Enzyme-linked immunosorbent assays take analyte detection to new levels

Enzyme-linked Immunosorbent Assays (ELISA): Recent Innovations Take Analyte Detection To New Levels By Dr John Comley Enzyme-linked immunosorbent assays (ELISA) have been around as one of the primary methods of analyte detection for more than four decades. Over the years many changes to the basic format have resulted in assay improvements, but some of the most recent look set to take the traditional ELISA to new levels. These include many vendors now addressing the automation of ELISA assays. Some automated platforms include new or alternative ELISA formats typically with miniaturised or microfluidic components. Other automated instrumentation is designed to support the creation/coating of ELISA assay plates or contains cartridge-based delivery of assay reagents to reduce set-up time. New developments in automated plate washing and the minimisation of wash steps are also impacting conventional ELISAs. Perhaps most significantly are the big increases in assay sensitivity and the widening of the dynamic range accomplished by using alternatives to the traditional colorimetric detection, or by the use of new platform technologies. Several of the new offerings utilise bead or particle-based components some of which are advantageous for multiplexing. These are supported by a wide range of ELISA reagents, assay and kits providing relative profiling or absolute quantitation of proteins across a wide range of study areas. When coupled with the breadth of existing global expertise in immunoassays, it is easy to see why ELISA still remains the methodology of choice by those labs tasked with analyte detection. Enzyme-linked immunosorbent assays (ELISA) have been one of the primary methods of detecting the presence of a substance, usually an antigen, in an aqueous sample for more than 40 years. For most of this time a heterogeneous, solid-phase microplate-based format utilising multiple washing steps and a colorimetric readouts has predominated. However, numerous variants of this format have been developed over the years (eg indirect, sandwich, competitive, etc) and a multitude of detection chemistries have been applied to ELISA, including chemiluminescence and various fluorescent readouts. ELISA has gained widespread acceptance across a very diverse range of fields (eg diagnosis of infectious diseases, food allergen detection, plant pathogens to biomarkers), but it never achieved significant adoption by early drug discovery labs. This initially reflected the fact that plate washing was perceived to be difficult to automate, and subsequently the industry’s preference for homogenous screening methods, that could be miniaturised. The immunoassay landscape has, however, dramatically changed in recent years with the development of reduced assay steps protocols, no wash ELISAs and new automated systems designed to run ELISA. More recently, bead-based methods and microfluidic platforms have started to impact immunoassays and by coupling with single molecule detection look set to take the sensitivity of traditional ELISA to new levels. This review article uses as its starting point some of the findings of HTStec’s ELISA Assay Trends 2012 survey and report, published in June 2012 (1). This report discusses end-users’ current practices and preferences in ELISA assays, and seeks to understand future user requirements. It also examines interest in adopting new or emerging ELISA or immunoassay platforms with potential to significantly improve performance. The article endeavours to give the reader a feel for the current vibrant state of ELISA and related immunoassay alternatives. Main application of ELISA assays The main (primary) application area for survey respondents’ ELISA assays was immunology (17% selecting). This was followed by cancer (12% selecting); infectious diseases (11% selecting) and then inflammation (9% selecting). Among those responding no interest was found for blood banking, stem cells, environmental or quality control as the main (primary) applications of ELISA assays (Figure 1). Samples sources analysed by ELISA Of the sample source analysed by ELISA most respondents (67%) were analysing serum. This was followed by plasma (48% analysing); cultured cells (41% analysing); whole blood (31% using) and then cell lysate (30% using). Least used sample sources were environmental samples (3% using); dried blood spot (3% using); water (1% analysing) and foetal cells (not analysed by any respondent) (Figure 2). Enzyme/detection chemistries used in ELISA The enzyme/detection chemistries most used by survey respondents today (2012) in their ELISA assays was HRP (horseradish peroxidase)-colorimetric (88% using); followed by AP (alkaline phosphatase)- colorimetric (31% using) and then HRPchemiluminescent (29% using). In contrast, only 13% were using electrochemiluminescent detection (Mesoscale Discovery) and 8% using AlphaScreen/AlphaLISA (PerkinElmer) (Figure 3). Level of automation applied to ELISA Most respondents (33% each) have either not applied any automation to their ELISA assays today (2012), or ELISA automation has been limited to plate washing only. Of the remainder, 13% have partially automated ELISA (ie sample/reagent additions, plate washing and incubation cycles); 12% have applied fully automated robotic processing to non-homogeneous ELISA (ie sample/reagent additions, plate washing, incubation and detection); 1% have partially automated ELISA by a homogeneous method (ie sample/reagent additions and incubation) and so far no respondents have applied fully automated robotic processing (ie including detection) to homogeneous ELISA (Figure 4). Types of plate washers used for ELISA Most (36%) of survey respondents were using a 96-well whole plate washer for their ELISA assays. The next most used washer type was an 8 or 12- channel strip washer (30% using), followed by manual/hand washed assays (22% using). The preferred manufacturer of these ELISA washers was BioTek (Figure 5). Primary assays format for ELISA The majority (51%) of survey respondents were using the 96-well microplate as their primary assay format for ELISA assays today (2012). The next most used format was 96-well strips with 39% of respondents using. This was distantly followed