls the Freedom EVOlyzer is a good example of this; we have seen definite improvements with successive generations of the software, and the latest versions are excellent. As well as simplifying control of the instrument, the software interfaces directly with customers’ internal laboratory information management systems (LIMS), simplifying reporting and auditing,” Sergi continued.
Even small laboratories can automate their ELISAs on Freedom EVOlyzer. Here is shown a simulation model of the small scale Freedom EVOlyzer 100 for loading up to 96 samples. The validated Inverness configuration with programmed ELISAs can be used on all platform sizes.
CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2010
The worktable layout showing sample section with the capacity of 96 samples, followed by the wash station, microtiter plate carrier, reagent troughs with sample diluents and decontamination solution, customized carriers for conjugates, stop solutions in the bigger round tubes and controls in the Eppendorf® tubes. The last carrier contains trays with the disposable tips.
“Since we first began offering Tecan instruments to our customers, we have seen rapid development of the Company’s automated liquid handling platforms into the flexible systems we use today. We supply Tecan instruments to the majority of our customers, for use with our kits and reagents, and have a range of different platform configurations, depending on the customer’s needs. The instrument we offer customers will obviously depend on their laboratory size and throughput, as well as the spectrum of tests they are offering. We support many different laboratories throughout Spain, from small hospitals up to very large regional facilities in Barcelona and
Madrid, and have a broad portfolio of Tecan workstations to meet this varied demand. For example we have both Freedom EVOlyzer 100 and Freedom EVOlyzer 150 workstations, fitted with two-, four- or eight-channel liquid handling (LiHa) arms, although the majority of customers are using Freedom EVOlyzer 100 platforms with 4-channel arms. This set-up is suited to a wide range of throughput needs, with many of our customers processing in excess of 100 samples a day for ELISA testing.” “Complete automation of ELISA processing is of great benefit to our customers, as it saves a lot of time and releases staff time for other applications. The reliability and flexibility of Tecan’s innovative and reliable
liquid handling platforms are a key part of this strategy, and ensure we have high levels of customer satisfaction. We have a number of long-standing customers, who have been with us for over 20 years, and the ability to offer complete solutions for automated ELISA processing is pivotal to retaining this customer loyalty.” Eppendorf is a registered trademark of Eppendorf AG.
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WASHERS TECAN JOURNAL 1/2010
HydroFlex™ offers a helping hand for automated bead washing
Erling Finne and Ingrid Holm with the HydroFlex washer
Scientists at Life Technologies’ Dynal® facility in Oslo, Norway, are using Tecan’s HydroFlex microplate washers in their R&D and QC departments for automated washing of Dynabeads® assays, reducing manual pipetting and improving consistency of results.
The use of magnetic beads as the solid phase for ELISAs offers several advantages over traditional direct coupling techniques, including shorter incubation times and improved sensitivity. These benefits have made magnetic bead-based technologies the gold standard for immunodiagnostic assays, as well as providing a versatile toolbox for researchers in academia and industry. Life Technologies’ Dynal division has been at the forefront of magnetic bead technology for over 20 years, and the Company’s range of Dynabeads is used in hundreds of laboratories around the world. Development of the Dynabeads range is carried out at Life Technologies’ dedicated Dynal facility in Oslo, Norway, allowing the Company to offer specialist support and services to customers, including customized development projects. Ellen Kleveland, industrial product manager, described Dynal’s work: “Although many established laboratories are already using Dynabeads, we work with various players in the in vitro diagnostics (IVD) sector, optimizing new bead-based assays to supersede existing immunoassays for improved consistency and sensitivity.
(Photos courtesy of Elisabeth Breivold)
An important aspect of this work is assisting customers with adapting our assay protocols to their specific requirements and equipment, using our expertise to help them achieve greater throughput and reproducibility.” A major benefit of the Dynabeads technology is that it allows much higher throughput and better sensitivity than traditional approaches, where antibodies are coupled directly to the wells. Many IVD laboratories rely on automated platforms to help them achieve the high throughput capacities required, and Dynal is using HydroFlex microplate washers to automate washing for magnetic bead-based ELISAs. Erling Finne, principal scientist in the Research and Development laboratory, explained why they chose Tecan instruments: “Our involvement with Tecan actually began as a collaboration to develop a washing protocol for a customer. We worked with Gerald Habenbacher from Tecan Austria to co-develop a protocol using Dynabeads with the HydroFlex microplate washer, coming together to help the customer get the best results from automated ELISA washing. Once we had successfully optimized the customer’s assay, we decided that we would
WASHERS TECAN JOURNAL 1/2010
Magnetic Dynabeads technology allows much higher throughput and better sensitivity than traditional approaches, by coupling the beads to the antibodies.
