Note 1. In B cell ImmunoSpot® assays there is no inherent lower limit of detection. The PBMC numbers plated per well into a 96-well plate should not exceed 1 x 106 cells per well, because with higher numbers the cells no longer form a monolayer on the membrane [34] and the resulting cell layering can interfere with the capture of ASC-derived antibodies. If, e.g., 10 million PBMC are plated at 1 x 106 PBMC across 10 replicate wells, 1 in 10 million is the detection limit, etc. Owing to increased Poisson noise occurring at such low frequencies, however, the number of replicate wells needs to be increased accordingly to obtain accurate measurements [35]. As shown in Figure 3 of the chapter by Lehmann et al. in this volume [12] antigen-specific memory B cells (Bmem) quite frequently occur in low frequencies.
Note 2. In ELISPOT assays, the enzymatic amplification of the signal leads to loss of strict proportionality between the detection antibody bound and the eventual substrate precipitation. Moreover, once the density of the substrate deposition on the membrane reaches a certain point, the spot’s optical density/appearance does not increase even if more substrate is deposited (much like applying many layers of paint). With fluorescent detection, however, the number of fluorescent tags bound is proportional to the number of detection antibodies retained on the membrane.
Note 3. As enzyme linked ImmunoSpot (ELISPOT) and FluoroSpot assays differ only in the modality of detecting secretory footprints of cells on membranes, we collectively refer to both as ImmunoSpot® assays. In the former, the detection antibody is tagged to enable the engagement of an enzymatic reaction that results in the local precipitation of a substrate that is visible under white light. In the latter, the plate-bound detection antibodies are visualized via fluorescent tags using appropriate excitation and emission wavelengths. Data provided in a chapter in this volume by Yao et al. [22] establish that ELISPOT and FluoroSpot assays have equal sensitivity for detecting numbers of antibody-secreting cell (ASC)-derived secretory footprints. However, they are not equally suited for high-content analysis (HCA) of spot morphologies (see Note 2).
Note 4. Our introduction of the PVDF membrane to ELISPOT assays [9,10], with its by far superior capture antibody adsorption properties [36], was key for improving our ability to detect secretory footprints to the point needed for transforming ImmunoSpot® into the robust immune monitoring platform it has become for detecting rare -- even very rare -- antigen-specific lymphocytes ex vivo, in freshly isolated PBMC or other lymphoid cell material. We refer to Figure 1 in [37] to appreciate the difference in assay performance using the PVDF membrane vs. the previously used mixed cellulose ester membrane.
Note 5. The shape of secretory footprints (spot morphologies) produced by T cells follow defined rules since the capture antibody’s (i.e., an anti-cytokine specific mAb) affinity for the analyte to be detected is high and fixed. Consequently, only the quantity of analyte (cytokine) produced by the T cell will define the morphology of the resulting secretory footprint [25]. Predictable (log normal [32]) spot sizes permit objective automated size gating [14,13].
Note 6. Although only B cells can secrete antibody, even in B cell ImmunoSpot® assays, there can be small background spots resulting primarily from aggregated detection reagents. Such artefacts can be reduced/eliminated by filtering or centrifuging the reagents at high speed to eliminate aggregates. To identify such spots, it is important also to include negative controls wells that are subject to the entire test procedure, but do not contain cells. They should and can be readily gated out during ImmunoSpot® analysis.
Note 7. We refer to Figure 3 of the chapter by Lehmann et al. in this volume [12] to convey the high degree of variability in frequency of antigen-specific Bmem in PBMC.
Note 8. ASC secrete Ig in an undirected fashion into 3D space above the membrane. In ImmunoSpot® assays, the antibody released towards the membrane will be captured as a secretory footprint while the remainder of the secreted antibody will diffuse away from the surface and will be diluted in the bulk of the culture supernatant. As the concentration of such diffused antibody increases in the culture medium, these antibodies are captured on the membrane distantly from the source ASC, increasing the background signal in the assay and undermining the resolution of individual secretory footprints. Such an elevated background in an ImmunoSpot® assay is termed an ELISA effect. If ASC –by chance—settle in clusters on the membrane, local ELISA effects can occur surrounding these cells resulting in regions with increased local background. The ImmunoSpot® software implements powerful local background correction, and therefore such ELISA effects do not interfere with the detection of SFU, however they affect threshold-based detection of spot outlines needed for HCA.
Note 9. Fine-tuning of parameters manually not only requires expert knowledge, but also takes considerable time, and thus it can rarely be done for analyzing an entire assay. Due to global and local ELISA effects in wells, the background level is variable in most assays preventing the accurate detection of outlines of secretory footprints. When using parametric counting for the initial machine reading of the plate, under such conditions well-by-well recounting in Quality Control mode is required for finalizing the results. IntelliCount™ greatly streamlines this process.
