ELISPOT Assays

ELISPOT Assays Visualize the Secretory Product of Individual Cells

"ELISPOT represents the most sensitive technology for the detection of immune cells secreting signature proteins, such as cytokines. It therefore represents the “state-of-the-art” for the detection, measurement, and functional analysis of immune cells."

ELISPOT assays measure the secretory activity of individual cells. The unique sensitivity of ELISPOT assays vs. supernatant-based measurements, (e.g ELISAs, protein/cytokine arrays, or cytokine bead arrays (CBAs) results from the fact that the analyte is captured directly around the secreting cell as it is being released, before it can be diluted in the supernatant, absorbed by receptors of adjacent cells, or degraded by proteases.

ELISPOT assays are also orders of magnitudes more sensitive for detecting rare cells than flow cytometry-based techniques such as tetramer or intracytoplasmic cytokine staining (ICS). Since antigen-specific lymphocytes frequently occur in PBMC in frequencies too low for detection by other techniques, ELISPOT assays have emerged as the most sensitive and robust technique for direct ex vivo monitoring of T and B cell immunity. By measuring the frequencies of the antigen-specific cells and the types of molecules these lymphocytes secrete, ELISPOT assays not only establish the magnitude (clonal size) but also the quality (effector class, e.g. Th1, Th2, Th3, etc.) of antigen-specific immunity.

Why ELISPOT

Accurate ex vivo frequency measurements down to the one-in-1,000,000 cell range.
This resolution is orders of magnitude higher than what can be reached with intra-cellular cytokine or tetramer staining or by ELISA measurements. Such added sensitivity is critical, since antigen-specific T cells typically occur in low frequencies in vivo.

High-throughput T cell analysis becomes feasible.

A trained team in a reference laboratory can test hundreds of samples for reactivity to dozens of antigens.

Fewer cells are required compared to other cellular assays.
For example, 45ml of blood is sufficient for testing reactivity to 600 different antigens/peptides. Determinants targeted by CD4 or CD8 cells can be defined. Because few cells are needed and the assay can be performed in high-throughput mode, our ELISPOT assays are ideal for screening peptide libraries for determinant mapping.

No Loss of Function
Cryopreserved human lymphocytes can be tested without loss of function. Pre- and post-treatment samples can be tested side-by-side and results can be reproduced.

CTL's founding scientists take pride in having established the technical and scientific foundations of cytokine ELISPOT analysis (see peer-reviewed publications listed by theme).

We have elevated ELISPOT to an exact science, transforming it to the robust and validated T cell diagnostic tool it is today. We would like to share this expertise with you.

Interested in learning more about Why ELISPOT? Download our ImmunoSpot brochure.

Why CTL

At the very heart of science are exact, objective and reproducible measurements (see figure 1). The precise evaluation of even such pristine ELISPOT results is impossible when the spots are counted visually, as shown in the lower right panel of the figure.

By introducing and patenting ELISPOT image analysis, we converted the previously time-consuming and subjective evaluation of ELISPOT results into an automated and objective process.

Figure 1: Improvement of IFN-ץ ELISPOT assay results using the ImmunoSpot® protocol. Freshly isolated peripheral blood mononuclear cells (PBMC) were cultured for 24h with media or the recall antigen tetanus toxoid (TT). Note the decreased background and increased signal with the ImmunoSpot® protocol ("New").

• Which cytokine spots are produced by antigen-specific T cells (that are to be measured) vs. those produced by bystander cells—being of no T cell diagnostic value?
• What do small vs. big spots mean?
• Where should the cutoffs be set for minimal/maximal spot size when counting and analyzing the different cytokine ELISPOT results?
• Are measurements made at single-cell resolution?
• In what frequency range are accurate measurements made?
• If there are antagonistic cytokines produced simultaneously, to what extent do they interfere with the results?
• How many replicates are required to produce significant results?

We Orchestrate the Largest Concentrated Effort Around ELISPOT

Starting from its CWRU-based academic roots, CTL has grown to become the world leader in cytokine ELISPOT assay development, automated image analysis, and data management. Over fifty employees at CTL dedicate their full-time efforts specifically to the ELISPOT technology. These efforts are complemented by over 15 academic scientists at CWRU and by close collaboration with many scientists worldwide.

CTL has been selected as the only ELISPOT reference laboratory by the Cancer Vaccine Consortium (CVC). Dozens of the finest immunology laboratories have entered collaborations contributing invaluable expertise. This alliance constitutes the largest group in the world to further the frontiers of ELISPOT technologies.

The ImmunoSpot® Product Line has Emerged as the Crystallization of Science

We provide scientifically-validated ELISPOT analysis equipment, including three analyzer models to meet different user needs and budgets, satellites, accessory software, and data management solutions. In addition, CTL offers image analysis services, ELISPOT Hands-on Workshops, image analysis training, and complete Contract Research Services for clinical trials. Join the community of those who do not compromise in elevating ELISPOT to an exact science!

CTL is the only entity in the world dedicated solely to All-around ELISPOT™!

ELISPOT vs Other Applications

Differences between ELISPOT assays and other approaches for measuring antigen-specific T cell immunity

Every naïve T cell expresses a unique T cell receptor (TCR) which is specific for a single antigen. In order to be able to recognize potential infinite numbers of microbial antigens and at the same time to distinguish them from self antigens, T cells system relies on a close to infinite number (~1012) of different possible T cell specificities.

Subsequently, the frequencies of T cells recognizing individual antigens are very low. As a result of the clonal expansion associated with infections, the frequency of antigen-specific effector cells can transiently rise to as high as 1:100, but these frequencies typically settle in the range of 1:10,000 for effector memory or memory cells. The frequencies of specific T cells in blood after immunizations with subunit vaccines (proteins/peptides), environmental sensitization and in long-term, anti-microbial immunity rarely exceed the 1:10,000 thresholds.

Therefore, techniques that measure T cell immunity need to be sensitive enough to reliably detect such rare cells. In addition to measuring the frequencies of antigen specific T cells, which delineates the magnitude of immunity, it is essential to understand the functions of these T cells. The quality of T cell immunity depends on whether these cells are Th1 or Th2 (Tc1/Tc2) type cells and whether they are capable of direct killing. Sophisticated assays therefore will also provide information on the effector lineage of the antigen-specific T cells.

Optimized ELISPOT assays measure the magnitude and the quality of T cell immunity at single-cell resolution by detecting individual events of antigen-specific T cells that engage in secretion of cytokines and effector molecules such as granzyme B and/or perforin. The following is a comparison of the performance and utility of ELISPOT assays with other T cell diagnostic techniques including bulk supernatant measurements, Tetramer/Pentamer, and ICS techniques.

Interested in learning more about the advantages and applications of ELISPOT?Contact us