by 5% using 384-well regular volume plates, and only 3% using either 96-well microfluidic channel plates or 384-well low volume plates (Figure 6). The need for commercially available ELISA kits to be miniaturised The majority (57%) of survey respondents do not see the need for commercially available ELISA kits to be miniaturised, indicating that the standard volume 96-well format meets their current needs. Of those seeking miniaturisation, a standard volume 384-well format (14% wanting) or a microchannel 96-well format (eg Siloam Biosciences Optimiser plates) were preferred (12% wanting) (Figure 7). The biggest issue with ELISA today Increasing sensitivity was ranked as the biggest issue (problem) with ELISA assays today. This was followed by reduced sample/reagent consumption and then improving precision by eliminating wash steps and increasing throughput. Ranked of least importance was the ability to miniaturise format (Figure 8). Awareness of some new improved ELISA platforms Of some new, emerging or alternative technologies or approaches, respondents were most aware of (including already investigated/implemented) Luminex microparticle-based flow tests for multiplexed assays. This was followed by qPCR-based alternative tests; PerkinElmer AlphaLISA beadbased homogenous no-wash format; and then MesoScale Discovery ECL MultiArray technology. Respondents were least aware of Douglas Scientific Nexar system optimised for inline homogeneous ELISA processing and Singulex High Definition Erenna Immuoassay microparticlebased system with single molecule detection (Figure 9). What is driving adoption of new improved ELISA platforms? Facilitates improved performance/greater sensitivity/ better uniformity was ranked as the main motivator/ driver for adopting a new improved ELISA platform. This was distantly followed by enhances speed, reduces assay time, enables higher throughput; and then facilitates miniaturisation, eg lower volume, less sample required. Ranked least motivating was avoids time-consuming wash steps (Figure 10). Latest developments in ELISA Abcam (www.abcam.com) offers a comprehensive catalogue of immunoassay kits suitable for relative profiling or absolute quantitation of proteins from a wide range of study areas. Consisting of easy-touse kits for more than 1,000 targets, Abcam offerings provide coverage of an extremely wide range of analysis including both extracellular and intracellular pathway targets such as cytokines, angiogenesis, adipokines, cell differentiation, growth factors, oncoproteins, tumour biomarkers, signal transduction and many more. A variety of ELISA kit formats and detection methods are available to suit any researcher’s preferred methods and instrumentation. Users can obtain consistent, accurate and sensitive measurements of pathway proteins with Abcam immunoassay kits based on standard platforms including sandwich, competitive, direct and indirect ELISA methods. Additionally, Abcam provides several assay platforms employing advanced methods and techniques offering improved performance and value for the users. Abcam’s PhosphoTracer ELISA offers simplified protocols and accelerated assay times, yielding sensitivities superior to Western blotting with only one wash step in less than 90 minutes. Novel metabolism focused MitoSciences In-Cell ELISA kits (sometimes referred to as In-Cell Westerns) enables measurement of analytes in fixed cell cultures after treatments, eliminating perturbation of the cells from further sample preparation as required by traditional Western blotting methods. This immunoassay kit method is more sensitive and offers higher throughput than Western Blotting and multiplexed measurements of analytes is achievable when using Infra-Red (IR) detection (Figure 11). When considering automating ELISA or other immunoassay workflows, several factors influence the automation system configuration. Agilent Technologies (www.agilent.com) offers a BenchBot Benchtop workstation for ELISA assays. In this workstation a dedicated liquid handler, such as the Agilent Bravo Platform, provides more flexibility than a regular dispenser. The Bravo Platform permits whole plate (well-by-well) transfers and can automate protein extraction and purification. The Bravo Platform also enables fast and accurate serial dilution for quantitative ELISAs. Typically, a variety of dispensers and microplate washers can be incorporated into the automated workflow depending on throughput requirements. A sensitive absorbance or multimode reader can also be integrated according to the assay needs. The benchtop workstation in Figure 12 accommodates all of the devices mentioned. The configuration offers a compact layout while providing sufficient space between devices for accessibility. With the understanding that batch size and processing times are a function of the configuration and protocol, Agilent application scientists can propose configurations and provide simulation data specific to your ELISA assays. Tested timings and optimised protocols can be provided with all configurations. Aurora Biomed’s (www.aurorabiomed.com) VERSA™ Liquid Handling Workstations automate ELISA/TRF applications while also catering to their customers’ diverse throughput, protocol and integration needs. Aurora offers four workstations capable of meeting a range of throughputs for ELISA/TRF-based applications. To accommodate high-throughput ELISA-based applications, Aurora recently launched the VERSA Integration Workstation, which integrates a VERSA 1100 Workstation with a range of third party devices including a plate washer, plate reader and highly sophisticated scheduler (Figure 13). This integrated workstation eliminates bottlenecks of a standalone workstation through the tower gripper, which efficiently accesses and presents ELISA plates to the various components of the integrated assembly, thus creating a complete solution for the ELISA/TRF process. The VERSA 1100 liquid handling head within the integrated assembly reformats samples from tubes to 96-well plates while the 12-channel ReagentDrop increases throughput by simultaneously dispensing the specified reagents into one 12-well row. This highly efficient bulk dispenser eliminates tip costs by its tip-less reagent addition to the target plate. The on-deck shaker