Note 10. In FluoroSpot assays, the total fluorescence intensity of a spot is proportional to the quantity of analyte captured within the secretory footprint of a given ASC. The total spot intensity is equal to the mean spot intensity (shown on figure 3) multiplied by the spot size. In antigen-specific direct assays, spot morphologies can include all possible variations of sizes and intensities (see Fig. 2). A multitude of morphological parameters are readily captured for each SFU, in FCS format, to perform in depth HCA.
Note 11. The so-called “Goldilocks” number is defined as the maximal number of cells that can be plated in a B cell ImmunoSpot® assay well while still being able to discern clearly secretory footprint boundaries derived from individual antigen-specific ASC. For HCA, i.e., for the accurate definition of secretory footprint boundaries, the Goldilocks number is lower than the breaking point for linearity in mere SFU counts. As it is assay-dependent, it needs to be experimentally established by serial dilution of PBMC in the respective assay, but ~50 SFU/well is a safe estimate.
Note 12. Frequently, the background membrane staining of individual wells is not perfectly even in ImmunoSpot® assays and that can interfere with accurate SFU detection, in particular when relying on fixed counting parameters. Lowering non-specific background staining, and reduction of “hot spots” in the center of the assay wells can be achieved through performing the “back to front” water filtration technique. Regarding regional and global ELISA effects, see Note 8.
Note 13. Antibodies occur in four classes (IgM, IgG, IgA and IgE), and in four subclasses (IgG1, IgG2, IgG3 and IgG4). ASC producing all four, can be detected simultaneously in multiplexed ImmunoSpot® assays using only 4 x 105 PBMC/antigen (see chapter by Yao et al. in this volume, [22]). The different Ig classes and subclasses are endowed with distinct effector functions and each contribute non-redundant roles towards maintaining host defense (reviewed in [38]). Stimulating optimal Ig class usage during an infection or following vaccination is vital to successful host defense and the avoidance of collateral immune-mediated pathology (reviewed in [39]).
Note 14. During the primary immune response, B cells can transition from IgM-expressing naïve B cells into effector cells (antibody-secreting plasma cells) and resting Bmem that have undergone class switch recombination (CSR). CSR is an irreversible process that involves the excision of DNA encompassing the exons of the Igμ heavy chain required for expression of IgM and the juxtaposition of upstream variable region genes with downstream exons encoding alternative Ig classes or IgG subclasses [40]. Class switching of the BCR to downstream Ig classes or IgG subclasses is an instructed process and can be influenced by the cytokine milieu and co-stimulation provided by CD4+ T helper cells. Thus far, we have not seen evidence for in vivo class-switched Bmem to undergo further during short-term polyclonal stimulation in vitro using R848 plus IL-2, as needed for their detection in ImmunoSpot® assays. Thus, it has to be assumed that the Ig calls subclass utilization of ASC observed in ImmunoSpot® assays ex vivo, reflects on the corresponding Bmem commitment for Ig class/subclass utilization upon antigen reencounter in vivo.
Note 15. Once activated by polyclonal stimulation, ASC are autonomous. Thus, the well-to-well variation in numbers of ASC in B cell ImmunoSpot® assays is dependent on their concentration in the test sample following the rules of a Poisson distribution: the rarer the cells, the higher the well-to well variation when a set volume is sampled/plated. This knowledge permits to precisely calculate the number of replicate wells needed to establish frequencies with precision when ASC frequencies are low [35].
Note 16. For low frequency antigen-specific B cell assay results, the conventional parametric approach can establish SFU counts (but to a lesser extent HCA-pertaining parameters) with a similar accuracy as IntelliCount™; however it requires expertise to set up parameters, whereas IntelliCount™ does it automatically.
Note 17. Subjective counting is a considerable challenge for count harmonization among individuals and laboratories [33].
Note 18. Even slight changes in assay conditions (e.g., incubation times and temperature) as well in reagents properties over time (e.g., storage dependent aggregation or decay) can have an effect on the SFU staining intensity seen in repeat ImmunoSpot® assays. By being less sensitive to such qualitative differences, IntelliCount™ helps the assay’s robustness in the evaluation phase.
Note 19. Activation of the PVDF membrane with 70% EtOH is instantaneous and can be seen visually as a graying of the membrane. It is important to be sure that the EtOH solution has spread across the entire membrane before adding the first wash of PBS. If needed, tapping of the plate can promote contact of the EtOH solution with the PVDF membrane. We recommend only pre-wetting one plate at a time with 70% EtOH to ensure that the contact time is ≤ 1min; longer contact times may promote leaking of the membrane and result in suboptimal assay performance.
