MULTIPLEXING JUST WENT DIGITAL!
UltraPlex™ Immunoassays
The UltraPlex™ system has already been used to multiplex a wide range of assays which demonstrate its broad applicability. The most advanced applications are in direct immunoassay, where Pronostics is in the process of launching a portfolio of products directed at clinical immunology laboratories. These products clearly demonstrate that UltraPlex™ assays yield sensitivities and specificities at least as good as conventional “one analyte at a time” ELISAs.
UltraPlex™ ANAScreen is available today and provides valuable time and cost savings compared to existing ENA/ANA experiments using Hep-2 screens and ELISA tests. It allows hospitals and laboratories to perform fully automated profiles of patient samples against 9 anti-nuclear antigens (ANA) simultaneously. This product is CE marked and is initially available in the UK and other European countries.
UltraPlex™ ANA - The World's First Barcoded Immunoassay for Simultaneous Testing of Autoimmune Disease Biomarkers
UltraPlex™ ANA is fully CE marked and is available in the UK and other European countries today.
Autoantibodies, including those against ANA (Anti Nuclear Antigens) occur with high frequency in the serum of patients with connective tissue disorders and rheumatic diseases. Results of serologic tests for detection of these autoantibodies, including antibodies to specific nuclear antigens such as double-stranded DNA (dsDNA), play an important role in the diagnosis of systemic rheumatic diseases. Most laboratories use indirect immunofluorescence (IIF) for ANA testing. Reading IIF patterns is time consuming, subjective, prone to misinterpretation and can be difficult even for the most experienced technologist. The difficulty in pattern interpretation highlights the need for autoantigen-specific immunoassays for diagnosis. UltraPlex™ ANA simultaneously tests for 9 key autoantibodies plus controls with multiplexed immunoassays, eliminating subjective errors and freeing up skilled technologist time.
The UltraPlex™ ANA multiplexed immunoassay identifies autoantibodies against the following nine antigens in patient samples: Jo-1, Scl-70, Sm, Sm/RNP, SSA, SSB, RNP-70, Centromere and dsDNA.
The nine ANA antigens are each individually tagged with uniquely barcoded microparticles. A blank barcoded No-Coat Control microparticle is also included and used as an internal reference control in the multiplexed test. This greatly reduces false positive results, particularly those seen in patients suffering rom SLE and other similar indications. The barcodes enable tracking of each microparticle and therefore allow mixing of all ANA antigens and the internal reference control. For ease of use and long-term stability, these reagents are supplied lyophilised as a single SmartBead in each well of a microtitre plate.
Product presentation
UltraPlex™ ANA is supplied in a 96-well plate format and includes all the necessary reagents and controls for processing 80 patient samples. The barcoded antigens and internal reference standard are supplied ready-to-use in a lyophilised Accusphere format.
UltraPlex™ ANA Automation
The assay procedure, including patient sample dilution and addition of reagents, and determination of results is fully automated.
The UltraPlex™ System includes the UltraPlex™ SmartStation robot and UltraPlex™ SmartReader, the combination of which provides a fully automated walk-away assay procedure and data acquisition process which eliminates sample handling and processing time. The UltraPlex™ SmartReader employs Pronostics' user-friendly SmartDecode software to accurately measure and report test results in a range of user-selected formats, which can be directly fed into the user's LIM system.
ANAScreen Fast facts and benefits
• No specialist training or experience required
• Consistent results, both within an assay and between users
• Generates full 9 antigen profile for 160 patients per 8 hour day
• No need for confirmation of results through ELISA (sensitivity >90%)
• Test employs recombinant and highly purified antigens (specificity >95% )
• Overall agreement >97% (IIF and ELISA, 700 samples UK Hospital)
• Multiplexed assay increases productivity and provides compelling economic benefits
• Miniaturisation reduces reagent and sample volumes
• Simultaneous assays performed under uniform conditions
• 96-well microtitre plate format with standardised protocols
• Multiplexed reagents ready for use in each well
• Lyophilised SmartBeads provide stability and reproducibility
• No-Coat Control microparticles greatly reduces false positives
• Minimal 'hands on' assay time facilitated by the UltraPlex™ SmartStation
• UltraPlex™ SmartReader provides automated generation of results
• Validated against CDC Human Reference Sera panel and multiple panels of clinical samples
• dsDNA standardised against WHO Wo/80 reference material
UltraPlex™ ANA brochure Version 1.0 29/4/08
Follow on UltraScreen Products
• Thyroid Available now
• Coeliac Available now
• Rheumatoid Q4 2008
• Liver Q1 2009
• Vasculitis Q4 2008
• Cardiolipin Under development
UltraPlex™ SmartReader
Pronostics have developed a dedicated reader, the UltraPlex™ SmartReader, running Pronostics' SmartDecode software, which is fully compatible with virtually all LIM systems.
