Immunoassay Selection Guide

Single-analyte ELISAs are the immunoassay of choice in many instances due to their superior accuracy, sensitivity, specificity, and ability to withstand harsh pretreatments or buffers. One advantage of single-analyte ELISAs is the ability to customize the assay for the target analyte and not having to worry about interference caused by an abundance of other antibodies and proteins working together.

While ELISAs have long been the standard for quantitative analysis of cytokines and other biomarkers, they generally are not well-suited for high-throughput multi-protein analysis. That is where multiplex immunoassays can deliver substantial value.

Growing demand for immunoassays and spiraling testing volumes has put pressure on scientists to rein in costs and optimize sample usage by designing multiplex assays to cover several analytes simultaneously. Factor in the time saved by running the equivalent of 20 or more different assays in a single immunoassay workflow, and the benefits of multiplexing are obvious.

There are other advantages as well. Assessing multiple analytes in a single assay allows scientists to be judicious with precious sample materials while alleviating the need to prioritize assays based on the sample volume available, allowing for future studies using those same samples.

Multiplex assays allows you to:

• Optimize limited sample volumes—collect data from just 25μl or less of undiluted sample.
• Minimize experimental variability—derive data points from a single manipulation.
• Optimize productivity—minimize sample preparation and processing.
• Save time and resources—Cover several analytes simultaneously.

The demand for fast, inexpensive analysis of multiple analytes in a single assay is helping to drive new innovations in multiplex assay capabilities. Antibody arrays are a good example.

With these assays, specific capture antibodies are spotted in duplicate onto nitrocellulose membranes and a simple antibody sandwich assay principle is utilized. If your lab is set up for Western blotting, then you already have all the equipment you need.

Each array offers a quick and inexpensive qualitative analysis of up to 119 proteins in a single sample. Captured analyte is detected using a cocktail of biotinylated antibodies and positive spots are visualized using an appropriate detection system.

Key benefits include:

• Rapid sample screening
• Simple, easy to use 
• Large number of targets per array 
• No specialized equipment required 
• Choice of detection substrates

From clinical research to drug development, there is a growing need to accelerate multiplexing workflows while eliminating the potential for cross-reactivity and interference. That’s where microfluidics-based immunoassay platforms are playing a vital role.

These fully automated assays deliver the same specificity as single-analyte ELISAs, but with greater sensitivity and a broader dynamic range. Setting up an assay simply requires loading diluted samples into the cartridge. Once loaded, the instrument performs every step of the immunoassay automatically, from wash steps to final quantification of results in just 90 minutes. 

Whether you are starting with a multiplex assay or single analyte, today's researchers have a broad range of flexible options from which to choose. For new or novel targets, you can also have outside experts build custom multiplex panels to meet your specific research needs.   

The unique binding characteristics of the antibody is the central factor in determining performance within a given immunoassay. Screening antibody pairs for optimal performance is a time-intensive process that requires access to a library of antibodies and a great deal of research to confirm the antibodies under consideration can produce consistent results.

A good first step is making sure you select antibodies from a reputable supplier that has been demonstrated to be fit-for-purpose in the application and sample type of interest. Validation protocols can help ensure that antibodies are producing appropriate results.

While there is no one-size-fits-all approach to antibody validation, there are some best practice measures that can help improve your outcomes.

• Always follow the manufacturer’s recommendations.
• Use a relevant model system to test the antibody.
• Employ proper controls, both for the target and for the reagent.
• Compare your results to other similar antibodies if available.
• If something appears amiss, contact the supplier and share your data.