Providing researchers with a convenient, off-the-shelf method for detecting a specific analyte, immunoassay kits have been used for decades to facilitate the generation of consistent and reproducible data with minimal up-front optimization. Yet as the demand to obtain more information from less sample volume continues to grow, along with the desire for greater ease of use and higher throughput, the technologies and applications of these products are becoming increasingly sophisticated.
“Researchers want to grab a kit and generate data,” says James Murray, director of immunoassay development at Abcam, “and since kits are essentially optimized assays they allow researchers to get straight to the science. The technologies employed within the immunoassay kits we make work on commonly used laboratory equipment, meaning that our products are instrument agnostic. This simplifies workflows and lets researchers use the tools they already have available to yield reproducible results on their platform of choice.”
Consistency matters
For singleplex experiments Abcam offer the SimpleStep ELISA® range of sandwich ELISA kits, while for multiplexing they provide FirePlex® and FirePlex®-HT. FirePlex immunoassays are run on flow cytometers, make use of 96 well plates, and provide analysis of up to 75 analytes from just 12.5 µL of sample. The high-throughput version, FirePlex-HT, runs on high content imagers, uses 384 well plates, and permits data collection at scan speeds of less than 20 minutes per plate. “By manufacturing fit-for-purpose antibody pairs and using the same pairs across all three platforms, it’s easy for researchers to move from a singleplex, low-throughput assay to a multiplex, high-throughput version, and vice versa,” notes Murray. “Furthermore, by using recombinant monoclonals wherever possible, our immunoassay kits provide unrivalled batch-to-batch consistency.”
Also keen to stress the importance of consistency when it comes to using off-the-shelf immunoassay kits, Jason Truskowski, director of marketing at Cayman Chemical, explains that for an end-user to build an immunoassay with the same level of validation that is adhered to by a specialist manufacturer could take months or even years. “Proper validation includes a full range of cross-reactivity testing, sample purification validation, sample parallelism from a variety of sample types, and, in some cases, correlation to LC-MS data,” he explains. “Every batch of reagents that is used to make up our ELISA kits undergoes rigorous testing to ensure both inter- and intra-lab reproducibility.”
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Although HRP has historically been the detection enzyme of choice for ELISAs, Cayman Chemical instead uses acetylcholinesterase in many of its immunoassay kits. “This offers several advantages,” says Truskowski. “Non-enzymatic hydrolysis of acetylthiocholine in buffer is essentially absent, yielding a lower background than the inherently unstable peroxidase substrate TMB. Also, in contrast to peroxidase, acetylcholinesterase is a completely stable enzyme so if a plate is accidentally splashed or dropped during development, it can easily be re-developed by washing and adding fresh Ellman's Reagent.”
Expert collaboration
By working in close collaboration with independent researchers, Hycult Biotech is able to have its ELISA kits tested externally prior to product launch. “Our focus is on innate immunity, with a specific interest in inflammation and cell- and tissue-damage,” says Kim Knol-Peeters, product manager. “We work with dedicated experts in, for example, the fields of antimicrobial peptides, complement, acute-phase proteins, TLR, LPS, and microbial toxins, and by pooling our knowledge we are able to develop and produce relevant, robust, and stable immunoassay kits for the wider scientific community.”
Hycult Biotech’s immunoassay kits have been extensively literature cited, with recent publications including a study that assessed whether the novel serological biomarker I-FABP could be used to improve the diagnostic accuracy of detecting acute mesenteric ischemia, and research to establish whether the presence of sCD59 in cerebrospinal fluid might have utility as a biomarker of demyelinating diseases such as multiple sclerosis. “Our immunoassay kits eliminate the need for researchers to spend valuable time identifying the right antibodies and proteins and then optimizing these components with the appropriate buffers, significantly speeding up the time to results,” notes Knol-Peeters.
Host cell proteins
The focus of Cygnus Technologies’ range of immunoassay kits is on bioprocess-related impurities, particularly the detection of host cell proteins (HCP), which are present in the different expression systems used to generate a biotherapeutic drug. “It is desirable to reduce HCP impurities to the lowest levels practical since they can result in adverse toxic or immunological reactions,” says Ken Hoffman, president. “We test the reactivity of the HCP antibodies with our immunoassay kits using a novel method, developed by and unique to Cygnus, known as Antibody Affinity Extraction (AAE).”
AAE is designed to evaluate antibodies in a more natural state than traditional techniques, providing a better indication of their coverage. “The comparison of duplicate gels by Western blot and silver stain has long been the method expected by regulatory agencies as part of an HCP ELISA validation package,” explains Hoffman. “Yet this is beset by problems such as epitope destruction by heat and chemical treatment, poor transfer, and the difficulty of aligning a silver stained gel with a Western blot membrane. During AAE, sample enrichment is performed by immobilizing the HCP antibody under study on a solid support, over which the HCP sample is extracted and eluted multiple times. Silver stained gels of unextracted total HCP and AAE HCP can subsequently be compared directly.”
Bridging genomic and proteomic tools
Launched toward the end of 2017, Thermo Fisher Scientific’s ProQuantum High Sensitivity Immunoassays leverage the use of oligonucleotides to provide detection and measurement of low-expressing proteins. Utilizing proximity ligation assay (PLA) technology, ProQuantum immunoassay kits provide a much larger dynamic range than an ELISA while consuming only a fraction of the sample material. They also have the additional advantage of being compatible with any qPCR instrument which, according to Dr. Thao Sebata, senior product manager, means that existing laboratory equipment can now be used for DNA, RNA, and protein measurement purposes.
“ProQuantum combines the analyte specificity of antibody–antigen binding with the signal detection and amplification capabilities of qPCR to achieve a highly sensitive assay,” says Sebata. “Although the system presently offers only single-target analysis, the technology is extremely powerful. Just 2 µL of serum sample can be used to measure up to five analytes.” While the range of detectable analytes currently consists of various human cytokine and chemokine proteins, rapid expansion is underway to keep up with customer demand for this exciting new technology. Thermo Fisher Scientific presented recently at AAI 2018 plans to include murine targets within the ProQuantum portfolio.
Image: ProQuantum immunoassays rely on antibody binding to the target, followed by ligation and signal amplification within a qPCR instrument. Image courtesy of Thermo Fisher Scientific.
Consensus regarding the future of immunoassay kits indicates that the focus will be to further increase sensitivity and reproducibility while minimizing sample volumes. This will, in part, be driven by the discovery of new biomarkers and emerging technologies, while the sample type and desired throughput will also influence the configuration of each new immunoassay kit. As manufacturers continue to develop better, faster, and more affordable tools for the research community, the number of researchers relying on these products is likely to grow accordingly.