The UltraPlex™ SmartReader is a self-contained unit, occupying approximately 0.3m2 of bench space, combined with a dedicated compact PC workstation. Although based around a fluorescent microscope, the unit accepts standard 96-well plates and yields up to 27,000 data points per hour at the press of a single button. Each well is scanned in turn and the microparticles are decoded from a phase-contrast image. Simultaneously, the output of the assay is quantified from a fluorescent image of the same field of view. At least 10 particles of each code are measured in each well, and the mean fluorescence reported.
Fig.4 The UltraPlex™ SmartReader
SmartReader is configured with high quality optics and alternative filter sets to read commonly used fluorescent reporters. Our next generation reader, now under development, will allow simultaneous measurement of multiple dyes further expanding UltraPlex™ multiplexing capabilites.
The reader's software, SmartDecode, already outputs the data matrix (which can contain more than 1,000 test results for a 16-plex assay) in a format compatible with all major LIM systems, or in Excel format for manual analysis, and custom output formats can readily be added.
Available off-the-shelf, the UltraPlex™ SmartReader is the ideal solution for most medium throughput applications. However, we have carried out proof-of-principle demonstrations decoding UltraPlex™ assays on collaborative partners high-end microscope based reader systems, and have shown that data accrual rates 10-fold higher than with UltraPlex™ SmartReader can readily be achieved. Similarly, real time decoding in a flow-based reader is also practicable and could be developed further for applications demanding very high throughput
UltraPlex™ SmartStation - Liquid Handling Robot
The UltraPlex™ system also incorporates a customised liquid handling robot, the UltraPlex™ Robot, which automates assay performance in medium to high throughput environments. For example, in clinical diagnostics laboratories 160 patient samples a day can be subjected to a 10-plex UltraPlex™ assay using a single integrated Robot and Reader, yielding 16,000 datapoints in a single day, exceeding the performance of many autoanalysers.
The UltraPlex™ SmartStation robot has a four-tip head, capable of dispensing 10-1000µl of liquid (1% CVar and maximum 3% inaccuracy), and incorporates liquid level detection technology to ensure reagent transfer. The system uses disposable plastic tips, but can be reconfigured to re-use tips if required for particular applications. Typically, three washes on a 96-well plate take about 10 minutes to perform. The Robot has four (or more, if required) separate liquid reservoirs suitable for complex protocols with multiple detection reagents, with a dead volume of less than 10ml conserving costly assay components. Importantly, the system has a shaking vacuum manifold optimised for handling UltraCodes™ in conventional filter plates.
Fig. 5 UltraPlex™ SmartStation
Samples can be taken from a wide range of tubes (1.5 to 10ml volume) or from 96-well plates. The UltraPlex™ SmartStation also incorporates a standard barcode reader (compatible with most standard barcode formats) for sample tube identification. Ideal for high throughput applications, this ensures that the sample ID and data subsequently obtained from it are permanently linked, effectively eliminating sample handling errors. The SmartStation is driven by an integrated PC workstation. In addition to delivering ‘one-button’ convenience to operate standard protocols, it also offers the user immense flexibility in designing new protocols for a wide range of applications.
Multiplexed Genetic Testing
Applications are not limited to immunoassays. We have demonstrated that biotinylated oligonucleotides can be readily coupled to streptavidin-coated UltraCodes™, and then be specifically hybridized with labelled probes, opening up a wide range of potential applications in multiplex genetic testing.