HydroFlex with smart-2 MBS magnetic carrier for bead washing
benefit from having the same instrument in-house, for both method validation and to help with our own research projects.” “In the Research and Development department we typically run between two and four plates a day, with a lot of variation in the types of beads and samples in each plate, so our work is not suited to using large automated platforms. The HydroFlex washer fits well between these systems and manual techniques, allowing us to perform a wide variety of Dynabeads immunoassays in a 96-well microplate format, with minimal manual pipetting operations. This reduces the time taken to perform experiments, and helps to minimize inter-operator variability, which can introduce bias into our results.” The improved consistency and time savings offered by the HydroFlex washer in the Research and Development laboratory has led to the purchase of a second HydroFlex for the Quality Control (QC) department. Ingrid Holm is responsible for quality and method development, and explained the advantages of the HydroFlex for QC: “My role is to develop and validate new methods, particularly for our QC activities. Although
our laboratory already had good results using manual washing techniques, automated washing reduces the pressure on QC staff, and offers even greater consistency than manual methods. Although we have only transferred a few of our methods to the automated washing protocol so far, we are already seeing time saving benefits, allowing staff to concentrate on other tasks. We will certainly be adapting more of our methods to use the HydroFlex washer in the future, and
look forward to working with Tecan again, to develop new solutions for customers.” To find out more on Tecan’s HydroFlex microplate washer, visit www.tecan.com/hydroflex To find out more about Dynabeads, visit www.invitrogen.com/dynabeads Dynabeads and Dynal are registered trademarks of Invitrogen Dynal AS.
Schematic illustration of a bead-based ELISA: the capture antibody is covalently attached to Dynabeads and the antigen detected on the solid phase using a labeled detection antibody.
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GENOMICS TECAN JOURNAL 1/2010
Automated DNA preparation pushes forward evolutionary biology research Reliable high throughput genotyping on the Tecan Freedom EVO® 200 platform has helped research in parenthood analysis and population genetics of the three-spined stickleback at the Max Planck Institute for Evolutionary Biology, Plön, Germany.
In evolutionary biology research, progress in molecular techniques has allowed the studies of individual lifetime reproductive success and how it is affected by environmental and genetic factors. The three-spined stickleback (Gasterosteus aculeatus) is an ideal model organism for these investigations (Gibson, 2005) because it is amenable to laboratorybased monitoring and its reproduction, immunology and evolution have been well studied. Three-spined sticklebacks have a complex mating system based on fitness-related traits and genes of the major histocompatibility complex (MHC), which encode for molecules that bind pathogen-derived peptides to trigger an adaptive and specific immune response. Studies have demonstrated that variation in MHC genes in the sticklebacks is ecologically relevant, through changes in resistance to parasite attack, suggesting a trade-off between parasite resistance and cost of immunity (Wegner et al., 2003a, 2003b). Dr Christophe Eizaguirre, Evolutionary Biologist at the Leibniz Institute of Marine Sciences at the Christian-Albrechts Universität zu Kiel (IFM-GEOMAR), and previously at the Max Planck Institute for Evolutionary Biology, Plön, SchleswigHolstein, Germany, has spent several years studying the population genetics of three-spined sticklebacks. He explained: “Our research requires extraction of high quality DNA from hundreds of sticklebacks for MHC genotyping. Yearly, we run a large experiment during the sticklebacks’ breeding season and we needed an automated, easyto-handle method to increase productivity;
in 2008 for instance, we collected almost 10,000 eggs to analyze in three months. Our Freedom EVO 200 platform from Tecan, equipped with an 8-channel liquid handling arm, meets our requirement for high throughput purification of genomic DNA from tissue and eggs. We use the Invisorb® DNA tissue HTS 96 Kit from Invitek GmbH, and it was very easy to establish an automated protocol.” For tissue DNA extraction, small dorsal spine samples are incubated overnight with continuous shaking at 52 °C in 96-well plates for lysis and protein digestion. The plates are then transferred to the Freedom EVO 200 platform for automated processing, including the addition of binding buffer, incubation, washing to remove protein, nucleases, PCR inhibitors and ethanol contamination, and elution of the purified DNA. For parenthood analysis, to determine the number of offspring produced by each individual (see figure), DNA is extracted from eggs, which is technically challenging because these eggs have an extremely tough shell. The shells are disrupted overnight with continuous shaking at 52 °C and, as with DNA extraction from tissue, subsequent steps are carried out on the Freedom EVO platform. Multiplex PCRs are performed on extracted DNA using 18 microsatellite loci and amplified products are analyzed by sequencing. However, the MHC Class II B loci are genotyped by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) or reference strand-mediated conformation analysis (RSCA), and DNA quality is critical for the success of these methods.
GENOMICS TECAN JOURNAL 1/2010
Microsatellite 1 Black
Microsatellite 4 Blue2 MOTHER
Microsatellite 2 Blue
Microsatellite 5 Black2
Microsatellite 3 Green FATHER
OFFSPRING1
OFFSPRING2
Christophe continued: “The main advantage of using this instrument is its autonomy; basically, we switch it on and walk away and, for two hours, we can do something else. Also, because the Freedom EVO system is capable of normalization of DNA concentration, the instrument is useful for other tasks. We do a lot of work with cloning, so we also use it for automated plasmid DNA extraction and DNA normalization.” “Automating high throughput DNA extractions on the Freedom EVO platform efficiently purifies high quality genomic DNA from small pieces of tissues or earlydeveloped stickleback eggs with minimal hands-on time. We can now address important questions in evolutionary biology such as individual reproductive success and ecological relationships between recently diverged species. Automated DNA sample preparation is saving us time and offering higher production rates, reliability and high quality DNA, which are all essential for sensitive downstream studies such as SSCP/ RSCA analysis,” Christophe concluded.
