When a researcher mentions that they’ve used an immunoassay to generate data, they may mean any experimental technique that relies on antibodies. In practice, the term immunoassay typically refers to a sub-set of antibody-based tests used to identify the presence and quantify the concentration of a target antigen. This sub-set is ELISA, an application that is becoming increasingly sophisticated through the use of advanced capture reagents and reporter systems. Yet despite improved levels of sensitivity and specificity, it’s vital to remember the basics—the measured analyte should be fit-for-purpose, and the immunoassay designed to monitor it should be both robust and highly characterized.
High-quality antibodies are essential
“The most important attributes of any assay are accuracy and precision such that there is a high degree of confidence in the data,” explains Peter Banks, scientific director at BioTek Instruments. “In the case of immunoassays, the antibodies are the main determinants of performance and should have high affinity to and selectivity for the analyte of interest. These two measures will help ensure accuracy and minimize any systematic error associated with antibody cross-reactivity.”
Adding that antibody availability can play a key role in dictating the design of an immunoassay, Banks notes that larger analytes are considerably more amenable to different assay formats. “If the analyte is a small compound such as a drug of abuse, a competitive immunoassay is required,” he says. “When the analyte is a larger biomolecule like a protein, the format can be more flexible. A sandwich immunoassay tends to provide improved sensitivity and selectivity relative to a competitive assay, yet this requires that antibodies to two different epitopes of the analyte are available.”
“Unfortunately, it can be very difficult to establish good antibody pairs from existing clones that have been selected for applications other than ELISA,” notes James Murray, Ph.D., director of immunoassay development at Abcam. “This is one of the greatest challenges researchers may face in developing an immunoassay in-house—rarely are available antibodies created specifically for sandwich ELISA. By selecting at the hybridoma stage antibody clones that work well in this format, we can subsequently validate the best antibody pairs for robust and accurate quantification of a target analyte in a variety of native biological samples.”
Immunoassay validation can be complex
The process of validating antibodies for ELISA considers many critical factors. “Specificity, sensitivity, and range are easily determined by measurement using samples containing a known concentration of the analyte, and use of selected positive, negative, and related control antibodies,” says Murray. “Recovery and linearity of dilution—measurements that demonstrate continued accuracy when interfering substances are present—are also key, especially when dealing with complex samples such as biofluids. These can contain varying amounts of unrelated substances that may skew assay results.”
Also noting that dilutional linearity and matrix effects represent key immunoassay design challenges, David P. Chimento, Ph.D., VP client solutions at Rockland Immunochemicals, explains that, if overlooked, these sample dilution issues can cause poor or non-functioning downstream assay performance. “A false negative or false positive result can have serious implications depending on the setting,” he says. “A poorly designed, ill-validated immunoassay will provide inconsistent results and may generate data that can be misinterpreted.”
Banks adds that other metrics that may be tested when developing an immunoassay include limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ). Epitope mapping and Levey-Jennings/Shewhart QC trending may also be employed. It is likewise important to decide upon direct or indirect detection, and to determine whether a homogeneous or heterogeneous format is best suited to the analyte in question. With so many different factors to consider, it’s perhaps unsurprising that researchers often choose to purchase off-the-shelf kits rather than designing and validating their own immunoassays in-house.
Off-the-shelf kits can deliver huge savings
According to Lizabethann Nolff, product development manager at Cayman Chemical, researchers may save thousands of dollars and months of time by sourcing fully validated, fit-for-purpose immunoassay kits. “Researchers are looking for sensitivity, reproducibility, use validation, lot-to-lot consistency, and efficient time-to-answer, all of which can be delivered commercially,” she says. “A good immunoassay kit will have been thoroughly characterized using the correct validation experiments to showcase key assay applications. It will also demonstrate performance in the desired sample matrices.”
Highlighting as an example Cayman Chemical’s citrullinated histone H3 (clone 11D3) ELISA kit, which was developed recently to analyze a novel target, Nolff describes the immunoassay design and validation process. “Since non-specific binding could potentially skew assay results, we explored various ways to mitigate this,” she says. “To verify our efforts, we reached out to researchers in the field to beta-test the kit on relevant samples. The aim was to ensure assay functionality and sensitivity met their needs. Following production of the initial large-scale batch, our QC team performed thorough functional validation, including monitoring for intra- and inter-assay precision utilizing matrix controls, analysis of lot-to-lot consistency, and sensitivity determination. This delivered a robust, relevant, and highly characterized product endorsed by researchers.”
“An off-the-shelf assay can be a tremendously cost-effective aid to a researcher looking to quantify a common analyte,” says Chimento, “yet with such a diverse set of end-users, these products must often deliver on very different objectives. In developing our BioQuantiPro™ CHO-HCP ELISA kit to support in-process testing of biopharmaceuticals during manufacture, it was abundantly clear to us that each user has a unique process with different sample histories and conditions. The challenge in assay qualification was to collect enough sample material from multiple end-users to demonstrate robust functionality under these various conditions.”
Murray notes that researchers in the discovery phase of a project often require a rapid, reliable answer to a specific question, and will typically purchase a ready-made immunoassay kit to provide this. “Further downstream, it may be desirable to build a more precisely tailored solution for an ongoing long-term requirement,” he says. “Our SimpleStep ELISA® kits employ our matched antibody pairs in a semi-homogeneous format to reduce assay times to just 90 minutes, swiftly delivering the answers researchers need, yet the paired antibodies can also be purchased separately for further immunoassay development.”
Image: SimpleStep ELISA™ kits streamline the sandwich ELISA process by using a semi-homogeneous format. Image courtesy of Abcam.
Future challenges
With time-to-answer a critical factor for many research groups, Nolff believes shorter, less labor-intensive protocols will become increasingly popular. Banks adds that BioTek is facilitating this through advanced liquid-handling automation. This includes microplate readers, liquid-handling devices, and stackers that link these together for sequential processing of up to 50 microplates at a time.
“Technology developments in sample processing, automated instrumentation, and improved detection systems will all make existing antibody performance better,” says Chimento. “The Quanterix system is a good recent example of this, whereby the performance of existing antibodies has been increased to the highest levels of sensitivity by improving several assay steps. Furthermore, the technology to produce and screen for high-end therapeutic antibodies is becoming economical for development of research antibody reagents. This should also contribute to better immunoassay performance.”
“The next challenge will be not how you collect immunoassay data, but how you analyze it,” notes Murray. “With the fields of AI and bioinformatics advancing, new approaches will help researchers more easily understand the immense amount of data they have available to them. It should not, however, be overlooked that appropriate immunoassay design and thorough validation are key to generating that data in the first place.”