Single cell versus bulk assay analysis
ELISpot, like flow cytometry-based intracellular cytokine staining (ICS), directly determine the frequency of antigen (Ag)-specific T cells, a core competency for immune diagnostics. Such resolving power is unattainable with supernatant-based assays, such as ELISAs or multiplex bead arrays, where measurements are based on bulk cytokine production by all cells in a given sample well.
Individual cell variation is biologically important
In acute HIV subjects, the frequency of cells producing IFNg in response to common recall antigens (e.g., TT or PPD) was comparable to healthy donors; however, spot size is dramatically reduced. This result suggests that HIV-specific T-cell function, and not cell number, was impaired. Similarly, T cells recently activated in vivo may show increased per cell cytokine production when compared to “older” memory T cells. The ability to distinguish between long-term memory and recently activated subsets has implications for T cell diagnostics of autoimmune disorders and chronic infections.
Individual cell analysis isolates noise
Results from bulk assays are often confounded by the contribution of background signal(s) from the innate immune system. Dilution of the Ag-specific response results in overall signal flattening; this issue is most relevant for detecting the presence of rare populations, such as circulating tumor cells (CTC) in PBMC or disseminated tumor cells in bone marrow, both early markers of metastasis.
ELISpot format is extremely sensitive
For the T cell repertoire to be capable of recognizing a potentially infinite number of infective agents while simultaneously distinguishing them from self, the total naive pool contains ≥ 1012 unique T-cell receptor (TCR) specificities. Consequently, in the absence of infection, the frequency of circulating memory cells with specificity to any one antigen is quite low, typically in the range of 1:10,000 -1,000,000.
ELISpot allows quantitative single cell analysis
Multiparameter ELISpot Assays
In some applications, for example tuberculosis (TB) diagnostics, multi-analyte signatures may provide a clear distinction between latent and active forms of an infection or other immunological response.
ELISpot assays are amenable to multiplex analyses carried out simultaneously (single well) or in parallel. Well established dual-color ELISpots, using both enzymatic and fluorescent approaches, are currently used in many research settings. Fluorescent ELISpots, or FluoroSpots, offer significant advantages over colorimetric formats, particularly in the areas of multiplexing and automated spot detection. Moreover, as spot development is not enzymatic, signal intensity is directly proportional to the amount of analyte within the spot and therefore far more quantitative.
See a detailed discussion, data, and images in our ELISpot Whitepaper.
ELISpot reduces samples and increases replicates
A note on ELISpot automation
The ease of ELISpot data acquisition and analysis makes it amenable to automation, and working with far fewer cells per assay also means that multiple replicates can be performed, thereby increasing statistical power and sensitivity.
96 or 384 well automated platforms promote the standardization of ELISpot data analysis and greater reproducibility across sites. Merck’s Immobilon®-P Membrane is a 384-well white plate typically requiring less cells per well for optimal set up conditions (see the graph below). This combination of automation and acquisition features also makes ELISpots the ideal choice for high-throughput testing applications, which could be applied in large-scale subject profiling. For example, IFNgamma ELISpots are commonly used as a correlate of vaccine efficacy to identify potential candidates for HIV and other diseases.
The data presented in the figure below is part of ongoing studies performed at Cellular Technology Limited (CTL) to validate the application of ELISpots to a 384-well format. In this example, IFNgamma ELISpots were performed on PBMCs following stimulation with CEF-7 peptide. Plates were imaged and analyzed using CTL’s ImmunoSpot® S6 Micro Analyzer. For the range of seeding densities tested, the assay demonstrated a strong linear relationship (R2=0.9866) between spot-forming units (SFU) and cell number (see graph above). Lastly, modifications to microplate design have increased compatibility with existing robotics systems, thereby also improving potential throughput. These plate adaptations include stricter dimensional specifications and rigid side walls. Plates are now fully compatible with standard fluidics platforms, plate washers, and devices for imaging and image analysis.
|Membrane & Plate Needs for Multiplexed ELISpot|
Increasing the multiplexing capacity beyond two colors requires certain considerations:
See a detailed discussion, data, and images in our ELISpot Whitepaper.|
|Diagnostic Grade 8-Well Strip Plates|
One long-standing problem with the 96-wellmicroplates has been the waste of unused wells in small-scale assays such as that occurring in diagnostic analysis of a single patient sample. Merck offers transparent 8-well strips designed for the diagnostic community. These strips are currently part of Oxford Immunotech’s T-SPOT.TB Test, an FDA-approved IFNg ELISpot test designed specifically for diagnosis of tuberculosis infection and should prove important in any resource-limited situation such as in devoloping countries where diseases such as TB and HIV are most devastating (Figure 4A).