References
Acknowledgements Dr Eizaguirre wishes to acknowledge the contributions of Prof Manfred Milinski, Dr Martin Kalbe, Dr Sven Becker and Ines Schultz, Max Planck Institute for Evolutionary Biology, as well as Ilka Dankert, Institute of Evolution and Biodiversity, University of Münster, Germany and Sonja Farhangi, Invitek GmbH, Berlin, Germany. To find out more visit www.tecan.com/freedomevo Invisorb is a registered trademark of Invitek, Germany. The Invisorb technology is covered by patents and patent applications.
Three-spined stickleback (Gasterosteus aculeatus)
Gibson G (2005) Evolution: The Synthesis and Evolution of a Supermodel: Science, 307, 1890-1891. Wegner KM, Kalbe M, Kurtz J, Reusch TBH Milinski M (2003a) Parasite selection for immunogenetic optimality. Science, 301, 1343. Wegner KM, Reusch TBH, Kalbe M (2003b) Multiple parasites are driving major histocompatibility complex polymorphism in the wild. Journal of Evolutionary Biology, 16, 224-232.
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LIQUID HANDLING TECAN JOURNAL 1/2010
New tools of the trade
MedImmune in Maryland, USA, is using its Freedom EVO® platforms as general-purpose tools for simple and routine tasks, rather than specific projects. This ‘tools’ concept has greatly increased use of the systems, saving much development time.
MedImmune LLC., based in Maryland, USA, is a global biopharmaceutical company offering a comprehensive range of pharmaceutical development services, from target validation through to preclinical and clinical trials. MedImmune acts as the biologics support business for parent company AstraZeneca, and has a broad expertise in healthcare, covering areas such as infection control, oncology, respiratory disease and inflammation, cardiovascular and gastrointestinal diseases, and neuroscience. In addition, MedImmune has several marketed products, including FluMist™, an intranasal live influenza vaccine, and Synagis™, for prevention of respiratory syncytial virus in premature babies. MedImmune uses automation as part of its research and development strategy, accelerating a variety of routine liquid handling tasks to reduce development times. Mr Nicholas Knoepfle, Associate Scientist in Analytical Biochemistry and leader of the Automation Group, explained MedImmune’s approach to automation: “The Analytical Biochemistry Group at MedImmune is responsible for development of ELISAs and cell-based assays for testing our products
during development. When I joined the Company in 2002, the group already had an older Tecan liquid handling workstation, but it was an under-used resource as nobody was taking the time to develop automated versions of our assays. I began to extend our use of this instrument, splitting my time between ELISA development and transfer of existing assays onto the automated system. The benefits of automation were quickly realized, and my role changed to focus purely on automated assay development using the Tecan platform. To cope with an increasing demand for automated assays, we began looking for new systems to improve our throughput, and chose to stay with Tecan because of the very positive experience we already had with its instruments.” “We now have a dedicated team for development of our automation capabilities on the Tecan systems, and the use of these platforms has expanded beyond automation of existing manual ELISAs to include cell-based chromatographic analyses and nucleic acid assays, as well as mass aliquoting of reference standards. We are also increasing our use of automated data transfer and analysis, and providing an
LIQUID HANDLING TECAN JOURNAL 1/2010
internal consultancy service on automation strategies and purchasing of systems.” “We purchased our first Freedom EVO 150 workstation to automate a suite of ELISAs for a specific project, but quickly realized that this did not offer the best return on investment in equipment, because the focus of projects often changes in the early stages of therapeutic agent development. This made it impractical to spend a lot of time developing complex protocols for specific applications, so we adopted a ‘tools’-based approach instead, automating routine laboratory functions. We began by targeting some of the simplest and most frequently performed tasks, such as serial dilution, fraction collection and microplate replication, creating simple scripts to allow analysts to rapidly pipette out reagents for multiple analyses by entering just a few variables. We created a whole suite of these scripts or ‘tools’, which could be used individually or linked together to create more elaborate processes, and this approach proved very popular with analysts. Use of the automated systems increased dramatically, leading to noticeable reductions in development times for new assays.”
