ELISA versus ELISpot: Which assay is right for your research?

Not sure whether to use ELISA or ELISpot? This guide breaks down the differences to help you select the most suitable assay for your project.

ELISA types

The four main types of ELISA are direct, indirect, sandwich, and competitive ELISA.

• Direct ELISA employs an enzyme-labeled primary antibody. This format offers a straightforward and fast workflow but typically has lower sensitivity due to limited signal amplification.
• Indirect ELISA enhances sensitivity by introducing a two-step detection system involving a primary antibody followed by an enzyme-labeled secondary antibody. This setup allows for signal amplification but may introduce cross-reactivity, especially when using a secondary antibody raised against the species of the primary antibody.
• Sandwich ELISA is highly specific and ideal for detecting analytes in complex samples. It relies on two antibodies: a capture antibody, coated to the plate bottom, that immobilizes the target antigen and a detection antibody that binds a different epitope on the same antigen. While it requires well-matched antibody pairs, its high specificity and sensitivity make it our preferred method, especially for quantifying low-abundance analytes in complex matrices.
• Competitive ELISA is best suited for small molecules or antigens with limited epitopes. In this format, sample antigen competes with a labeled antigen for antibody binding. It provides high sensitivity but involves a more intricate setup and inverse signal readout (i.e., signal decreases with increasing antigen concentration).

When using ELISA to detect immunoglobulins, the assay design should be tailored to the specific research question. Learn more.

In summary, each ELISA format has its strengths and limitations. Choosing the right type depends on your experimental needs and available reagents.

When to choose ELISA

ELISA is your go-to assay when your goal is to quantify the concentration of a specific protein in solution. Whether you're working with serum, plasma, cell culture supernatants, or other fluids, ELISA provides a reliable and well-established platform for detecting soluble analytes.

Choose ELISA when:

• You want quantitative data. (ELISA results come in units like pg/ml or ng/ml.)
• You're analyzing secreted proteins, such as cytokines, antibodies, or hormones.
• Your experiment involves comparative analysis across multiple samples, such as treatment groups or time points.
• You need a high-throughput, scalable solution with compatibility for automation.
• You’re working with limited or no access to live immune cells, or simply don't need cellular resolution.

Typical applications include:

• Profiling cytokine secretion in response to stimuli.
• Measuring antibody titers in infection or vaccine studies.
• Monitoring biomarkers in clinical samples.
• Validating protein expression levels in recombinant systems.

When to choose ELISpot

ELISpot is the method of choice when you need to evaluate functional immune responses at the single-cell level. It’s designed to detect and count individual cells that secrete a target protein, offering unmatched sensitivity for identifying rare responder cells.

Choose ELISpot when:

• You want to measure the number of protein-secreting cells (e.g., IFN-γ–producing T cells).
• You need to assess cell-mediated immunity, particularly in response to antigens or pathogens.
• Your study involves vaccine development, cancer immunotherapy, or T cell monitoring.
• You're working with PBMCs, isolated immune cells, or splenocytes from human or animal sources.
• You require high sensitivity.

Typical applications include:

• Monitoring T cell responses in vaccine trials.
• Evaluating immune activation in infectious disease or oncology studies.
• Tracking B cell antibody production at the single-cell level.
• Detecting rare immune events not visible in bulk protein analysis.

Practical consideration

When deciding between ELISA and ELISpot, it's also important to factor in practical aspects of your experiment: 

• Sample type
  ELISA requires a liquid sample (e.g., serum, plasma, or supernatant), making it a good fit when viable cells are unavailable. ELISpot, on the other hand, needs freshly isolated or properly frozen immune cells, which may limit its use depending on sample access.
• Sensitivity 
  ELISpot offers high resolution and can detect rare antigen-specific responses. ELISA is less sensitive but provides robust concentration data for proteins that are abundantly secreted.
• Readout equipment
  ELISA results are typically read on a standard plate reader, which is widely available in most labs. ELISpot requires a specialized plate reader and analysis software to detect and count individual spots (cells).
• Assay complexity
  ELISA protocols tend to be simpler, particularly when using pre-coated plates. ELISpot requires careful cell handling, making it slightly more technically demanding.
• Results
  ELISA gives you a quantitative concentration (e.g., pg/ml), while ELISpot gives you a number of secreting cells (e.g., SFU per 10⁶ cells). Think about which type of data best supports your experimental goals.