ELISpot Assay Kits

ELISpot kits are sensitive assays for the detection and visualisation of cytokine or antibody secreting cells at the single-cell level. The ELISA Genie ELISpot assays are highly sensitive and therefore useful for detecting low frequency cytokine-secreting cells or antibody-secreting cells in vitro.

What is an ELISpot assay?

The enzyme-linked immunospot (ELISpot) assay is a universal method for monitoring immune responses in vitro. ELISpot assay is an effective immunoassay tool due to its high sensitivity. It is used for the ex vivo measurement of cytokine or antibody secreting cells at the single-cell level following activation with a suitable stimulus in vitro. The ELISpot assay allows for the visualization of the secretory product of individually activated or responding cells. Each cell can be detected by the assay as long as a characteristic protein is released and specific high affinity antibodies recognizing the protein are available.

The cells of interest are both cultured and activated on plates immobilised with antibodies that capture the associated target analytes released by the stimulated cells. Following target analyte capture, the presence and distribution of these molecules are detected through a sandwich ELISA. A reporter enzyme is added where the analyte is captured. Each spot that develops in the assay represents a single reactive cell.

As a result, ELISpot assay gives both:

    1. Qualitative analysis, by providing the type of immune proteins present.
    2. Quantitative results, by the number of responding cells data.

ELISpot principle

In the ELISpot assay, cells are cultured on a 96-well plate coated with the appropriate capture antibody to the protein of interest, in the presence or absence of stimuli. Once the cells are stimulated, they release proteins (e.g. cytokines). These secreted proteins are then trapped by the immobilized antibodies coated on the surface. Following a period of incubation, the cells are washed away and the secreted proteins are detected using a detection antibody. The ELISpot detection process employs the same immunochemical 'sandwich' principle as an enzyme-linked immunosorbent assay (ELISA). In contrast to the ELISA method, ELISpot is a combination of both an immunoassay and bioassay. This is due to living cells being cultured directly within the wells of the ELISpot plate.

The detection antibody is typically a biotinylated polyclonal antibody specific for the analyte of interest. The biotinylated antibody is added to each of the wells and the wells are then washed to remove any unbound detection antibody. Next, a streptavidin-enzyme conjugate is added or the antibody is simply conjugated to the enzyme. Unbound streptavidin-enzyme is then washed away and a precipitating substrate is added. When a precipitating substrate is used instead of a soluble product, the final result appears as visible spots. Each spot corresponds to an individual cytokine or antibody-secreting cell. The resulting spots can be quantified using an automated ELISpot reader system or manually, using a stereomicroscope.

ELISpot Features & Benefits

Advantages Description

Highly Sensitive

ELISpot can allow for the detection of a single cell out of 100,000

Fast Procedure

Rapid detection time, easy to analyse ‘spots’

Accuracy & Reliability

Reagents validated according to ISO 9001:2000 quality systems

No cross reactivity

With other human cytokines tested

Inexpensive Analysis

ELISA Genie ELISpot does not require expensive equipment or analysis software

ELISpot Applications

ELISpot techniques is one of the most-sensitive methods available (up to 400x more sensitive than conventional ELISA).

  • ELISpot assays are quantitative and measure key cellular functions of both innate and adaptive immune cells.
  • ELISpot assays indicate the type of cytokine response that has occurred and therefore can be used to determine the immune response that has taken place.
  • An ELISpot assay is commonly used for
    • Vaccine development
    • Diagnostic and prognostic analysis of autoimmune diseases
    • Allergy research
    • Monocytes/macrophages/dendritic cells characterization.

ELISpot vs ELISA

ELISpot ELISA

Sensitivity

Highly sensitive (up to 400 times more sensitive)

Less sensitive

Available time for analysis/re-analysis

Weeks - months

Hours

Detection and Colour Reaction

In the cell culture (ELISpot) plate

In the supernatant in a separate ELISA plate

Data Analysis

Computerised visual analysis; one spot equals one cell

Standard curve to determine concentration

Result

Detection of a single cell that secrets a protein of interest

Total concentration of secreting cell proteins of interest

ELISpot Step-By-Step Guide

Figure 1: Schematic of ELISpot assay protocol

ELISpot protocol

Steps