(l to r) Amanda Dodds, William Ivory, Nicholas Knoepfle and Dorian Zoumplis with one of their Tecan Freedom EVO 200 platforms
“As a result of this rapid expansion, we now have two additional Freedom EVO platforms in Gaithersburg. We have one Freedom EVO 200 that is equipped for cell-based and ELISA-type assays, as well as DNA/RNA PCR preparations, and a second which is primarily set up for a range of bulk pipetting operations, including aliquoting of all reference standards used by MedImmune. However, in keeping with the ‘tools’ concept, both platforms are equipped with a wide range of devices and readers, giving us the flexibility to explore many different assays without having to buy in new equipment.” “Overall, we have been very impressed with our Tecan workstations. The hardware
is high quality and has been immensely reliable, and the Freedom EVOware® software is also excellent, being easy to use, program and customize. We are very happy with the progress we have made so far, and plan to extend our use of these platforms to include more routine laboratory tasks,” Nicholas concluded. For further information on MedImmune LLC., visit www.medimmune.com/ To find out more on Tecan’s Freedom EVO workstations, visit www.tecan.com/freedomevo MedImmune and Synagis are registered trademarks of MedImmune, Inc. FluMist is a registered trademark of MedImmune Vaccines, Inc.
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LIQUID HANDLING TECAN JOURNAL 1/2010
Plug and play electrophysiology Fluxion Biosciences is using Tecan’s Freedom EVO® platforms to offer high throughput solutions for patch clamping studies, based on its advanced Well Plate Microfluidics™ system.
Fluxion Biosciences in South San Francisco, California, provides cellular analysis tools for the pharmaceutical and biotechnology industries. Compound screening for drug discovery requires high throughput techniques, allowing large libraries of potential therapeutic agents to be assessed in parallel. Manual patch clamping methods are the ‘gold standard’ for live cell electrophysiology studies, yet these techniques are time-consuming and labor-intensive, making large-scale studies infeasible. Fluxion uses its proprietary Well Plate Microfluidics (WPM) technology to offer automated patch clamping for electrophysiology, helping researchers investigate ion channels as possible therapeutic targets.
The Company’s IonFlux™ HT automated electrophysiology system offers high performance patch clamping in an automation-friendly plate format, permitting high throughput electrophysiology studies. Mike Schwartz, Marketing Director at Fluxion, explained: “Ion channels are an important yet underrepresented class of therapeutic target, due to the unsuitability of most patch clamping techniques for candidate drug screening. The IonFlux system – with its 64-channel amplification circuitry – offers automation of 32 separate electrophysiology experiments in parallel. The WPM design means that, once the plate has been loaded into the reader, all steps of the experiment are automatically performed, in duplicate, helping to improve reliability and minimize variation.”
To further increase the throughput capacity of the IonFlux system for its customers, and minimize the need for user intervention, Fluxion is working with Tecan to integrate the system into the Freedom EVO series of liquid handling workstations. “Automated plate preparation using the Freedom EVO platform can significantly enhance the efficiency of electrophysiology workflows,” Mike continued. “Several IonFlux instruments can be multiplexed on a single workstation, with the platform handling all the plate preparation, reagent pipetting and logistical needs of all the instruments. The system’s liquid handling (LiHa) arm is able to perform all the pipetting operations required to fill each plate with cells and reagents, and the robotic manipulator (RoMa) arm can load and unload plates from the IonFlux readers. The Freedom EVO can then prepare the next plate while the IonFlux analysis cycle is running, offering customers virtually continuous operation if required, and a much higher throughput than could be achieved manually.” “When selecting a liquid handling platform to automate plate preparation for the IonFlux system, we were keen to offer a highly integrated solution that could be tailored to the needs of individual customers. The Freedom EVO series of workstations is highly flexible and customizable, allowing fully integrated electrophysiology workflows for a range of throughputs. Our standard Freedom EVO set-up has a four-channel liquid
LIQUID HANDLING TECAN JOURNAL 1/2010
Representative application for the IonFlux HT. Response of a cell ensemble exposed to 1.0 µM GABA co-applied with increasing concentrations of diazepam (marketed as Valium®). Diazepam acts on the GABA ion channel receptor to decrease neuronal activity for clinical indications such as anxiety and epilepsy. Each diazepam concentration is pre-incubated for 1 min before the GABA co-application. The positive modulation of mean GABA-sensitive currents (n = 4) during exposure to increasing diazepam concentrations is shown. A hill fit provides an EC50 of 425 nM.
handling arm for plate formatting, a robotic manipulator arm to load and unload plates from the IonFlux instruments, and hotels for microfluidics plate storage. In addition, there are various optional modules to enhance automated cell manipulation, including a microplate centrifuge below the deck of the workstation. For customers requiring very high throughput, up to four IonFlux systems can be integrated onto a single Freedom EVO 100 platform – two at the front and two at the rear – with an 8-channel liquid handling arm providing high speed plate formatting.” “As well as the broad flexibility and extensive integration options offered by the Freedom EVO systems, we chose to work with Tecan because of the Company’s reputation for quality and reliability. This gives our customers a high level of confidence in the laboratory automation platforms we recommend, as well as helping to maintain high levels of performance for our IonFlux systems, through consistent and reproducible plate preparation and formatting. Tecan’s application specialists have an in-depth knowledge of customers’ needs, and this has been an important aspect of our very successful collaboration to develop this integrated solution for high throughput electrophysiology.”
IonFlux system for high throughput electrophysiology. The IonFlux system is designed to operate much like a plate reader. Plates are preloaded with cells, compounds, and reagents using the Freedom EVO platform. Once filled, plates are fed automatically into the instrument. All fluidic control necessary to trap cells and deliver compounds is handled within the instrument and integrated electrodes record ion channel currents.
