Detecting Vaccinia Virus Response in Human Blood Cells Using ELISPOT

Introduction of ELISPOT Detecting Vaccinia Virus Response

Vaccinia virus is the prototypic orthopoxvirus that has been widely used in the twentieth and twenty-first centuries to vaccinate individuals against smallpox (variola) as it induces cross-protective immunity against variola. Although routine vaccination of individuals with vaccinia virus ceased during the mid-1970s in the USA, President George W. Bush initiated the US National Smallpox Vaccination Program in 2002 to vaccinate healthcare workers, first responders, and mission-critical forces with smallpox vaccine due to the possible threat that smallpox or another orthopox-virus could be weaponized and used as a bioterorrist weapon. The resurgence of individuals receiving smallpox vaccination as a result of the US National Smallpox Vaccinization program has fueled the development and implementation of high-throughput assays that are designed to rapidly and succinctly assess the immune status of large populations postsmallpox vaccination.

Figure 1. Schematic overview of PBMC isolation for IFN-γ ELISpot Assay. (Coughlan L, et al.;2015)

Successful vaccination against smallpox requires both humoral and cell-mediated immune (CMI) responses. The vaccinia specific humoral response can be measured using several methods; however, a neutralizing antibody (Ab) assay is considered the gold standard by the greater scientific community to quantify circulating vaccinia-specific Ab titers in sera. While no single gold standard assay exists to measure vaccinia-specific CMI response, an enzyme-linked immunospot (ELISPOT) assay is a high-throughput assay that assesses vaccinia-specific CMI response following smallpox vaccination. ELISPOT assays can be used to functionally assess recall immunity, to determine the frequency of vaccinia-specific cytokine-secreting lymphocytes at a single cell level, and to determine the Th-type immune response profile of a vaccinated individual. Mayo Clinic's Vaccine Research Group has optimized the use of both IFNγ and IL-10 ELISPOT assays to accurately assess both Th1-like and Th2-like cytokine profiles, respectively, for subjects previously vaccinated with vaccinia virus based on frequency of cytokine-secreting lymphocytes following in vitro stimulation with vaccinia virus. In addition, we also describe an ELISPOT assay to measure IFNG secretion of vaccinia-specific CD8+ T cells that does not require any expansion, presorting, or isolation of T cells. The antiviral CD8+ T cell-specific IFNG ELISPOT allows for a more precise measurement of vaccinia-specific IFNG response to vaccinia antigens by removing lymphocytes that are not specific to vaccinia, but still have the potential to secrete IFNG in response to vaccinia virus stimulation (e.g., NK cells). Moreover, a CD8+ -specific IFNG ELISPOT allows for a measurement of vaccinia-specific cytotoxic T lymphocyte (CTL) response in individuals previously vaccinated with vaccinia virus.

Most importantly, ELISPOT assays allow for a per cell measure of vaccinia-specific CMI response without in vitro expansion of the T-cell population or purification of T-cell subsets; thus, ELISPOT assays are a rapid, reliable in vitro method to monitor immune response postsmallpox vaccination. Here, we describe in detail vaccinia-specific IL-10 ELISPOT, IFNG ELISPOT, and IFNG CD8+ T-cell ELISPOT assays as well as techniques that are essential for measuring human vaccinia virus-specific CMI response using ELISPOT assays.

Methods of ELISPOT Detecting Vaccinia Virus Response

The following methods describe the steps required to detect human IFNG and IL-10 secreting lymphocytes (1) infecting HeLa cells with vaccinia virus, (2) harvesting vaccinia virus, (3) titrating vaccinia virus, (4) inactivating vaccina virus, (5) collecting and isolating PBMCs, (6) thawing cryopreserved PBMCs, (7) resting PBMCs in the presence of IL-2, (8) infecting PBMCs with vaccinia virus for IL-10 secretion, (9) infecting PBMCs with vaccinia virus for IFNG secretion, (10) detecting IL-10 secreting cells, (11) detecting IFNG secreting cells, and (12) detecting CD8+ IFNG secreting T cells.