mixes samples and reagents at desired temperatures for efficient interaction of biomolecules. The plate hotel/incubator offers basic humidity, air and CO2 control while housing more than 100 plates. The ViBE® workstation from Bioscale (www.bioscale.com) is powered by Acoustic Membrane Microparticle (AMMP®) assay technology. It integrates simple sample preparation; walk-away operation; broad application flexibility; and novel, non-optical detection to deliver sensitive, reproducible protein analysis from complex biological samples. When looking for added decision- making data from ELISA assays, researchers routinely use AMMP assays to measure biomarkers as they search for early warnings of disease, screening new drugs, or monitoring tumour or patient response to therapies. Homogenous AMMP assays require no time-consuming wash steps and typical assay sample prep time is less than 30 minutes. The assay works in microplate wells where samples are mixed with a target-specific antibody linked to microparticles and a second paired-antibody linked to a hapten. The resulting complex is passed over vibrating acoustic sensors and drawn to the surface by a magnetic field. Complexes that bind to the affinity-coated surface remain and when eluted are measured as a frequency shift that is converted into highly precise and quantitative measurement of biomarker concentration. The noted lack of optical detection makes AMMP assays indifferent to sample matrix effects. The nature of the assay technique improves sensitivity even with low abundant proteins by using direct measurement of proteins in their native state and avoiding variability due to dissociation caused by multiple wash steps. In addition, AMMP assays require less sample. Recent results presented at the 2012 AACR show the ability to measure multiple kinases in their native states in a single AMMP assay plate using 100-2,000 cells per assessment. Assay development is straightforward and fast with antibody screening, assay optimisation and matrix testing accomplished in less than a week compared to 2-3 months for other technologies (Figure 14). A new product for delivering 96 unique reagents into 96-well format ELISA plates has recently been developed by Apricot Designs (www.apricotdesigns.com). The MultiWell plate dispenser consists of a 96-channel dispense head that can access 96 individual source reagent reservoirs or bottles and deliver microlitre volumes to the 96-well plate, standard height up to deep well. Benefits have been realised by ELISA kit manufacturers and high volume ELISA customers that would like to create assay plates with 96 different tests, assay conditions or dilutions. Delivery volumes can range from 2μL and above. The system also has a backflush feature to ensure that reagent usage is minimised. Automation for higher throughput and/or unattended operation can be provided by BioSero (www.bioseroinc.com) (Figure 15). The general process of ELISA remains constant with most actions performed manually by the technician, from managing assay timing steps to physically moving the sample microplates between commonly- used instrumentation such as dispensers, washers and detection systems. When many samples need to be processed routinely, manual methods are impractical and inefficient. BioTek (www.biotek.com) and Thermo Scientific Laboratory Automation have collaborated to define a modular bench top workstation to automate the ELISA process – to free up technician time, allow continuous processing of multiple plates and to provide excellent assay performance and ensure sample consistency plate to plate. The main component of this workstation is the Thermo Scientific Orbitor® Momentum Workstation integrated seamlessly with BioTek instruments such as a MultiFlo™ Dispenser, 405™ Touch Washer and an Eon™ Spectrophotometer around the periphery. Using a modular Thermo Scientific mezzanine to stack the dispenser and washer, multiple devices fit neatly into a compact 30in x 55in bench space, with plenty of room for device and plate stack access. The Orbitor RS has a 360° rotating lift that moves a bidirectional telescoping arm up and down to the component instruments, quickly and efficiently. The workspace can also be extended with a BenchTrak to incorporate additional dispensers, washers and readers. The BioTek instrument plate carriers are all designed to easily receive plates from the Orbitor gripper without requiring tedious alignment. Increased throughput and productivity are the outcome with equal or better results compared to manual ELISA processing (Figure 16). ELISAs have been the mainstay of immunoassay technology for more than four decades. However, the advent of proteomics has created a widespread need for multiplexed analysis, and using parallel ELISAs has proven to be expensive and impractical. Unfortunately current multiplexing approaches including multiplexed ELISAs provide compromised assay performances, and suffer from numerous operational drawbacks that relegate these technologies to early research applications. CyVek (www.cyvek.com) has developed a novel microfluidic, multi-analyte immunoassay platform (CyPlex™) to expedite biomarker research, enable translational medicine, and transform medical diagnostics. The CyPlex™ system consists of a disposable test cartridge (mimics an ELISA plate) and a bench-top analyser; typical data show a >4 log dynamic range and fM detection limits. CyPlex™ is the only multi-analyte technology that offers traditional ELISA-like data quality because each antigen- specific antibody reaction occurs within segregated proprietary reaction vessels (μTubes™). This obviates cross-reactivity concerns, improves data quality, simplifies development and expedites the validation of multi-analyte assays. CyPlex’s microfluidic architecture allows complete automation; the user adds samples into the cartridge, inserts the cartridge into the analyser and hits the ‘go’ button to obtain analysed results within 10 to 60 minutes. CyPlex™ allows a modular approach for assay-panel development: μTubes™ validated for a given antigen, are easily swapped into unique microfluidic cartridges to assemble novel multianalyte assays without concern for cross-reactivity. CyVek provides a versatile platform that is easily configured for diverse