Choosing an Immunoassay

Immunoassays have different capabilities depending on the format that is chosen. This article offers guidance for selecting an immunoassay that best meets your experimental needs.

Essential tools for cellular and molecular biology

Immunoassays are a mainstay of scientific research, enabling the detection and/or quantification of many different biomolecules. Over the years, established methods like western blot, ELISA, and flow cytometry have been augmented by newer techniques, including approaches based on antibody proximity. One such example, known as a Lumit^® Immunoassay, uses antibodies linked to complementary fragments of the enzyme NanoBiT^® Luciferase.

“When the antibodies bind to their target protein, the NanoBiT fragments come into close proximity, forming an active luciferase enzyme that produces a luminescent signal in the presence of the substrate furimazine,” explains Hicham Zegzouti, Ph.D., Senior Research Scientist at Promega. “Because Lumit Immunoassays are homogeneous, they are both easier and faster than conventional ELISAs.”

Other available methods include mass cytometry, bead-based assays such as Luminex xMAP^® Technology, and label-free techniques like surface plasmon resonance (SPR). Deciding which immunoassay to choose depends on a multitude of factors.

General considerations for immunoassay selection

“One of the first things to consider when selecting an immunoassay is the sample type, since this will dictate which immunoassays you can perform,” explains Amber Miller, Ph.D., Flow Cytometry Scientist and IHC Supervisor at Bethyl Laboratories (a Fortis Life Sciences brand). “For example, if you have tissue, you could 1. generate lysate to perform assays like western blot or immunoprecipitation, 2. make sections for IHC or mass cytometry, 3. form a single-cell suspension to perform flow cytometry, or 4. culture the tissue and collect supernatant for ELISA. But, for other sample types, not all of these options may be feasible.”

“You will also want to think about the answer you are looking to get,” adds Jessica Tracy, Ph.D., Manager IP/Western blot Antibody Validation at Bethyl Laboratories. “Are you investigating one protein or multiple proteins? Do you want to know abundance or location? Are you trying to study interacting partners? Do you have an idea who those partners are or are you trying to identify them? Each immunoassay is suited for answering questions in specific ways, often using different types and amounts of starting material. Understanding these caveats will increase your likelihood for success when running the assay.”

Adam Buckle, Ph.D., Chief Scientific Officer at Arrayjet, notes that a further important consideration is the complexity of the immunoassay, not only in terms of how easy it is to perform and optimize, but also when it comes to making sense of the results. “Modern immunoassays can produce a huge amount of data that can be challenging to analyze and interpret without specialist knowledge,” he says. “This is certainly worth bearing in mind for immunoassay selection.”

Key immunoassay capabilities

Once you’ve established your sample type and the general aims of your research, you will want to delve further into the capabilities of different immunoassays. This should include investigating the following:

• Sensitivity

Sensitivity refers to the lowest concentration of an analyte that can be accurately differentiated from background. “Immunoassay sensitivity is important for detecting low abundance targets,” says Vanitha Margan, Global Product Manager, Immunoassays, at Bio-Rad Laboratories. “Techniques that are known for their excellent sensitivity include ELISA, especially the highly sensitive variants like chemiluminescent ELISA or ultra-sensitive ELISA, as well as xMAP bead-based assays, fluorescence-based assays, and surface plasmon resonance (SPR).” Buckle agrees, commenting that Arrayjet favors fluorescence-based approaches for its antibody/protein microarrays, as when coupled with modern imaging technologies they offer very high levels of sensitivity.

• Type of readout—quantitative, semi-quantitative or qualitative

The type of readout determines the type of information obtained, which could be a precise analyte concentration measurement (quantitative), an approximation (semi-quantitative), or a simple presence/absence result (qualitative). “For quantitative data, techniques such as ELISA, SPR, xMAP assays, and mass cytometry are widely used,” says Zegzouti. “Additionally, Lumit Immunoassays can provide quantitative results.” Techniques that are considered semi-quantitative or qualitative include western blot, IHC, and lateral flow immunoassays.

• Throughput and compatibility with automation

“In many application areas, especially in high-throughput screening for research and clinical diagnostics, the ability to process a large number of samples efficiently is critical,” says Margan. “Here, automation helps streamline the workflow, minimizes human error, and increases reproducibility. Assays offering high throughput include xMAP multiplex assays and microarray-based assays. ELISA can be easily automated using robotic liquid-handling systems.”

“Of all the methods discussed so far, microarrays are the option capable of processing the highest number of samples in a simple and automated manner,” reports Buckle. “At Arrayjet, we have applied our microarraying technology to ELISA miniaturization, such that the core benefits of the well-established ELISA method are kept, but the throughput is massively improved. This promises significant cost savings, which can be further enhanced with multiplexed detection.”

• Capacity for multiplexed detection

Multiplexing saves time, reduces sample volume requirements, and provides more comprehensive information than single-analyte detection. “This makes it particularly useful for biomarker discovery, disease profiling, and drug development,” notes Margan. xMAP Technology is synonymous with multiplexing, allowing for as many as 500 different analytes to be detected simultaneously, and antibody/protein microarrays can often detect an even greater number of targets. However, almost any immunoassay technique can be multiplexed to some extent, with western blot limited to 2–3 targets, flow cytometry routinely used for studying 10–15 markers, and mass cytometry capable of detecting 50 or more analytes.

• Tips for immunoassay selection

Before finalizing your immunoassay selection, heed our experts’ advice. “My number one tip is to thoroughly define the specific goals and limitations of your experiment,” says Zegzouti. “Understand what you need to measure in what sample type, the required sensitivity and specificity, and any logistical constraints such as time, cost and available reagents and equipment.”

“Identify the types of data you need to answer your scientific questions,” advises Miller. “This will help you to not only design appropriate experiments but feel confident that you will have the information you need to progress your projects forward. Also, try to avoid choosing an assay just because it is the hot new thing—make sure that it will do what you require.”

“Determine the application of the research and the criteria for your results,” recommends Margan. “For example, if you are engaged in biomarker discovery and you need quantitative measurements, high sensitivity to detect proteins in low abundance, reproducibility, high throughput, and preferably multiple analyte detectability, then xMAP assays can be a great fit.”

“Always use high-quality, well-validated antibodies from trusted suppliers,” says Buckle. “Antibodies have certainly improved over the last decade, with curated antibody databases like The Human Protein Atlas, Antibodypedia, and Biocompare giving researchers more confidence in selecting an appropriate product.”

Lastly, Tracy stresses the importance of familiarizing yourself with each assay’s pros and cons. “When you understand the strengths, weaknesses, and limits of each assay, you can determine which one best meets your experimental needs,” she says. “Choosing the wrong immunoassay can result in incorrect data interpretation and will waste both time and money.”

Factors to consider for immunoassay selection

• Sample type
• The answer you are looking to get
• Complexity of the output data
• Sensitivity
• Type of readout—quantitative, semi-quantitative or qualitative
• Throughput and compatibility with automation
• Capacity for multiplexing
• Specificity of the antibodies used
• Reproducibility
• Continuous availability of the antibodies or immunoassay kit
• Dynamic range
• Time-to-result
• Ease of use
• Cost, time, and labor involved
• Need for specialist equipment