Infecting HeLa Cells with Vaccinia Virus

1. Seed 1 × 10⁶ to 5 × 10⁶ HeLa cells per T75 culture flask.
2. Incubate flasks at 37°C in a 5% CO₂ humidified incubator until cells are 80–90% confluent (approximately 2–3 days).
3. Dilute virus stock to MOI = 0.05–0.1 in 2 mL of medium per tissue culture flask.
4. Aspirate all medium from tissue culture flask using a sterile Pasteur pipette connected to a vacuum flask.
5. Wash flask once with HBSS and aspirate.
6. Infect each sterile T75 flask containing HeLa cells with 2 mL of virus suspension.
7. Swirl flask gently to ensure that virus suspension covers all cells.
8. Incubate for 2 h at 37°C in a 5% CO₂ humidified incubator swirling flask every 15 min.
9. After 2 h, add approximately 20 mL of complete medium to the flask.
10. Incubate cells for 2–3 days checking for the formation of plaques [i.e., cytopathic effect (CPE)] daily until >90% CPE is observed.

Harvesting Vaccinia Virus

The method below is a modified protocol for culturing and purifying vaccinia virus. HeLa cells should be harvested only after >90% CPE is observed.

1. Remove all medium from flask and pipette into a 50-mL sterile polypropylene conical centrifuge tube.
2. Centrifuge medium at 500 ug for 10 min at room temperature, and aspirate supernate without disturbing the cell pellet. Save the cell pellet (see Note 1).
3. Using cell lifters, scrape the remaining cells from the flasks into 2 mL of 10 mM Tris–Cl, pH 9.0. If harvesting multiple flasks, scrape cells into 2 mL of 10 mM Tris–Cl, pH 9.0 per flask.
4. Pool cells harvested from each flask with the cell pellet collected in step 2 into a 50-mL sterile polypropylene conical centrifuge tube, and centrifuge the conical tube at 500 ug for 10 min at room temperature.
5. Aspirate supernate then resuspend the cell pellet in 14 mL of 10 mM Tris–Cl, pH 9.0.
6. Transfer cell/virus suspension to a 15-mL U bottom tube and place on ice.
7. Sonicate cell/virus suspension for 30 s at full power (wear proper protective equipment during sonication including a laboratory coat and eye protection).
8. Allow suspension to cool down for 1–2 min on ice, then resonicate cell/virus suspension for 30 s.
9. Layer sonicated cell/virus suspension onto 17 mL of 36% sucrose solution in a sterile ultra centrifuge tube. Place cell/ virus suspension on ice without disturbing the virus suspension-sucrose interface.
10. Centrifuge suspension at 15,800 rpm, 4°C for 80 min. Place ultra centrifuge tube on ice.
11. Aspirate supernatant and resuspend pellet in 4 mL of 1 mM Tris–Cl, pH 9.0, in a U bottom tube.
12. Sonicate suspension for 1 min as described in steps 7 and 8. 13. Store viral suspension at 4°C until the virus is titrated (see Note 2).

Titrating Vaccinia Virus

The method presented below is slightly modified from established protocols for determining the titer of vaccinia virus in pfu/mL. Two separate aliquots of virus should be titrated in parallel to ensure that the observed titer is reflective of the complete viral stock. The titer of each aliquot should have a pfu/mL within 0.5 log of each other. If both titers are within 0.5 log, average the pfu/mL to determine titer of vaccinia viral stock. If the observed titers are greater than 0.5 log apart, repeat titration with a third aliquot.

