8 February 2013

ELISpot assay principle

The ELISpot assay is a sensitive method for quantification of the number of cytokine secreting cells


The enzyme-linked immunospot (ELISpot) assay is a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. In this assay, cells are cultured on a surface coated with a specific capture antibody in the presence or absence of stimuli. Proteins, such as cytokines, that are secreted by the cells will be captured by the specific antibodies on the surface. After an appropriate incubation time, cells are removed and the secreted molecule is detected using a detection antibody in a similar procedure to that employed by the ELISA. The detection antibody is either biotinylated and followed by a streptavidin-enzyme conjugate or the antibody is directly conjugated to an enzyme. By using a substrate with a precipitating rather than a soluble product, the end result is visible spots on the surface. Each spot corresponds to an individual cytokine-secreting cell.

Schematic illustration of the principle of the ELISpot assay.

Sensitive

The ELISpot assay captures the presence of cytokines immediately after secretion, in contrast to measurements that are skewed by receptor binding or protease degradation. The assay is considered as one of the most sensitive cellular assays available. The limit of detection typically achieved can be 1 in 100,000 cells. The high sensitivity of the assay makes it particularly useful for studies of the small population of cells found in specific immune responses.

Mabtech has worked extensively for more than 20 years to optimize the ELISpot protocol. New proteins are continuously added to the range of analytes that can be analyzed by ELISpot.

Easy to perform

The ELISpot assay 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. It is also robust and suitable for large-scale trials and for field studies. Plates pre-coated with the capture antibodies and one-step detection reagents offer additional advantages. Standardization of the ELISpot assay in specific settings is well described and the method is the basis of an FDA-approved diagnostic test, the T-spot test for tuberculosis.

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.

ELISpot kits

Mabtech supplies high quality ELISpot reagents for small frontline research groups as well as for large vaccine trials. Mabtech’s platform of high quality monoclonal antibodies enables research on your own terms. They know that all researchers have different needs – to accommodate this, they offer ELISpot kits in many different formats.

ELISpotBASIC

Tailor the ELISpot assay for your needs.

Mabtech’s ELISpotBASIC provide reagents suited for users who want to set up and optimize their assay. The reagents have been developed for ELISpot and include matched monoclonal antibodies and Streptavidin-enzyme conjugate. For B-cell ELISpot, polyclonal activators are included.

ELISpotBASIC products are available in 4 plate format and can also be supplied in larger quantities on request.

Please note that while plates and substrate are not included in ELISpotBASIC, these can be purchased separately.

Kit contents

  • Capture mAb
  • Biotinylated detection mAb
  • Streptavidin-enzyme conjugate (ALP or HRP)
  • Polyclonal activators (B-cell ELISpot only)

ELISpotPLUS kits

Mabtech’s ELISpotPLUS kits are ideal for users who wish to obtain the necessary components for a convenient ELISpot assay, including plates pre-coated with capture antibody.

Mabtech offers ELISpotPLUS kits with pre-coated PVDF plates for optimal performance. These plates have the capture antibody bound to the membrane of the wells, which allows for a more rapid assay as it eliminates the ethanol pre-treatment and antibody coating steps from the assay protocol. In addition, the use of pre-coated plates reduces variability between assays. For some analytes, ELISpotPLUS kits are also available with pre-coated strip-plates. The use of pre-coated strip-plates is ideal when a limited number of wells are needed, e.g. when analyzing few samples.

Mabtech’s ELISpotPLUS kits are available in 2 and 10 plate format.

Kit contents

  • Pre-coated PVDF plates or PVDF strip-plates
  • Biotinylated detection mAb
  • Streptavidin-enzyme conjugate (ALP or HRP)
  • Substrate (BCIP/NBT-plus or TMB)
  • Anti-CD3 mAb Included as a positive control for selected systems

ELISpotPRO kits

Kits with pre-coated plates and one-step detection.

The ELISpotPRO kits are Mabtech’s most innovative products and combines top performance with a rapid assay procedure in complete kits featuring pre-coated plates and ALP or HRP-conjugated detection antibodies.

The ELISpotPRO kit is the optimal choice for an easy and rapid performance with high reproducibility. With minimal intra- and inter-assay variation it is also the preferred choice in large scale vaccine trials. The detection antibodies are conjugated to ALP or HRP which allows for one-step detection making the commonly used biotin-Streptavidin step redundant. The ELISpotPRO kits are also available with pre-coated strip-plates for increased flexibility. The use of pre-coated strip-plates is ideal when a limited number of wells are needed, e.g. when analyzing few samples.

Mabtech’s ELISpotPRO kits are available in 2 or 10 plate format.

Kit contents

  • Pre-coated PVDF plates or PVDF strip-plates
  • One-step detection mAb (ALP or HRP conjugated)
  • Substrate (BCIP/NBT-plus or TMB)
  • Anti-CD3 mAb Included as a positive control for selected systems

Find the product you need

All product groups

Related articles

Newsletter for researchers

We gladly support you by keeping you updated on our latest products and the developments around our services.