To learn more about Fluxion Biosciences, visit www.fluxionbio.com To find out more on Tecan’s Freedom EVO workstations, visit www.tecan.com/freedomevo Valium is a registered trademark of Roche Group IonFlux and Well Plate Microfluidics are trademarks of Fluxion Biosciences Inc.
Microfluidic device for cellular electrophysiological recordings. Ion-channel expressing cells move through a main flow channel, then get trapped at the junction of smaller fluidic channels under negative pressure. Electrodes in contact with the intra- and extra-cellular space of the cells provide a voltage across the cell membrane. Ion channel activity is measured in the form of ionic current going through the membrane-bound ion channels.
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MICROPLATE READERS TECAN JOURNAL 1/2010
Infinite® offers the MIDAS touch Avacta is combining physics and biology to develop innovative technologies for the pharmaceutical and clinical diagnostics markets, and its Infinite M200 microplate reader is playing a key role in its analytical services provision.
Avacta Group in York, UK, offers innovative analytical services to the biopharmaceutical sector, exploiting its specialist knowledge in biophysics to develop structural analysis solutions for pharmaceutical development. Avacta’s range of services includes comparability studies, formulation/stability studies and aggregation problem solving, working closely with industry to develop new technologies that address the needs of the market.
Ed Quinn with the Infinite M200
High quality and reproducible data acquisition is crucial to Avacta’s services, and the Company has chosen an Infinite M200 microplate reader from Tecan for this pivotal role. Russell Hodgetts, MD of Avacta’s Laboratory Services, explained: “The Infinite M200 allows us to provide quality, high throughput services to our clients, and is an important support instrument for many of our laboratory procedures. It is also integral to the development of solubility screening assays, which are the starting point for our formulation development service.
MICROPLATE READERS
The multiple detection methods and environmental control available with the Tecan instrument make it extremely versatile.” Avacta’s technologies also have applications in the clinical diagnostics sector, and Avacta has recently developed MIDAS (multi immuno diagnostic assay system), a pointof-care platform designed to automate medical diagnostic tests usually performed manually in a laboratory. Initially the MIDAS system will be launched for veterinary use
through Avacta’s Animal Health division, with future plans being developed for pointof-care testing in human health applications. The Infinite plate reader has been central to bioassay development for the MIDAS platform, as Product Manager Dr Ed Quinn, explained: “The multimode detection capabilities of the Infinite reader allow us to carry out a wide range of assaying techniques – using absorbance, fluorescence and luminescence detection – on one instrument, and the built-in temperature
TECAN JOURNAL 1/2010
control and shaking mean that the complete assay can be performed in situ. The easy-touse i-control™ software allows us to rapidly set up experiments, defining our own plate parameters to improve the resolution of our assays. Data is automatically exported to our laboratory information system, seamlessly integrating into our research and development program.” To find out more on Tecan’s Infinite M200 reader, visit www.tecan.com/infinite200 or visit www.avacta.com
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PROTEOMICS TECAN JOURNAL 1/2010
Automation greatly speeds up protein production for X-ray diffraction experiments ACEMBL is the first fully automatable pipeline for the production of multi-protein complexes, developed by researchers at the European Molecular Biology Laboratory (EMBL) on the Freedom EVO® platform from Tecan. This system offers possibilities for experiments that were previously impossible to contemplate using manual methods. Running many simultaneous experiments in parallel has dramatically increased the likelihood of producing crystallizable molecular complexes suitable for X-ray diffraction analysis.
