25 November 2025

ELISPOT Assay: From ELISA To Single-Cell Immune Monitoring

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The enzyme-linked immunospot (ELISPOT) assay, first described in 1983, is a powerful evolution of the classic ELISA immunoassay. While ELISA measures bulk cytokine or antibody levels in a sample, ELISPOT detects and quantifies individual cytokine‑secreting T cells or antibody‑secreting B cells at the single‑cell level.

Because plate‑bound antibodies capture proteins immediately as they are secreted, ELISPOT offers a sensitivity that surpasses conventional ELISA and intracellular cytokine staining. Rare antigen‑specific cells can be detected even at frequencies as low as 1 in 100,000 cells, making ELISPOT a preferred read‑out in many immunology workflows.

Today, ELISPOT assays are widely used in:

• Autoimmune disease research
• Cancer immunology and immunotherapy
• Infectious diseases (e.g. viral and bacterial pathogens)
• Transplantation immunology
• Vaccine development and evaluation

For researchers already using ELISA assays, ELISPOT is a natural next step when more granular, single-cell information is needed.

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From Red To Blue: A Smarter ELISPOT Staining System

In 2025, U-CyTech transitioned from the traditional red ELISPOT staining to a blue staining ELISPOT system in our portfolio of U-CyTech biosciences kits. This is more than just a cosmetic change.

The new blue ELISPOT staining offers:

• Less toxic reagents, providing a safer assay workflow in the lab
• Faster assay performance, helping you save time and energy without compromising sensitivity

For laboratories dependent on robust, reproducible ELISPOT read‑outs, this upgrade combines improved safety with streamlined processing, while maintaining the high quality and specificity associated with U-CyTech ELISPOT assays.

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T Cell ELISPOT Assay: High-Sensitivity Cytokine Profiling

The T cell ELISPOT assay is a highly sensitive method for detecting individual T cells that secrete specific cytokines or cytotoxic effector molecules. By capturing secreted proteins directly around each cell, the assay allows precise quantification of antigen‑specific T cell responses.

U-CyTech T Cell ELISPOT Kits

U-CyTech biosciences offers T cell ELISPOT kits for:

These kits cover a broad panel of cytokines and effector molecules relevant to cellular immunity, including:

• IFN‑γ
• IL‑2, IL‑4, IL‑10, IL‑17A, IL‑21
• GM‑CSF
• Granzyme B
• TNF‑α

The 2‑ and 5‑plate kit formats include all necessary reagents for blue spot detection, simplifying implementation in both routine and high‑throughput settings.

Monkey ELISPOT kits are validated for both Old World and New World species, including rhesus macaques, pig‑tailed macaques, cynomolgus monkeys, baboons and marmosets, making them suitable for preclinical and translational studies.

Why Choose ELISPOT Over Traditional T Cell Assays?

Compared with mixed lymphocyte reactions (MLR), cytotoxic T lymphocyte (CTL) assays or RT‑PCR:

• ELISPOT avoids radioactivity
• Measures secreted proteins directly, rather than mRNA levels
• Offers single‑cell resolution and high sensitivity

This makes the T cell ELISPOT assay an excellent choice for:

• Vaccine response monitoring
• Cancer immunotherapy development
• Longitudinal follow‑up of T cell immunity in clinical trials

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B Cell ELISPOT Assay: Quantifying Memory B Cell Immunity

While ELISA remains a standard method to quantify serum antibody levels, it does not reveal how many antigen‑specific memory B cells or plasma cells underlie the response. Long‑lived memory B cells and plasma cells are responsible for durable humoral immunity after vaccination or infection.

The B cell ELISPOT assay fills this gap by directly quantifying single antibody‑secreting cells (ASCs) and activated memory B cells.

U-CyTech B Cell ELISPOT Kits

U-CyTech biosciences provides B cell ELISPOT kits for:

These kits are designed to detect ASCs in blood or tissue samples such as peripheral blood, lymph nodes and spleen.

When To Use B Cell ELISPOT Instead Of ELISA

Because memory B cells are typically quiescent and do not secrete antibodies under resting conditions, their presence cannot be measured by ELISA alone. In contrast, the B cell ELISPOT assay:

• Activates antigen‑specific memory B cells ex vivo through a 2–5 day polyclonal stimulation
• Allows expansion and subsequent detection of these cells at the single‑cell level
• Provides a more direct correlate of long‑term, vaccine‑induced protection

B cell ELISPOT is particularly useful in:

• Vaccine development and optimization
• Infection research (e.g. herpes zoster, COVID‑19, hepatitis B)
• Drug treatment studies
• Autoimmune disease and allergy research

B Cell ELISPOT – Assay I vs Assay II

The B cell ELISPOT assay is performed on plates coated either with:

• Assay I: antibodies directed against an immunoglobulin class (e.g. IgG) to quantify total ASCs
• Assay II: antigen of interest to quantify antigen‑specific ASCs

After incubation of activated B cells in the coated wells, secreted antibodies are captured. Cells are washed away, and spots are developed using biotinylated anti‑immunoglobulin detection antibodies and enzyme‑labeled streptavidin, followed by substrate addition.

The resulting colored spots correspond to individual antibody‑secreting cells. By running Assay I and Assay II in parallel and comparing total ASCs to antigen‑specific ASCs, researchers obtain an accurate estimate of the frequency of antigen‑specific memory B cells.

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FluoroSpot Assay: Dual Cytokine Detection At Single-Cell Level

For researchers who need to dissect complex T cell functional profiles, the FluoroSpot assay offers an advanced alternative to conventional ELISPOT. This dual‑color assay enables simultaneous detection of two cytokines secreted by the same cell using fluorophore‑labeled antibodies.

Advantages Of FluoroSpot

• Simultaneous measurement of two cytokines at the single‑cell level
• Higher information content per sample, ideal when PBMC availability is limited
• Highly suitable for clinical trials and longitudinal immune monitoring
• Enables characterization of T cell subsets based on distinct cytokine profiles

In immunology studies, FluoroSpot is particularly attractive for:

• Detailed vaccine response profiling
• Studies in autoimmune diseases (e.g. type 1 diabetes)
• Research in both immunized and non‑immunized individuals

U-CyTech FluoroSpot Kits

U-CyTech biosciences offers a broad panel of human and monkey FluoroSpot kits, including dual detection of IFN‑γ with a secondary cytokine such as:

• IL‑2
• IL‑10
• IL‑13
• IL‑17A

Monkey FluoroSpot kits are designed to cross‑react with multiple macaque and baboon species. Each kit includes:

• All required reagents
• PVDF membrane‑bottomed plates optimized for FluoroSpot assays

In the FluoroSpot read‑out, green and red fluorescent spots indicate cells producing individual cytokines, whereas yellow spots identify cells producing both cytokines simultaneously.

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Why Choose U-CyTech ELISPOT & FluoroSpot Kits For Your Research?

For laboratories already working with ELISA or considering upgrading to single‑cell immune assays, U-CyTech ELISPOT and FluoroSpot kits offer a reliable and user‑friendly solution:

• High sensitivity for detecting rare antigen‑specific T and B cells
• Validated species coverage (human, monkey, mouse, rat)
• Comprehensive cytokine and effector molecule panels
• Safer, faster blue ELISPOT staining system from October 2025
• Ready‑to‑use kit formats with all essential reagents included

Whether you are working in vaccine development, oncology, infectious disease, autoimmunity or transplantation research, ELISPOT and FluoroSpot assays provide the level of resolution required to understand functional immune responses.