applications such as rapid diagnostic tests, multi-sample-multi-analyte tests, or routine ELISA-like tests, for use in research and clinical settings (Figure 17). Immunoassays have been widely adopted for analysis and quantification of low concentration proteins. The immunoassay is selective and sensitive; however, its utility has been limited in high throughput systems due to a cumbersome process involving multiple incubation and wash steps. Microplate-based automation has yielded some success; however, it failed to meet the requirement of continuous automation for high throughput (HT) applications. The ideal system should provide: continuous automation, reproducible and rapid analysis of biological samples, easy set-up and use, potential for broad deployment and cost savings per data point. To address these needs, Douglas Scientific (www.douglasscientific.com) has optimised Array Tape™ Platform for automated inline homogenous ELISA processing. The Nexar® is a fully automated, modular, inline system using Array Tape™, a thin and flexible microplate replacement, serially embossed with reaction wells. The system enables assay processing in miniaturised reaction volumes with walkaway operation. Inline modules include: random access sample storage, liquid handling, incubation and Alpha Technology detection. Detection speeds are less than three minutes per 384-well array and enable the generation of more data, faster. Experimental runs to validate were completed using PerkinElmer’s AlphaLISA® assay, human insulin, Immunoglobulin G, human necrosis factor TNF- . The assays were performed in accordance with the kit protocol in a miniaturised reaction volume. Additional assays were then completed using bead dilutions of 1 in 6 and 1 in 3. The results were equal to those reported in the kit. Findings from the experiments clearly show the sensitivity of the assay has been achieved in Array Tape at reduced volumes, thus offering a significant reduction in the assay cost (Figure 18). Dynex Technologies (www.dynextechnologies.com) is a leading manufacturer of open, fully automated ELISA systems, offering the two-plate DS2® and four-plate DSX® platforms. To make existing ELISA assays more automation-friendly, Dynex has designed the SmartKit™ packaging format. SmartKits include an insert for holding reagent bottles and a 2D barcode with lot-specific assay information. Once caps are removed, the SmartKit is placed directly into a compatible open system for testing. Through the combination of packaging and instrument compatibility, SmartKit technology dramatically removes the set-up that takes up the bulk of ELISA technicians’ time. This year, Dynex will launch the Agility™ automated ELISA processor, which can hold 16 SmartKits and run up to 12 plates per worklist. Another innovation, Dynex’s M2™ multiplex technology, builds upon proven ELISA methodologies to provide a flexible option for maximising productivity while reducing both sample volume and manual labour. Multiplexing is achieved by using a positionally determined array of 2mm diameter polystyrene beads in microplate wells. Pre-adsorbed beads (up to 10 analytes per well in initial configuration) are pressed into cylindrical pockets in the bottom of each custom well. The chemiluminescent signal is read by a high-resolution camera capable of capturing and analysing data from an entire plate in two minutes or less. M2 data consistently demonstrates a high dynamic range and sensitivity within femtogram/mL levels. Dynex plans to integrate M2 technology in its ELISA automation systems beginning in 2013, providing completely integrated automated multiplex systems at a cost equal to or less than existing standalone multiplex readers (Figure 19). eBioscience (www.ebioscience.com), a leading provider of immunoassays, recognises the diverse needs of both the research and drug discovery/development environments. eBioscience offers six immunoassay platforms, with thousands of immunoassay options over six species. Each platform was developed to meet the challenging demands that researchers face today. Its Ready- SET-Go coat-it-yourself ELISA kits are a highlyvalidated but economical alternative to pre-coated ELISA kits. Platinum ELISAs are a line of traditional pre-coated sandwich ELISAs, with extremely low inter- and intra- CVs that researchers can trust time and time again through and across various projects. As the traditional sandwich ELISA does not always address the limitations and complexities of biomarker analysis, eBioscience offers innovative alternatives to allow for either increased sensitivity with its High Sensitivity ELISAs, or increased throughput with the Instant ELISA. Instant ELISAs are ideal for researchers who either want to increase throughput or reduce hands-on time. All that is required is the addition of a sample to the pre-coated ELISA well already containing both the capture and detection antibodies, followed by simple incubation, wash and detection steps. For those researchers looking at signalling and phosphorylation differences, the InstantOne ELISA is the perfect solution with its sensitivity and easy one hour/one wash assay. InstantOne allows for detection of total/phosphorylation levels or different pathway members all on the same plate. Finally, as the market continues to demand multiplex immunoassays that provide more information in less time with less sample, eBioscience is offering Luminex-based assays with the recent addition of Procarta multiplex immunoassays covering approximately 300 analytes in six species. Procarta includes the largest commercially available multiplex panel with the human cytokine panel simultaneously measuring 54 analytes in a single well with only 25μL of sample (Figure 20). Immunoassay technology based on microfluidics principles, allowing integration of the chemical operations involved in conventional analytical processes, such as mixing, reaction and separation, has been gaining momentum in recent years. Working at nanolitre scale, this technology can drastically cut consumption of sample and reagents. The Gyrolab™ xP workstation from Gyros (www.gyros.com) uses microfluidic compact discs (Bioaffy CDs) consisting of microcolumns pre-packed with Streptavidin-coated particles, and a complex network of microchannels