Maxime Chaillet (left), engineer, and Imre Berger (right), Group leader, with the Freedom EVO at EMBL
Metabolic processes in eukaryotic cells depend on large multi-subunit assemblies where proteins combine with one another and with other molecules. Transcription factor complexes, the subject of gene expression studies by Dr Imre Berger and his group at EMBL in Grenoble, France, are good examples of such assemblies. Imre explained: “X-ray diffraction analysis is the only method that is capable of producing the high resolution necessary to elucidate how these very large protein complexes operate during transcription and gene expression processes, including binding to DNA and chromatin, and recruiting RNA polymerase II. For X-ray diffraction analysis, the multi-subunit complexes have to be crystallized first; to ensure that we have sufficient quantity and quality for this, we produce the proteins by a recombinant over-expression method based on baculovirus-infected insect cell cultures that we developed, called MultiBacEMBL. However, producing and purifying protein complexes does not guarantee successful crystallization, so creating many variants of each of the complex’s subunits is essential in obtaining complexes that will crystallize. As an example, a complex with four protein subunits, each with five genetic variants, would produce over 600 different combinatorial permutations. This is impossible to handle manually as I realized
PROTEOMICS TECAN JOURNAL 1/2010
X-ray diffraction involves the interpretation of complex diffraction patterns to elucidate protein structures
some years ago, due to the essentially sequential nature of experimentation by hand. This is when we had the idea to harness the power of automation by using robotics for our purposes. Automation is the essential solution for protein complex expression and production, when many fold variations in the individual proteins is required for experimental success. With a robot, many experiments can be done simultaneously and in parallel, with a precision that is unattainable in manual mode.” ACEMBL is an automatable multi-gene combination and expression system developed by Imre and his colleagues, to generate multi-gene baculoviral expression vectors by tandem recombination procedures. Firstly, one or several genes encoding proteins of interest are introduced into small plasmids; these so-called ‘donor’ and ‘acceptor’ plasmids recombine in vitro at specific sites to produce larger multigene vectors. These vectors, in turn, are combined into the baculovirus genome by transposition. The resultant recombinant baculoviruses, containing fluorescent reporter genes to track virus amplification and protein production, are used to infect insect cell cultures where the proteins of interest are produced. ACEMBL has great potential for revealing currently unknown
molecular mechanisms of health and disease that could be vital in drug discovery. “Before moving to the EMBL in 2007, I researched at Zurich, and visited the Paul Scherrer Institut (PSI), Villigen, Switzerland, where I familiarized myself with the Tecan systems in Michel O Steinmetz’s Biomolecular Research Group,” Imre continued: “We designed and developed modules for the Freedom EVO platform at the PSI to automate protein-complex expression. We are still closely collaborating on multiprotein complex production technology, producing joint publications. Our Tecan system in Grenoble consists of a Freedom EVO 200 platform with three identical liquid handling modules for, respectively, gene integration into small plasmids, multi-gene plasmid fusion and integration of plasmids into baculoviral genomes. This creates a pipeline with all the modules executing routine and easily scripted plasmid manipulation and transformation steps simultaneously. Additionally, the platform has a sterile, covered compartment for growing cell cultures, which allows eukaryotic cell cultures to be distributed into deep well plates, before they are transformed with the recombinant baculovirus.” “The two main advantages of automation are diversification and throughput. We
are introducing diversity through parallel processing into many genes, coding for variants of the same subunit of the protein complex we are studying. This vastly increases the chances of a successful outcome compared to manual procedures. Instead of one candidate that may or may not crystallize, we obtain, in a few days, 96 variants of one protein complex that will probably include at least one or two that will crystallize. Our research and optimization of the procedures are ongoing, with the aim of producing a routine and robust high throughput application in the foreseeable future, with several automated platforms running in parallel, repeating the ACEMBL procedure many thousands of times with up to 95 % efficiency.” ”We have an excellent working relationship with Tecan, which is a collaboration of mutual benefit. We are continuously in contact, and often have discussions for method development. From our experience, it is essential to have a dedicated and well-trained technician to operate the automated systems, to get the best out of them. Investing a little extra time and effort from the beginning really has paid dividends later on,” Imre concluded. ACEMBL has recently been described in an article published in Nature Methods, vol 6 (June 2009), pg 447-450.
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MICROARRAY PRODUCTS TECAN JOURNAL 1/2010
The missing link for autoimmune disease Researchers at McMaster University, Ontario, Canada, are developing protein microarrays for serum-based diagnostic testing. These microarrays, developed using a Tecan laser scanner, are able to combine autoimmunity, bacterial and viral antigens on a single array, and are helping researchers to investigate the link between infection and autoimmune disease.
Muscle and joint pains commonly occur as side effects of common infectious diseases, such as colds and influenza. These symptoms are the result of the body’s inflammatory response to infection, and are generally transitory. However, in some cases they appear to precede the onset of progressive autoimmune disease affecting joints, muscles, nerves, connective tissues and organs. Researchers at McMaster University in Ontario, Canada, are using protein microarrays to explore this potential link between infection and autoimmunity in adults. Dr Sandeep Raha, an Assistant Professor in the Department of Pediatrics, explained the focus of the project: “Although we are primarily involved in pediatric research, we are very interested in the development of clinical diagnostic tests based on microarray technology, due to the minimal specimen requirements for serodiagnostic testing. Use of microarrays allows parallel testing of a patient’s serum sample for a wide variety of pathogen and autoimmunity antigens from less than a milliliter of serum, significantly reducing costs and allowing ‘finger prick’ testing in a primary care setting.”
“The link between autoimmunity and infection offers a good demonstration of the versatility of protein microarrays,” continued Dr Tom Ewart, a Visiting Professor at McMaster University who is spearheading the development of this technology. “Analysis of this relationship requires a high degree of parallel screening, generating large quantities of complex data for each individual. This type of analysis would be virtually impossible using traditional ELISA techniques, due to the high costs, laborintensive processing requirements and need for large amounts of patient material. By contrast, a patient’s serum can be screened for a broad spectrum of target antibodies using a protein microarray.”
“Tecan’s LS Reloaded™ laser scanner is perfect for analysis of these microarrays, offering rapid data acquisition with minimal user input. It has excellent autofocusing and is a very fast scanner, with a typical scan taking just two minutes at 10 μm resolution. This allows complete analysis of an autoimmunity array in under eight minutes, ideal for use in a primary care setting. The scanner is also very good for microarray development, as it is able to handle both transparent and opaque substrates, and offers the flexibility to work with a variety of microarray formats. We used the scanner extensively for assessing silicon substrates during development and optimization of the array, assessing various binding chemistries for both peptides and DNA.”