Note 20. We refer to the chapter of Yao et al. in this volume [22] for detailed procedures covering the isolation of peripheral blood mononuclear cells (PBMC), their cryopreservation and thawing, as well as the polyclonal in vitro stimulation culture needed to trigger antibody production by resting memory B cells.
Note 21. PBMC, or other primary cell material, collected acutely following known antigen encounter, which may contain spontaneous (in vivo differentiated) ASC can also be evaluated in such assays.
Note 22. If the cells are not washed thoroughly, antibody in the cell suspension(s) can compete with the binding of ASC-derived Ig in the assay, resulting in elevated membrane staining that can interfere with the accurate detection of individual ASC’s secretory footprints.
Note 23. Using a serial dilution approach, an ideal starting cell input of 2 x 104 is appropriate for typical pan (total) IgA/IgG/IgM measurements following in vitro differentiation of PBMC. However, higher cell inputs may be more appropriate for measurements of spontaneous (in vivo differentiated) ASC.
Note 24. Serial dilutions involving single wells for each cell dilution, progressing in a 1+1 (2-fold) dilution series, is a valid option for establishing accurate SFU frequencies and greatly reduces the cell numbers and reagents required (see the chapter by Yao et al. in this volume, [22]. In Figure 5A of that chapter the recommended plate layout for such a serial dilution assay is shown.
Note 25. For automated washing, the pin height and flow rate should be customized to avoid damaging the assay membranes, which is the case for the CTL 405LSR plate washer. Plate washes may also be performed manually. See also Note 22.
Note 26. Optimal removal of background staining, fibers and other debris, along with reduction of “hot spots” in the center of the assay wells, is achieved through performing the “back to front” water filtration technique.
Note 27. To completely dry plates, blot assay plate(s) on paper towels to remove residual water before either placing them in a running laminar flow hood at a 45° angle for >20 min or placing face down on paper towels for >2hr in a dark drawer/cabinet. Do not dry assay plates at temperatures exceeding 37°C as this may cause the membrane to warp or crack. Fluorescent spots may not be readily visible while the membrane is still wet and the background fluorescence may be elevated. Scan and count plates only after membranes have dried completely.
Note 28. Direct application of an antigen to the PVDF membrane can result in variable and often low-affinity absorption to the membrane owing to weak, non-specific binding forces (primarily hydrophobicity). Alternatively, our recent introduction of affinity capture coating [11] enables specific and high-affinity binding of antigen to the assay membrane.
Note 29. Optimizing the concentration of His-tagged protein(s) used for affinity capture coating is recommended. A concentration of 10 µg/mL His-tagged protein has yielded well-formed secretory footprints for most antigens, but increased concentrations of the anti-His affinity capture antibody and/or His-tagged protein may be required to achieve optimal assay performance.
Note 30. Using a serial dilution approach, a starting cell input of 2-5 x 105 is appropriate for typical antigen-specific ImmunoSpot® tests following in vitro differentiation of PBMC. However, higher cell inputs may be more appropriate for measurements of spontaneous (in vivo differentiated) ASC.
Note 31. Owing to polyclonal stimulation of Bmem to trigger their terminal differentiation, a large majority of IgG+ ASC will not be antigen-specific yet will compete for “real estate” on the lawn of anti-IgG capture reagent used for coating. Consequently, inverted assays aimed at studying lower frequency ASC specificities are directly limited by the maximal number of total IgG+ ASC that can be input into a single well while still maintaining the ability to resolve individual antigen-specific secretory footprints.
Note 32. Prior to performing an inverted ImmunoSpot® assay using limiting quantities of antigen detection probe, it is recommended to first determine the Goldilocks cell input to achieve ~50 SFU/well using an aliquot of cryopreserved cell material.
Note 33. In instances when the frequency of antigen-specific ASC is low among all ASC, we recommend increasing the number of replicate wells and seeding at lower cell inputs. Moreover, to conserve on cell material required, increasing the fold dilution of the antigen probe and/or testing only at pre-determined concentrations are both valid options.
Note 34. If the Goldilocks cell number input is already known, and the intent of the assay is to assess the affinity spectrum of the antigen-specific ASC compartment, the relevant assay procedures are described in detail in the chapter by Becza et al., [8].
Note 35. Using a serial dilution approach, a starting cell input of 1 x 105 is appropriate for an antigen-specific, inverted ImmunoSpot® tests following in vitro differentiation of PBMC. However, higher cell inputs may be more appropriate for measurements of spontaneous (in vivo differentiated) ASC.
Note 36. Shorter B cell ImmunoSpot® assay incubation times are suggested if using an enzymatic-based detection approach to avoid merging of spots and/or elevated membrane background staining.
Note 37. The optimal concentration of affinity (His)-tagged antigen probe used for detection of all antigen-specific secretory footprints (i.e. SFU), low- or high-affinity alike, should be determined empirically.