with hydrophobic barriers through which samples and reagents flow under the influence of centrifugal and capillary action as the CDs are spun in the workstation. By altering rotational speeds of the CD, precise volumes of capture reagents, sample and detection reagents can be delivered through the microcolumns. Implementation of microfluidic principles ensures controlled flow of capture reagents, sample and detection reagents, so that all structures within a single CD are processed in parallel. This eliminates the occurrence of timedependent artifacts that may be observed in a typical plate-based ELISA, and ensures samples are processed under uniform conditions. It also means that different analytes can be studied simultaneously without introducing cross-talk. Quantification is achieved by detection of fluorescence within each microstructure, which is then analysed by the Gyrolab Viewer software (21 CFR part 11 compliant). The Bioaffy CDs are compatible with a number of conventional immunoassay formats, facilitating the quantification of any target protein for which an immunoassay can be developed. Assay development and optimisation is performed using pre-programmed template methods, or assay protocols, taking only days to select optimal conditions, compared with weeks for ELISA. Specialised assays have also been developed, for example, the Gyrolab ADA (anti-drug antibody) assay is a unique solution that improves drug tolerance and offers improved technical performance compared to alternative techniques (Figure 21). Hudson Robotics (www.hudsonrobotics.com/elisa) provides a number of automated solutions that support low throughput to high throughput ELISA assays. All software control and support of the third party instrumentation described in this paragraph are offered by Hudson. One component of the Hudson solution is the new ultra-compact RapidWash. The RapidWash works with most SBS microplate formats, both standard and deep well. It can be used as a standalone washer, or can be integrated into any automated system. The system shown in Figure 22 supports ELISA assays as well as serial dilutions. It includes the SOLO automated pipettor integrated with the Hudson Micro10x bulk reagent dispenser as well as the RapidWash. This system is equipped with a 12-channel pipette head that can move liquid between the five available nest positions on the deck of the SOLO as well as those on the Micro10x and RapidWash. The complete system is compact and can easily fit into a standard hood. In the layout shown (Figure 22), the SOLO contains disposable tips, two reagent reservoirs and a rack of sample tubes. The SOLO moves samples to the plate on the Micro10x nest, and the dispenser fills the remaining rows with diluent. The SOLO then carries out a serial dilution, adds aliquots of the serial dilution and reagents to the ELISA assay plate on the deck of the RapidWash, which carries out a series of wash steps. The new Novex® chemiluminescence ELISA kits from Life Technologies (www.lifetechnologies.com/elisakits) offer better sensitivity and a larger dynamic range of detection than conventional colorimetric ELISA kits. These advantages enable detection of proteins at low levels and minimise the need for sample dilutions. Validated for use with serum, plasma or tissue culture supernatant samples, these kits incorporate the easy-to-use Novabright™ CSPDEmerald II AP substrates that minimise hands-on time and protocols steps. As a part of the Novex® ELISA family portfolio, these kits are developed to quantitate disease-related proteins. With excellent sensitivity, proven specificity and consistent performance, these optimised ELISA kits offer you a choice when it comes to getting physiologically relevant and quantitative results (Figure 23). Matrical Bioscience (www.matrical.com) recently developed an instrument for ELISA assays, intended for washing unbound material from high density microplates in 96, 384, 1536-well format. The SQUIRT washer design uses a knife-edge stream of buffer sprayed across the plate and a stream of air to displace buffer from the wells. The washer is intended for washing microplates of any density and for ELISA, biochemical or cell-based assays. A stream of buffer rinses the tiny wells more reliably than old nozzle-based technologies, which are known to leave behind small amounts of liquid, resulting in variability and background noise. The SQUIRT washer features variable wash pressure settings, including a gentle ELISA protocol to prevent dissociation of antibody bound analyte, resulting in high signal and low %CVs. To complement the Squirt washer, Matrical offers 96, 384, and 1536-well plates, available with a variety of high quality coating options, including streptavidin, collagen, poly D lysine and super coating, used when passively binding antibodies and proteins (Figure 24). The Mercodia (www.mercodia.se) ELISA is a robust, flexible technology that is adaptable to any volume laboratory, carrying science from bench to bedside. ELISA can close the gap between efficiency and reliability of quantitative data through validation on novel automation platforms. Advancements in automation, including hardware with multiple robotic arms that operate simultaneously, have produced systems that are 2.5 times more efficient than their archaic counterparts. On a daily basis Mercodia’s Oxidized LDL ELISA can offer hundreds of data points, with a coefficient of variation well below the laboratory’s cut point. Many laboratories can reduce duplicate analysis to one single measurement, thereby reducing time and expense. Despite the addition of new technologies such as multiplex testing, ELISA still offers superior sensitivity and specificity. Mercodia’s Ultrasensitive C-Peptide ELISA, for example, provides a multi-fold improvement in sensitivity over existing commercial methods. This allows an opportunity to better understand the progression of diseases such as diabetes, resulting in improvements in drug efficacy studies and clinical standards of care (2). In addition, ELISA technology can provide simple solutions to complex questions in today’s drug development environment. For 20 years Mercodia assays have been relied upon for the robust measurement of rodent insulin in early drug discovery. Today, the Mercodia