Neil Winegarden, Head of Operations at The Microarray Centre, University Healthy Network in Toronto (right), shows their new Tecan LS Reloaded scanner to Tom Ewart, McMaster University, Hamilton (left). The two long time colleagues have a mutual interest in proteomic microarrays. “We are interested in the 4 color laser scanning of the LS Reloaded since it would double our array multiplexing capability. We could quantitatively capture all four IgA, D, G and M class antibody responses to infectious and autoimmune diseases in the same sample image,” said Dr Ewart. The Microarray Centre (www.microarrays.ca) is the major shared resource for microarray studies serving the University of Toronto and six core area universityaffiliated hospitals of Toronto. It is located in the expansive new MaRS (Medical and Related Sciences) research complex on the original Toronto General Hospital grounds.
MICROARRAY PRODUCTS TECAN JOURNAL 1/2010
The LS Reloaded 200 scanner generates concurrent color scaled IgG (left) and IgM (center), and combined response images (right) for an autoimmune patient (top) and a control subject (bottom). The autoimmune patient shows generally higher IgM acute phase responses to the bacterial and viral pathogens printed in the array as well as high rheumatoid factor (white boxes in the printed IgG calibration area).
Tom continued: “Robust data analysis and interpretation is a major factor of transferring this technology to a clinical environment, and the Array-Pro® Analyzer software Tecan supplies is very easy to use. We currently have around 80 different antigens complexed onto the microarray, split approximately equally between pathogen and autoimmunity targets. In addition, the microarray features internal calibration standards to compensate for changes in dilution, and the presence of interfering factors such as rheumatoid factor or immnuosuppresive therapeutic agents. Inclusion of these standards, in addition to accurate gridding and quantitation of the array, is crucial for meaningful interpretation of results, and the software certainly provides this.” “We have already validated the array using a relatively small number of samples from ‘control’ patients confirmed not to have autoimmune deficiencies, and have begun to analyze samples from patients with diagnosed autoimmune disorders. Our preliminary findings have been very interesting – indicating that patients with autoimmune disease have a much higher reactivity to infectious disease antigens – and we are now recruiting more patients from both populations to allow us to verify these results.” “Although development of this technology has so far focused on exploring the links
between infection and autoimmunity, due to the local clinical interest and the availability of a relevant patient population, this platform has a wide range of potential applications. Microarrays offer a low cost alternative to ELISA techniques, with approximately 100 assays in microarray format costing the same as a single ELISA assay. This, combined with the minimal requirements for patient material, makes microarrays an attractive prospect for future point-of-care diagnostic tests,” Tom concluded. Array Pro Analyzer is a registered trademark of Media Cybernetics, Inc.
By dividing the average autoimmune group responses to each pathogen by those of the control group, we obtain the fold ratio bar chart. This is a measure of the extent to which the autoimmune patient group is more reactive to each infectious disease. IgM (red) responses outside the ± 1 range (yellow lines) are significantly higher, suggesting active or unresolved infections.
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CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2010
Fully automated ELISA testing in veterinary diagnostics The Institut Galli-Valerio in Lausanne, Switzerland, has chosen a Freedom EVO® platform to fully automate all ELISAs for the Swiss authorities’ ambitious program to eradicate bovine virus diarrhea virus (BVDV), which can cause mucosal disease in cattle.
The Institut Galli-Valerio is the veterinary diagnostic laboratory of the canton of Vaud, Switzerland, offering a broad range of diagnostic tests for infectious diseases of domestic and wild animals. The institute’s routine BVDV testing forms part of the Swiss authorities’ ongoing program to rapidly eliminate BVDV from the domestic cattle population. The IDEXX HerdChek* BVDV Antigen/ Serum Plus ELISA was chosen for BVDV testing, offering high diagnostic sensitivity and specificity for reliable detection of persistently infected (PI) animals. In the initial phase, all cattle were individually tested to identify and slaughter PI animals. Since the beginning of 2009, all newborn calves are being tested using ear notch
samples taken as part of the regular tagging process. The resulting need for reliable high throughput screening has been met using a Tecan Freedom EVO liquid handling workstation to fully automate all ELISA testing. The Freedom EVO 150 platform is equipped with an 8-channel liquid handling arm using fixed ceramic tips, a robotic manipulator arm, shelves and incubators. A Sunrise™ absorbance reader and a Power Washer 384™ plate washer (using a 96-channel head) have been integrated onto the side of the platform. Tecan has also designed a special carrier to allow sample racks containing ear notch samples to be installed on the worktable. This platform has a maximum throughput of 26 plates per day for BVDV.
