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.
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.
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.
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.
Flow cytometry: Detection of such rare events can present a significant challenge to flow-based platforms, where the lower limit of sensitivity is reported to be 0.02%.
ELISA: Relative to ELISpot, the sensitivity threshold for cytokine measurements in culture supernatants is further diminished by analyte dilution in the surrounding milieu, absorption by bystander cells, and enzymatic degradation.
ELISpot: ELISpot assays demonstrate a detection threshold of less than 25 IFNg-producing T cells per million PBMC (0.0025%); this equates to a near 10-fold increase in detection sensitivity.
The ELISpot assay’s high sensitivity is also important for allergy research, where identifying the very low frequency Th2 cytokine-producing cells is critical for both disease monitoring and development of immune therapies. Specifically, both flow cytometry and ELISA platforms demonstrate insufficient detection of IL-4, the predominant indicator of a Th2-driven response.
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.
The unique design and sensitivity of ELISpot and available plates allows for significantly reduced sample volumes and better use of available wells.
Every cell in an ELISpot assay is measured, so no loss due to instrument priming occurs.
ELISpots, on average, require one-tenth as many cells per test than other formats, which provides a crucial advantage under conditions where samples are precious (remote settings) and/or limiting (pediatric or immunosuppressed test subjects).
ELISpot assays can be run in 96-well strip plates, reducing wasted wells and allowing more replicates per plate.
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.
The Lysispot Assay
The Lysispot assay is a modified ELISpot capable of enumerating Ag-specific cytotoxic CD8+ T cell effector function through direct target cell lysis. Until the development of the Lysispot assay, since most cytotoxicity assays are performed on bulk cultures, IFNg ELISpots were commonly used as correlates of CD8+ cellular immunity. Use of the Lysispot in the study of HIV revealed that not all IFNg producing cells were capable of killing. This finding also highlights the need for greater multiplicity of detection in single cell immunoassays.
Membrane & Plate Needs for Multiplexed ELISpot
Increasing the multiplexing capacity beyond two colors requires certain considerations:
Choose membrane surfaces with minimal fluorescent background signal. Due to their highly porous nature, membrane surfaces are very rough. For this reason, they scatter light and exhibit high fluorescence background.
While PVDF membrane (e.g. Immobilon®-P membrane) is purported to be a better surface than nitrocellulose for FluoroSpots, choose membranes specifically designed for fluorescence detection. For example, the Immobilon®-FL PVDF membrane variant was designed specifically for fluorescence detection in Western blotting applications and exhibits background fluorescence signal that is nearly 1/100 that of standard PVDF.
Plate color can also greatly impact the success of the FluoroSpot. White plates may show high background signal (relative to clear or black), making spot detection difficult. This high background is likely due to increased reflectance or transmission of light through the surrounding plate material.
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).