ELISpot and FluoroSpot

Cytokine secretion at the single-cell level

The enzyme-linked immunospot (ELISpot) assay is a highly sensitive method for quantification of the number of cells secreting cytokines, immunoglobulins, or other target proteins. Simultaneous detection of more than one isotype is possible in fluorescent FluoroSpot assays.

The ELISpot assay captures the presence of cytokines immediately after secretion, in contrast to measurements that are skewed by receptor binding or protease degradation.  With detection levels as low as one cell in 250,000, ELISpot is one of the most sensitive cellular assays. It is carried out in a 96-well plate, and an automated ELISpot reader is used for analysis. The assay is therefore easy to perform and allows rapid analysis of a large number of samples. 

ELISpot in Summary:

 

•  Sensitive immunoassay
•  Measures secretion of cytokines
•  Easy to perform
•  Robust and easy to standardize
•  Suitable for large-scale trials also in field settings

 

FIG: Schematic illustration of the principle of the ELISpot assay

 

Our partner Mabtech provides a large range of ELISpot and FluoroSpot assays for different applications:

 

- Quantification of cytokine secreting cells

- Quantification of B cells secreting antigen-specific immunoglobulin

- SARS-CoV-2 peptide specific FluoroSpot and ELISpot kits

 

 

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Quantification of cytokine secreting cells with ELISpot

ELISpot is a sensitive method for investigating specific immune responses and is able to discriminate between subsets of activated T cells. For example, Th1 cells are characterized by their production of IFN-γ, IL-2, and TNF-α, whereas Th2 cells produce other cytokines such as IL-4, IL-5, and IL-13. This is widely applied for example in studies of infectious diseases, cancer, allergies, and autoimmune diseases. 

In vaccine research, ELISpot is a standard tool that is used to define vaccine efficacy by measuring the capacity to elicit potent T cell responses, for example IFN-γ secretion.

 

Identification of T-cell clones reactive to cancer

T cells have been identified as promising mediators of cancer immunotherapies. After recognizing their specific antigen, T cells communicate by secreting cytokines. These secreted signalling molecules can be detected using the ELISpot and FluoroSpot techniques. With detection levels as low as one cell in 250,000, ELISpot and FluoroSpot are ideal for studying tumor-specific T cells, typically found at very low levels. Importantly, the assay also allows the definition of the most immunogenic epitopes by screening against individual peptides. The multiplexing capacity of FluoroSpot enables investigation of the cytokinesecreting profiles of tumor-directed T cells.

More information on ELISpot and FluoroSpot applications in cancer research (Mabtech White Paper)

 

Mabtech's ELISpot Assays are available for the full range of cytokines:

 

GM-CSF	IFN-alpha	IFN-gamma	TNF-alpha
IL-1alpha	IL-2	IL-3	IL-4
IL-5	IL-6	IL-8 (CXCL8)	IL-10
IL-12 (p70)	IL-12/-23 (p40)	IL-13	IL-17A
IL-21	IL-22	IL-23	IL-27
IL-31

 

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Quantification of B cells secreting antigen-specific immunoglobulin

The ELISpot technique is not limited to measurement of cytokines; it is also suitable for almost any secreted protein where single-cell analysis is of interest. The method was originally developed to quantify immunoglobulin-secreting cells. The B-cell ELISpot assay is a powerful tool to analyze antibody immune responses and is one of few assays directly measuring immunoglobulins upon secretion. 

B cells secreting antigen-specific immunoglobulin, or indeed immunoglobulin of all specificities, can be quantified using this assay. The number of antigen-specific B cells is most often compared to the total number of secreting B cells. B cells that secrete antigen-specific antibodies can be detected in the circulation from 6 to 9 days after antigen exposure (e.g. vaccination). Memory B cells may require polyclonal stimulation before detectable amounts of antibodies are found.

 

FIG: Schematic illustration of the principle of the B-cell ELISpot assay. A and B show the two ways of performing the antigen-specific B-cell ELISpot assay using either antigen for coating (A) or biotinylated antigen for detection (B). C shows the ELISpot assay to determine total IgG.

 

ELISpot assays for human Immunoglobulins:

Art. Nr.	Product Description
MAB-3860-2A / MAB-3860-2H	Human IgA ELISpot BASIC kit (ALP / HRP) for 4 plates
MAB-3810-2A / MAB-3810-2H	Human IgE ELISpot BASIC kit (ALP / HRP) for 4 plates
MAB-3850-2A / MAB-3850-2H	Human IgG ELISpot BASIC kit (ALP / HRP) for 4 plates
MAB-3851-2A / MAB-3851-2H	Human IgG1 ELISpot BASIC (ALP / HRP) for 4 plates
MAB-3852-2A / MAB-3852-2H	Human IgG2 ELISpot BASIC (ALP / HRP) for 4 plates
MAB-3853-2A / MAB-3853-2H	Human IgG3 ELISpot BASIC (ALP / HRP) for 4 plates
MAB-3854-2A / MAB-3854-2H	Human IgG4 ELISpot BASIC (ALP / HRP) for 4 plates
MAB-3880-2A / MAB-3880-2H	Human IgM ELISpotBASIC kit (ALP / HRP) for 4 plates

Other Species also available.

 

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SARS-CoV-2 peptide specific FluoroSpot and ELISpot kits

Mabtech's SARS-CoV-2 S2 N defined peptide pool (MAB-3620-1) is complemented with a SARS-CoV-2 S1 scanning peptide pool (MAB-3629-1) containing 166 peptides synthesized as 15 mers overlapping by 11 amino acids covering the S1 domain of the Spike protein.

For your convenience, Mabtech now provides these pools together with ELISpot and FluoroSpot kits in 1-plate formats: 

Human IFN-γ/IL-2 SARS-CoV-2 FluoroSpotPLUS kit (MAB-FSP-0102-P1-1)

Human IFN-γ SARS-CoV-2 ELISpotPLUS kit (MAB-3420-4AST-P1-1)

These kits with strip plate enable enumeration of cells secreting IFN-γ (ELISpot), IFN-γ, IL-2 or both cytokines (FluoroSpot) in response to SARS-CoV-2 peptides.

 

Mabtech also provides ELISpot and FluoroSpot Kits for enumeration of B cells secreting IgG antibodies specific for the SARS-CoV-2 Receptor Binding Domain (RBD) / S1:

ELISpot Path: Human IgG (SARS-CoV-2, RBD) ALP / HRP (MAB-3850-4APW-R1-1 / MAB-3850-4HPW-R1-1)

FluoroSpot Path: Human IgG (SARS-CoV-2, RBD) (MAB-FSP-05R-R1-1)

FluoroSpot Path: Human IgG (SARS-CoV-2, Spike) (MAB-FSP-05R-S1-1)

 

 

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