When it comes to measuring proteins such as cytokines, antibodies, or growth factors, there’s no shortage of options. ELISA, ELISpot, and FluoroSpot all have important roles to play. While each assay relies on antibody-based detection, knowing which one to use (and when) can mean the difference between simply collecting data and gaining real insight.

As a Product Manager at Millennium Science, I speak with researchers every day who are deciding between these techniques. In this blog we will explore each assay to help you spot the difference and help you choose the right assay for your next experiment.

ELISA (Enzyme-Linked Immunosorbent Assay) is one of the most widely used immunoassays for detecting and quantifying soluble proteins, such as cytokines, antibodies, and hormones, in serum, plasma, or cell culture supernatants. It’s robust, scalable, and ideal for high-throughput analysis when you need accurate, reproducible concentration data.

How it works (Sandwich ELISA):
A capture antibody is coated onto a high-binding plate and binds the target protein in your sample. A biotinylated detection antibody binds a different epitope, followed by a streptavidin–enzyme conjugate. Addition of a colourimetric substrate produces a measurable signal proportional to protein concentration.

Why researchers choose it:

• Delivers quantitative results (e.g., pg/ml or ng/ml)
• Compatible with high-throughput and automation
• Well-established, with widely available equipment
• Ideal for comparative analysis across multiple samples

Limitations:

• No information on the number or type of cells producing the protein
• Sensitivity can be lower than single-cell assays

ELISpot (Enzyme-Linked ImmunoSpot) detects and counts cells that secrete a specific protein. It’s particularly valuable when studying immune responses where the frequency of antigen-specific T or B cells is low – for example in vaccine research, oncology, and autoimmune disease studies.

How it works:
Live immune cells are added to a PVDF plate pre-coated with a capture antibody. When stimulated, the cells secrete the target protein, which is immediately bound near the cell. After washing away cells, a detection antibody and enzyme conjugate are added. A precipitating substrate forms a visible spot at each secretion site – each spot representing a single responding cell.

Why it stands out:

• Extremely sensitive – can detect one responder cell in >100,000
• Functional readout of immune activity
• Ideal for rare antigen-specific responses
• Widely used in T-cell response monitoring, vaccine trials, allergy research, and immuno-oncology

Pairing with Mabtech’s ASTOR2 automated ELISpot reader delivers rapid, high-precision spot counting with consistent, reproducible results across entire assay plates, and when used with Mabtech monoclonal antibody pairs and ready-to-use ELISpot kits, ensures reliable performance across species and sample types.

FluoroSpot builds on the ELISpot principle but uses fluorescently labelled detection antibodies to identify multiple proteins secreted by the same cell – typically 2-4 proteins. This allows you to measure not just whether a cell responds, but how it responds.

Why it’s powerful:

• Detects polyfunctionality – cells secreting multiple cytokines
• High sensitivity with low background
• Enables simultaneous detection of different functional subsets
• Excellent for vaccine research, infectious disease, and cancer immunotherapy

Pairing with Mabtech’s IRIS2 ELISpot, FluoroSpot and FociSpot reader enables precise, high-resolution spot detection across multiple fluorescence channels.

• ELISA → When you want to know how much protein is present in a sample.
• ELISpot → When you need to know how many cells are producing a protein.
• FluoroSpot → When you need to know which protein a cell is producing, and whether it’s producing more than one at the same time.

In many projects, using ELISA and ELISpot/FluoroSpot together can give the clearest picture — combining quantitative bulk measurements with functional single-cell insights.

If you’d like help selecting the right assay for your research, get in touch with our team at Millennium Science. We’re local experts, and Mabtech’s range covers everything from research use to clinical-grade applications.

Until next time… happy experimenting!

Joe Roberts, PhD
Product Manager
Millennium Science

Hi, I’m Joe Roberts, PhD – Product Manager here at Millennium Science, immunologist at heart, and someone who’s passionate about helping researchers get the most out of their tools, assays and experimental design. My career has taken me through PhD, post-doctoral, and senior scientist roles, including roles within flow cytometry core facilities across the UK, Ireland and Australia. That journey has shaped a deep appreciation for the technologies that enable great science.

Here at Millennium Science, our mission is to ignite life science discovery with revolutionary technology!

This blog series – which I’m calling Joe Blogs – is my way of sharing insights, practical tips, and a few behind-the-scenes stories from the technologies I work with every day. From immunoassays and cell-based analysis to real-time monitoring and flow cytometry, I’ll be unpacking the platforms and reagents that drive discovery across Australia and New Zealand’s life science community.

And here’s the best part: Joe Blogs will land every fortnight on Monday morning – your dose of lab-friendly advice to start the research week right!

Whether you’re running your first experiment on a new platform or refining an advanced workflow, Joe Blogs is designed to help you:
✅ Understand what your instruments can (and can’t) do
✅ Compare assay options with clarity and context
✅ Get practical insights from someone who’s been there
✅ Stay on top of emerging techniques and technologies

Think of it as a conversation, where we talk shop about the platforms you’re using, the questions you’re asking, and the science you’re working to advance.

Here’s a taste of what’s in the pipeline:

• Spot the Difference: ELISpot vs FluoroSpot vs ELISA
• Real-Time Results: What xCELLigence Really Tells You About Cell Health
• NovoCyte or NovoCyte Opteon? Choosing the Right Flow Cytometer
• Setting Up Your First ELISpot Assay: Tips for Success from Day One

Got a burning question, a technical challenge, or a topic you’d love to see unpacked in a future post?
Let me know! I’d really like this blog to reflect the real conversations and challenges happening across our local research community.

Whether it’s about assay design, data interpretation, instrument comparisons or workflow optimisation, your ideas will help shape future editions of Joe Blogs. You can reach me directly via email or through the team at Millennium Science.

jroberts@mscience.com.au
customerservice@mscience.com.au

As part of Millennium Science, we’re proud to partner with leading life science suppliers to bring world-class tools to Australian and New Zealand researchers, with the local support to match. My goal with Joe Blogs is to give you a bit of that support upfront: advice, clarity, and maybe even a shortcut or two.

So, whether you’re measuring T cell responses, analysing cell proliferation, or exploring new approaches to data-rich experiments, you’re in the right place.

Until next time…happy experimenting!