ELISpot assays are widely used to monitor immune responses in both humans and animals. The method was originally developed from the standard ELISA assay by Cecil Czerkinsky1 as a method to enumerate antibody secretion from B cells. The assay has now been adapted to also detect secreted cytokines or antibodies from individual cells.
Essentially, an ELISpot assay comprises the following steps:
Each coloured spot represents a cytokine secreting cell. The spots can be counted by eye or by using an automated plate-reader. Many different cytokines can be detected using this method including IL-2, IL-4, IFN gamma, TNF alpha and Granzyme B. The size of the spot is an indication of the per cell productivity and the avidity of the binding. The higher the avidity of the T cell recognition the higher the productivity resulting in large, well-defined spots. It is also worth noting that Granzyme B, TNF alpha and IL-10 assays frequently produce small spots.
For detection of CD8+ and CD4+ T cell responses by ELISpot, the optimal peptide design will depend on the type of response being analyzed. MHC Class I epitopes are generally shorter than MHC Class II epitopes. For most mouse and human Class I alleles, the peptide sequences are between 8 and 11 amino acids in length. Class II epitopes are generally longer, between 11 and 25 residues in length. Further information about peptide design can be found on the Design of Peptide Libraries page.
The advantage of using the ELISpot method is that it is a more sensitive assay than other techniques such as ELISA. The data generated from ELISpot can give better quantitative and qualitative data when compared with other methods such as intracellular cytokine staining.
Carrying out ELISpot assays in-house, particularly with inexperienced staff, can require significant effort and can be time consuming with variable results. You can save time and resources, and minimize risk by relying on the expertise of ProImmune’s experienced team, who perform these assays routinely using optimized and standardized protocols with minimal variation: Cellular Analysis Services, including ELISpot assays and HLA tissue typing.
The levels of responses for class II epitopes tend to be much smaller than for class I epitopes. By depleting samples of CD8+ cells before carrying out the ELISpot assay, the chances of detecting a small class II response will be increased.
A plate scan readout of a typical IFN gamma ELISpot assay is shown below. In this assay, 3x105 cells were plated per well and stimulated for 18 hours with each of a panel of peptides (quadruplicate wells) from a PEPscreen® peptide library. Negative control wells (negative peptide) are A1 & A2, with PHA positive control in wells A3 & A4.
The cells used have a strong response to the NLVPMVATV peptide (CMV; wells C1 - C4), with a borderline response to GILGFVFTL (Influenza; A9 - A12). In this assay, a response was defined as positive if it was greater than or equal to twice the negative response following deduction of the negative response counts.
The above plate scan also shows examples of undesired results in the form of marks caused by cell clumps (e.g. wells A5, E10, F9 & F10). Thus, it is important to filter cells prior to application on the plate.
The tracking of immune responses following drug administration or immunization is a useful way of monitoring the effectiveness of treatments. ELISpot assays are an integral part of many clinical trials or basic immune monitoring research projects. They can be used in conjunction with other functional assays such as MHC multimer staining, intracellular cytokine staining and proliferation or killing assays to gain a better overall picture of an immune response.
An important point to consider with immune monitoring is the standardization and validation of assays. The Cancer Vaccine Consortium is a collaboration between many laboratories world-wide that is trying to bring about a harmonization in large-scale immune monitoring. In a proficiency panel comparing the results of ELISpot assays there was a wide variation in results when laboratories were given general guidelines but allowed to use their own protocols and reagents2. The recommendations from this study were that laboratories should establish a standard operating procedure (SOP) for ELISpot testing and that only trained users should be allowed to carry out the assays with regular re-assessment. Variations in cell counting methods and plate reading should also be accounted for and thereby minimized.
In most cases, ELISpot assays for immune monitoring will focus on a few key epitopes and so will involve individual peptides rather than pools of peptides. Individual custom peptides are better suited to this application than peptide libraries which yield only small amounts of peptide. See Custom Peptides for further information on peptide design, scale and purity.
The aim of epitope mapping projects is to identify and characterize novel epitopes from a protein that are recognized by the immune system. By identifying novel epitopes, strategies can be developed to produce immunotherapies or vaccines for a wide range of disease areas such as cancers or infectious diseases. Another important application of epitope discovery projects is to identify novel biomarkers for immune monitoring purposes. In this way immune responses can be tracked and compared in patients pre- and post-treatment.
ELISpot is a key methodology used in the identification of novel epitopes. The most comprehensive studies involve the creation of a library of overlapping peptides with a single amino-acid offset. The most economic way of analyzing samples with such a large number of peptides, both in terms of cost and time, is to generate pools of peptides. Using pools of overlapping peptides can reduce the number of samples required for testing, which can be an important factor when using rare patient samples. There is no optimal number of peptides that can be used in one pool; this will be determined by a number of factors including the number of peptides to be tested and the cell samples available for testing. However, a peptide pool of less than 30 peptides is probably desirable as higher numbers of peptides can lead to cytotoxic effects.
There are several strategies used to identify 'hit' peptides from pooling experiments. For example, if using peptides in ELISpot assays, one method that can easily identify responder peptides is to use a checkerboard method. In the diagram below, there are 24 pools of peptides, each containing 12 individual peptides. In this way each peptide is represented in 2 separate pools and cross-referencing the hits will identify the antigenic peptide (highlighted in orange).
Another method is to create a pool of overlapping peptides which will then identify 'hot-spot' areas in the protein of interest. Further experiments can then be carried out using individual peptides from responder pools to identify the minimal/optimal epitope.
There are many considerations when designing peptide libraries for epitope mapping experiments. These include factors such as optimal peptide length, overlap, scale of synthesis and purity. The size and depth of analysis of a study will affect these decisions. See Design of Peptide Libraries for further information.
One way to increase the number of spots seen is to sort the samples to eliminate unwanted cell types. For example if the study involves looking for MHC Class I responses, CD8+ cells could be sorted by flow cytometry (FACS) or magnetic bead separation in order to increase the chances of seeing a clear positive response to a peptide.
1Czerkinsky C. et al (1983) A solid-phase enzyme-linked immunospot (ELISpot) assay for enumeration of specific antibody-secreting cells. J. Immunol Methods 65: 109-21 [PubMedID: 6361139]
2 Janetzki S. et al (2008) Results and harmonization guidelines from two large-scale international ELISpot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI) Cancer Immunol Immunother 57: 303-315 [PubMedID: 17721781]
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