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 Protocol

1.

Add 100µl of PBS 1X to every well.

2.

Incubate plate at room temperature (RT) for 10 min.

3.

Empty the wells by flicking the plate over a sink & gently tapping on absorbent paper.

4.

Add 100µl of sample, positive and negative controls cell suspension to appropriate wells providing the required concentration of cells and stimulant.

5.

Cover the plate and incubate at 37°C in a CO2 incubator for an appropriate length of time (15-20 hours).

Note: do not agitate or move the plate during this incubation

6.

Empty the wells and remove excess solution then add 100µl of Wash Buffer to every well.

7.

Incubate the plate at 4°C for 10 min.

8.

Empty the wells as previous and wash the plate 3x with 100µl of Wash Buffer.

9.

Add 100µl of diluted detection antibody to every well.

10.

Cover the plate and incubate at RT for 1 hour 30 min.

11.

Empty the wells as previous and wash the plate 3x with 100µl of Wash Buffer.

12.

Add 100 µl of diluted Streptavidin-AP conjugate to every well.

13.

Cover the plate and incubate at RT for 1 hour.

14.

Empty the wells and wash the plate 3x with 100µl of Wash Buffer.

15.

Peel of the plate bottom and wash both sides of the membrane 3x under running distilled water, once washing complete remove any excess solution by repeated tapping on absorbent paper.

16.

Add 100µl of ready-to-use BCIP/NBT (5-bromo-4-chloro-indolyl-phosphate/nitro blue tetrazolium) buffer to every well.

17.

Incubate the plate for 5-15 min monitoring spot formation visually throughout the incubation period to assess sufficient colour development.

18.

Empty the wells and rinse both sides of the membrane 3x under running distilled water. Completely remove any excess solution by gentle repeated tapping on absorbent paper.

19.

Read Spots: Allow the wells to dry and then read results. The frequency of the resulting coloured spots corresponding to the cytokine producing cells can be determined using an appropriate ELISpot reader and analysis software or manually using a microscope.

Plate should be stored at RT away from direct light, but please note that colour may fade over prolonged periods so read results within 24 hours.

Available ELISpot Products

ELISpot Kits

Human Kits

SKU Protein Target

Fas Ligand (CD178)

Granzyme B

 IFN-γ

IL-10

IL-12p70

IL-13

IL-17A

IL-17A/F

IL-17F

IL-1β

IL-2

IL-4

IL-5

IL-6

Perforin

TNF-α

Murine Kits

SKU Protein Target

 IFN-γ

IL-2

Rat Kits

SKU Protein Target

 IFN-γ

TNF-α

ELISpot Pairs

Human Kits

SKU Protein Target

Fas Ligand (CD178)

Granzyme B

 IFN-γ

IL-10

IL-12p70

IL-13

IL-17A

IL-17A/F

IL-17F

IL-1β

IL-2

IL-4

IL-5

IL-6

Perforin

TNF-α

Murine Kits

SKU Protein Target

 IFN-γ

IL-2

Rat Kits

SKU Protein Target

 IFN-γ

TNF-α

ELISpot FAQs

What's the difference between an ELISpot and ELISA assay?

An Enzyme-Linked Immunosorbent Assay (ELISA) determines the total concentration of the secreted signaling protein or antibody. In contrast, an ELISpot assay analyses individual cytokine or antibody secreting cells. ELISpot gives a quantitative result of the frequency of the secreting cells.

What cell types can be analysed by ELISpot?

ELISpot assay is typically used to identify cytokine secretion by antigen-activated T cells and antibody secretion by B cells from peripheral blood or spleen cells.

Which assay should I use? ELISpot or ELISA?

ELISpot is the appropriate assay for determining the frequency of secreting cells, since it detects cytokines or antibody secreting cells at an individual level. Therefore ELISpot is most suitable when used in addition to an ELISA. An ELISpot assay can be 100-400 times more sensitive than a standard ELISA.

How many cells should be put into each well?

The results of an ELISpot asssay depends on high cell density. A high number of cells increases the likelihood of contact between stimulating and responding cells. The cell density should typically range between 1-2 X 105 cells per well, and this may require some optimization.

Which research area can ELISpot be used in?

An ELISpot assay is commonly used for vaccine development, autoimmune disease, infectious disease, allergy research, veterinary research and monocytes/macrophages/dendritic cells characterization.

What are the storage instructions for an ELISpot assay?

Store kit reagents between 2°C and 8°C. Immediately after use, remaining reagents should be returned to coldstorage (2°C to 8°C). Expiry of the kit and reagents is stated on box front labels. The expiry of the kit components can only be guaranteed if the components are stored properly, and if in the case of repeated use of one component, the reagent is not contaminated by the first handling.

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