Rat and Mouse FIA applies a fluorescence detection system, offering more than a two-fold increase in the dynamic range commonly noted in competing immunoassays. This removes the guesswork that occurs when deciding what dynamic range is appropriate for the animal model; saving the laboratory much needed resources (Figure 25). As reflected in HTStec’s survey, many labs are planning to switch from traditional colorimetric ELISA assays to more modern biochemical techniques, such as ELISA with fluorescent labelling, AlphaLISA or chemiluminescent detection. Molecular Devices’ (www.moleculardevices.com) SpectraMax Paradigm Multi-Mode Detection Platform technology features cartridge modularity, making it ideally suited for this scenario. The system is available with basic read modes such as Absorbance, and can then be easily upgraded by the user to a variety of detection methods, including those mentioned above, with a simple twominute cartridge installation. SpectraMax Paradigm’s highly sensitive and versatile cartridges perform both basic and high-throughput detection. For example, the latest addition, the TUNE Detection Cartridge, offers the wavelength flexibility of traditional monochromator technology but with 10 times more fluorescence sensitivity, and also supports fluorescence intensity, time-resolved fluorescence and luminescence detection. For superior sensitivity of specific assays, application-specific optics in individualised cartridges for assays, such as AlphaScreen or HTRF, provide superior sensitivity. Overall, the system is ultra-sensitive and extremely flexible. Molecular Devices recently introduced the latest version of the SoftMax Pro 6 Software for image capture and analysis, which accompanies the system. This latest update automatically maximises signal-to-noise ratio by scanning through excitation and emission wavelengths simultaneously via the Wavelength Optimizer Wizard, reducing the assay optimisation time by up to 50%. The latest release of SoftMax Pro 6 GxP Software also makes this an easy decision for QC labs (Figure 26). PerkinElmer (www.perkinelmer.com) offers alternatives and ELISA-enhancing technologies – including Alpha Technology (AlphaLISA® and AlphaScreen®), DELFIA® and TSA/ELAST to ensure that your immunoassays perform with sensitivity, efficiency and accuracy. Alpha Technology is an alternative detection method for ELISA. The protocol is simple, taking fewer than three hours to complete, thus allowing for easy adoption, decreased variability and results in half the time of ELISA. In addition to offering time and cost savings, Alpha provides 10-100-fold wider dynamic range than ELISA allowing a higher level of sensitivity. Washing and separation steps are not required providing more robust and reproducible results. The required sample size is scalable and typically only 1-5μL are required, thus conserving precious sample volume while allowing multiple analyses and extensive target data. DELFIA (Dissociation-Enhanced Lanthanide Fluorescent Immunoassay) is a robust, high-performance immunodetection platform that utilises the unique chemical properties of lanthanide chelates in concert with time-resolved fluorescence (TRF) detection to offer higher sensitivity (femtogram-level), two-fold wider dynamic range, superior stability and excellent flexibility versus traditional ELISA. ELAST signal amplification kits improve the sensitivity of any colorimetric ELISA by eight to 32- fold. Improvement is attained by the addition of a short incubation and wash step. ELAST is ideal for any challenging ELISA where detection is limited by low abundance of the target antigen or a weakly associated detection antibody. To complement its ELISA alternatives portfolio, PerkinElmer offers the EnSpire® multimode label-free plate reader with Alpha technology and automated liquid handling workstations (Figure 27). Quanterix (www.quanterix.com) is developing an ultra-sensitive platform capable of measuring individual proteins at concentrations 1,000 times lower than the best immunoassays available today. The Single Molecule Array (Simoa™) technology at the heart of this platform enables the detection and quantification of biomarkers previously difficult or impossible to measure, opening up new applications to address significant unmet needs in life science research, biopharma and in vitro diagnostics. The ultrasensitivity of Simoa assays sets it apart from all other immunoassays available today, offering PCR-like limits of detection with both existing and novel protein biomarkers. This sensitivity is made possible by obtaining digital measurements of bound immunocomplexes. Because the signal generation volume in a Simoa assay is two billion times smaller than the conventional ELISA, a single target molecule in a sealed microwell quickly generates enough fluorophores to be measured using conventional fluorescence imaging. When concentrations of the target analyte reach levels above which digital calculations are meaningful, the system’s proprietary algorithm converts to an analogue measurement, ensuring accuracy across a wide dynamic range (>4 logs). Simoa provides multiplexing options up to a 10-plex on a variety of analyte panels with sensitivity comparable to single-plex assays, while also maintaining excellent precision across a broad dynamic range. The Simoa HD-1 Analyzer has been designed to provide users with a completely automated experience to ensure consistent results and the greatest sensitivity possible. A robust menu of ultrasensitive assays will be available for proteins important in a variety of applications including inflammation, metabolism, neurology, oncology and cardiovascular disease (Figure 28). Siloam Biosciences’ (www.siloambio.com) Optimiser ™ microfluidic technology represents a major overhaul to microplate design (which has remained largely unchanged for more than five decades). Optimiser™ microplates (and companion OptiMax™ microplates for automation) use a ~5μL microfluidic reaction chamber to complete all the solid-phase immunoassay reactions. With an ANSI/SBS compliant 96-well architecture – Optimiser™ microplates are compatible with all conventional microplate liquid handling equipment and can even be read using conventional fluorescence readers. Scaling the immunoassay