TALK TO TECAN TECAN JOURNAL 1/2010
Siegfried Sasshofer, Head of Sales & Marketing at Tecan Austria
The Freedom EVO platform at the Institut Galli-Valerio
In addition to BVDV testing, the laboratory performs a range of other ELISA tests from IDEXX Laboratories using the Freedom EVO workstation, including infectious bovine rhinotracheitis (IBR), brucellosis, enzootic bovine leukosis (EBL) and caprine arthritis encephalitis (CAE), as well as an in-house ELISA for Salmonella abortus-ovis. Close cooperation with IDEXX Laboratories has allowed fast and efficient transfer and validation of all ELISA tests on the fully automated system. The flexibility of the Freedom EVO platform has allowed the laboratory to cope with the demanding sample numbers of the Swiss BVDV eradication program. Acknowledgements We would like to thank Dr Belloy and Dr Waldvogel at the Institut Galli-Valerio in Lausanne, Switzerland, for sharing their expertise using the Tecan Freedom EVO platform and IDEXX Laboratories for the development of customized error handling and tracking, and for setting up applications in Freedom EVOware® for the complete portfolio of IDEXX ELISA tests performed at the institute. * HerdChek is a trademark or registered trademarks of IDEXX Laboratories, Inc. in the United States and/or other countries.
Leading the debate In case you hadn’t yet realized, 2010 is Tecan’s 30th birthday! 30 fast-moving years in the life science and clinical arenas, and 30 ground-breaking years of continuing innovation in laboratory automation. For the detection business unit, major innovation began in earnest in the late 90s with multimode readers and, soon after, technology took another big step forward with the introduction of high-level monochromator systems. A completely modular approach to reader technology, complete with an upgrade path, has improved flexibility, giving customers the option to define the product they want now and in the future. So what’s next? Where will innovation take us in the next 30 years? The early signs suggest that, for basic research, convenience and ease-of-use at a low cost will remain paramount. We will continue to focus on keeping our software as simple as possible yet still as powerful as necessary. Just a simple issue like language can make all the difference to a laboratory user; however good your English, an instrument or software that talks your language makes life so much simpler. For more advanced applications in academia and industry, two main factors are coming to the fore. The first is multiplex assays; the importance of getting more information out of just one assay, saving samples, reagents, costs and time. Microarrays are a perfect example of one investigation giving literally thousands of data points. The second is relevance; knowledge has passed the limits of biochemical assays and researchers now need to work on a higher biological level, on cells or tissue etc. Cellbased assays are becoming more relevant and as new methods and technologies bury into deeper levels of understanding, users will demand high performance instruments. Whatever happens, in these or other methodologies, the common factor stays the same; the key is to stay close to laboratory users, allowing them to drive innovation according to what they need in their applications.
Email talk@tecan.com to tell us what you think about the direction of innovation or any other life science topic of your choice.
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EVENTS 2010 TECAN JOURNAL 1/2010
Meet Tecan at these events in the coming months Americas Lab Automation 2010
Palm Springs
23 – 27 Jan 2010
MD&M West 2010
Anaheim, CA
9 – 11 Feb 2010
AAFS American Academy of Forensic Sciences 2010
Seattle, Washington
22 – 27 Feb 2010
Pittcon 2010
Orlando, Florida
28 Feb – 05 Mar 2010
ASM American Society for Microbiology 2010
Miami
22 – 26 Mar 2010
SBS Society for Biomolecular Sciences 2010
Phoenix
11 – 15 Apr 2010
Expofarma-Interphex 2010
Mexico City
21 – 23 Apr 2010
Japan Society for Bioscience, Biotechnology and Agrochemistry
Tokyo, Japan
28 – 30 Mar 2010
Lorne Protein Structure and Function,
Erskine, Victoria
7 – 11 Feb 2010
Lorne Cancer
Erskine, Victoria
11 – 13 Feb 2010
CHUV Research Day
Lausanne, Switzerland
28 Jan 2010
30. Spurenworkshop
Zurich, Swizterland
5 – 6 Feb 2010
Lausanne Genomics Days
Lausanne, Switzerland
18 – 19 Feb 2010
ELRIG
Silverstone, United Kingdom
4 Mar 2010
Analytica 2010
Munich, Germany
23 – 26 Mar 2010
Gen2bio
Saint Malo, France
30 Mar 2010
Asia and Pacific
Europe, Middle East and Africa
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Tecan Journal, Customer Magazine of Tecan Trading AG., ISSN 1660-5276 Design: OTM/London www.otmcreate.com Photography: Marc Wetli/Zürich www.wetli.com, Günter Bolzern/Zürich www.bolzern.net, Susanne Völlm/Zürich www.susannevoellm.ch Editor in Chief: Tecan Trading AG, Cornelia Kegele Project Lead: Tecan Trading AG, Cornelia Kegele/Aline Weiss Editor: kdm/UK www.kdm-communications.com Print: DAZ Druckerei Albisrieden AG/Zurich www.daz.ch Address: Tecan Trading AG, Marketing Communications, Seestrasse 103, CH-8708 Männedorf, Switzerland, journal@tecan.com, www.tecan.com Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication, however, it is possible that omissions or errors might have occurred. Tecan Group Ltd. cannot, therefore, make any representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in this publication. Changes in this publication can be made at any time without notice. All mentioned trademarks are protected by law.
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