reaction down to a microfluidic scale delivers all the benefits of microfluidics to ELISA users. Optimiser™-based assays allow researchers to select for their desired assay output simply by adjusting sample size. All other steps in the assay protocol are fixed. Scientists can ‘tune’ their assays to select for higher assay sensitivity, low sample volume/high throughput; or select across a range of sensitivities, sample volumes and speed. For instance, a 5μL sample for the detection of IL-4 in a two-hour Optimiser™ assay yields similar sensitivity to standard plates using a 100μL sample in a ~6-8-hour assay. Alternately, adjusting sample volumes to 100μL (in aliquots of 5μL) results in a 3.5- hour Optimiser™ ELISA with femotgram level sensitivity. The use of yet more sample drives sensitivity down to the attogram level (~1,000-fold gain in sensitivity over the same assay in a 96-well microplate). Optimiser™-based ELISAs provide a significant improvement in performance and can allow researchers to expand on their research objectives while still utilising the well-established and reliable ELISA method (Figure 29). Singulex (www.singulex.com) has pioneered digital single-molecule counting (SMC) technology that can quantitatively measure proteins and metabolites directly in complex biological samples. Proprietary digital detection technology and high precision immunoassays are combined in the Erenna® Immunoassay System to provide higher sensitivity and broader dynamic range over traditional immunoassay platforms. The Erenna System utilises a bead suspension sandwich assay to directly capture the analyte, and the eluate is then read on a separate detection plate. This unique assay approach, along with Singulex’s proprietary Sgx link™ software algorithm, provides a lower background and increased slope, enabling quantification of low abundance biomarkers and monitoring of levels over time. Typical plate readers excite the bulk of a sample in a well and then attempt to measure the fluorescence in the entire well. This works well when the target concentration is high enough, but a deficiency arises at low concentrations, as the bulk sample has some intrinsic emission, and thus the sample is lost in the background. This inability to quantify low level samples makes it impossible to obtain baseline endogenous measurements or to stratify healthy and normal individuals, which is critical for many study designs. In a direct comparison with a traditional ELISA-based assay, Singulex single-molecule counting technology shows improved assay sensitivity. This head to head comparison demonstrates significant improvement in detection capabilities by migrating it to the Singulex high precision digital immunoassay platform (Figure 30). The ELISA demands of customers are driven by their needs for increasing productivity, sensitivity and quicker results. Tecan (www.tecan.com) has provided automated workstations for ELISA assay for many years, with the advantage of having liquid handling, plate washing and detection in the product portfolio which can be integrated with the powerful Freedom EVOware® software to provide a complete solution for variable ELISA assay throughput. A recent development has seen Tecan and Siloam Biosciences announce their partnership to automate low-volume, high-sensitivity microfluidic ELISA. The system is based on Tecan’s Freedom EVO® liquid handling platform and Siloam Biosciences’ OptiMax™ microplate. Together with Tecan’s fully integrated Infinite® M200 PRO reader this solution offers rapid, sensitive and specific chemi-fluorescence-based ELISA procedures using exceedingly small (~1.5μL) sample volumes. The speed, sensitivity and small sample requirements are achieved as a result of the unique microfluidic design of the Optimiser™ technology. Researchers and clinicians are also looking to increase sensitivity on existing ELISA platforms based on HRP amplification for Biomarker discovery and broadening the diagnostic window of clinical assays. To address this requirement Tecan collaborates with Sword Diagnostic in marketing a new ultrasensitive immunoassay detection platform exclusively for use on Tecan’s Infinite M1000 PRO and 200 PRO multimode readers in benchtop mode or fully integrated in a Tecan automated ELISA workstation. The novel Sword™ Peroxidase Assay platform provides improved performance with enhanced signal intensity, enabling improved sensitivity and precision in biomarker measurement. Researchers will appreciate the ease with which the Sword Peroxidase Assay substitutes into most existing protocols that use horseradish peroxidase (HRP) with minimal optimisation (Figure 31). Thermo Scientific Nunc Immobilizer Streptavidin plates (www.thermoscientific.com/dxplates) have Streptavidin covalently coupled to the surface via a polyethylene glycol spacer arm and are optimised for easy and sensitive detection of various biotinylated biomolecules such as oligonucleotides, peptides and proteins. Using biotinylated capture antibody and horseradish peroxidase (HRP) labelled detector antibody, human IgG in plasma samples is detected using a clear Immobilizer Streptavidin plate in a non-competitive sandwich ELISA. In this type of assay, the amount of bound detector antibody is proportional to the amount of analyte present in the sample. Bound HRP detector antibody is measured by incubating the wells with the substrate OPD and reading the resulting absorbance. The illustration (Figure 32) shows that an analyte can be successfully and easily quantitated via a sandwich immunoassay that employs a biotinylated capture antibody and a streptavidin coated plate. The signal is proportional to the amount of plasma added to the well. The controls indicate that the signal is specific. This assay is easy to perform, the plates were coated rapidly (one hour), and a low background was observed even though no specific blocking step was employed. Immobilizer Streptavidin plates provide a flexible and sensitive immunoassay platform. Immobilizer Streptavidin plates have the following specifications: clear 96- well plates, Streptavidin coated area 100μL/well, total binding capacity for biotin 5ng/well (20pmol/well), stable at room temperature for 18 months after manufacturing, co-efficient of variation (CV) <5% well-to-well. The binding capacity may vary depending on the biotinylated biomolecule being used. Thermo Scientific also offers 96- well plate sandwich ELISA kits, reagent kits and antibody pair kits to assay cytokines, growth factors and soluble receptors in serum, plasma, media and cell culture supernatant samples from human, mouse and other species. It developed matched pair antibodies to specifically ensure maximum sensitivity and specificity to the given target. Each kit comes with an Instruction Booklet to guide customers through the step-by-step protocol and how to achieve the best results possible. The specifications for each kit (specificity, assay range, precision, etc) are clearly described and include attractive figures of example standard curves. Coupling the quality and technical information of Thermo’s kits with its superior technical support service, customers who purchase its kits have the best opportunity to get sensitive and accurate results with their immunoassays. Choosing a kit on the Thermo Scientific ELISA Kit web pages (www.thermoscientific.com/ELISA) is simple as the content is organised by target name (eg, IL-6), then by species (human, mouse, etc) (Figure 33). Laboratories performing ELISA assays have relied on Titertek washer and dispenser technology for many years. Coating of microplates for ELISA at rates of 200 plates per hour is performed by very compact coating instruments which are easy to install and maintain. Zoom Washers automate the washing of 96 or 384-well microplates at highest speed. The MAP-C2 platform facilitates automation of the entire assay process, in batches of 50 microplates or more. Today, being part of the Titertek-Berthold group (www.titertek-berthold.com), the ELISA product portfolio benefits from additional R&D resources. The latest product launch is the Crocodile miniWorkstation for complete automation in smaller scale. Crocodile miniWorkstation provides walk-away automation of all steps of ELISA processing. Pipette standards, controls and samples, place the plate in the workstation, click start and come back to review results. It will follow assay procedure exactly, thus eliminating operator error and improving productivity. Crocodile combines five individual instruments in one 26.5 x 62 x 27cm housing. Use it as an assay processor, or as a washer, four-reagent dispenser, incubator/shaker or reader. It will deliver a superior performance in every mode. Crocodile is manufactured according to DIN ISO 13485 (Figure 34). Discussion - Enzyme-linked Immunosorbent Assays (ELISA) In putting together this review quite a large number of companies came forward indicating that ELISA and related immunoassays assays were still an area of significant new investment and perceived market opportunity. In Table 1 the ELISA product offerings of these companies are compared with respect to their provision or support of ELISA reagents, assays and kits; coated microplates; plate washers; assay detection; assay automation; and new and alternative ELISA formats. The table shows a large number of the vendors addressing the automation of ELISA assays. This automation ranges from simple workstations, to fully automated ELISA platforms that perform the entire process from start to finish (Agilent, Aurora Biomed, Bioscale, BioTek, Hudson, Tecan, Titertek-Berthold). Quite a number of these automated platforms include new or alternative ELISA formats (Bioscale, Dynex, Singulex), typically with a miniaturised plate (Douglas Scientific) or microfluidic components (Bioscale, CyVek, Gyros, Quanterix, Siloam Biosciences). Some of the automated instrumentation described is designed to support the creation/coating of ELISA assay plates (Biosero, Titertek-Berthold) or contains cartridgebased delivery of assay reagents (Bioscale, Dynex, CyVek). New developments in automated plate washing also impact conventional ELISAs (BioTek, Hudson Robotics, Matrical, Titertek- Berthold), as does the reduction/removal of the washing requirement of some new ELISAs (Douglas Scientific, eBioscience, PerkinElmer). Big increases in assay sensitivity and broader dynamic range over traditional ELISAs are attained by using alternatives to colorimetric detection, or the use of new platform technologies (Abcam, Bioscale, Douglas Scientific, Life Technologies, Mercodia, PerkinElmer, Quanterix, Siloam Biosciences, Singulex). Several of the new offerings utilise bead or particle-based components (Bioscale, Dynex, Gyros, Quanterix, Singulex) some of which are used to facilitate multiplexing. Others achieve multiplexing through use of different detection technologies (Abcam, Molecular Devices). These are supported by an ever-expanding range of ELISA reagents, assay and kits providing relative profiling or absolute quantitation of proteins from a wide range of study areas (Abcam, Dynex, Gyros, Life Technologies, Mercodia, PerkinElmer, Quanterix, Siloam Biosciences, Tecan, Thermo Scientific). In summary, significant new innovations have recently been, and continue to be, applied to traditional ELISAs and related immunoassays that have greatly extended assay sensitivity, its range of capabilities and suitability to enable automated processing. When coupled with the breadth of existing global expertise in ELISA it easy to see why the approach still remains the methodology of choice in those labs undertaking analyte detection. --- This article originally featured in the DDW Fall 2012 Issue --- Dr John Comley is Managing Director of HTStec Limited, an independent market research consultancy whose focus is on assisting clients delivering novel enabling platform technologies (liquid handling, laboratory automation, detection instrumentation and assay reagent technologies) to drug discovery and the life sciences. Since its formation nine years ago, HTStec has published more than 80 market reports on enabling technologies and Dr Comley has authored more than 40 review articles in Drug Discovery World. Please contact info@htstec.com for more information about HTStec reports. References 1 ELISA Assay Trends 2012 Report, published by HTStec Limited, Cambridge, UK, June 2012. 2 Wang, L et al (2012). Persistence of prolonged Cpeptide production in type 1 diabetes as measured with an ultrasensitive C-peptide assay. Diabetes Care 35(3):465-70.