1. Seed 12-well tissue culture (TC) plates with 3 × 10⁵ Vero cells per well 1–3 days before titrating. Cells should be >90% con- fluent before infection with virus (see Note 3). One 12-well TC plate is sufficient for testing one dilution of the virus in quadruplicate along with the negative controls and standard reference virus.
2. Dilute 2.5% trypsin (10× trypsin) to 1× concentration (0.25%) with calcium and magnesium free 1× PBS.
3. Mix equal volumes (1:1) of 1× trypsin and virus stock in a microcentrifuge tube. Incubate mixture in a 37°C water bath for 30 min.
4. After incubation, perform a tenfold series of dilutions of the virus using medium. Generally, 10^-3 to 10^-11 dilutions are used for determining virus titers.
5. Dilute reference standard virus, obtained from ATCC or well characterized vaccinia virus with known pfu, to 500 pfu/mL.
6. Remove medium from the wells of one TC plate leaving 50–100 ML of medium in each well.
7. Add 100 ML of diluted viral test samples to the top four wells of the 12-well TC plate.
8. Add 100 ML of standard reference virus to the middle four wells of the 12-well TC plate.
9. Add 100 ML of medium to the bottom four wells of the 12-well TC plate for negative controls (see Note 4).
10. Rock plate vigorously to ensure the inoculum covers the entire surface of each well.
11. Incubate plates at 37°C in a 5% CO₂ humidified incubator for 1 h. Rock TC plates every 15 min during the incubation period.
12. After 1 h, add 1 mL of medium to each well.
13. Incubate plates at 37°C in a 5% CO₂ humidified incubator for 72 h.
14. After 72 h of incubation, remove medium from the plate (see Note 5).
15. Gently add 1–2 mL of 1× PBS to each well of the TC plate.
16. Gently swirl PBS and then aspirate.
17. Add approximately 1 mL of 1% crystal violet in 70% methanol to each well. Allow the plate to incubate at room temperature for at least 20 min to fix and stain the remaining Vero cells as well as inactivate the virus.
18. Dump crystal violet solution into the sink. Wash wells in a gentle stream of tap water until the water runs clear.
19. Allow plates to dry.
20. Count plaques per well in the wells with the highest number of countable plaques (usually no more than 120 plaques) per well to calculate titer.
21. Titer in pfu/mL average plaques/well × dilution × 10 (100 μL added per well) × 2 (virus is diluted 1:1 with trypsin).
22. Count plaques in the next lower dilution and average the titers to get the overall titer in pfu/mL (see Note 6).

Inactivation of Vaccinia Virus

Inactivated virus protects laboratory workers from live virus exposure while not damaging the surface antigens that stimulate a host response. Importantly, inactivated virus has been shown to stimulate cells in assays measuring recall immunity comparable to live virus.

1. Dilute virus stock to proper pfu/mL (1 × 10⁸) with 0.1% bovine serum albumin (BSA) in HBSS.
2. Add 5.0 μg/mL psoralen to the virus stock.
3. Place virus suspension in a 35-mm Petri dish.
4. Incubate at room temperature for 10 min.
5. After 10 min, place 35-mm Petri dish under cross-linker and UV irradiate for 60s at 365 nm.
6. Remove virus suspension from cross-linker and aliquot 0.3 mL of inactivated virus suspension into prelabeled cryogenic freezing tubes.
7. Repeat titration assay to determine pfu/mL of inactivated virus. Inactivation should result in a 7–8 log reduction in titer.
8. Store aliquots at -80°C.

Collecting and Isolating PBMCs

The method below is used to separate PBMCs from whole blood that is collected in tubes treated with heparin or EDTA to prevent coagulation. This procedure is based on the manufacturer's protocol for separating PBMCs in Accuspin^TM tubes.

Using cryopreserved PBMCs rather than fresh PBMCs to perform ELISPOT assays has several benefits. Freezing isolated PBMCs allows ELISPOT assays to be performed at a later date postblood draw. This minimizes assay drift because subjects can be tested in consecutive tests and saves money because the entire microplate can be filled with samples. Furthermore, using cryopre served PBMCs allows for the use of multiple recruitment sites to collect the subject's specimens, but have ELISPOT assays performed at a single site thus minimizing variability. Importantly, cryopreserving human PBMCs does not affect the ability of lymphocytes to produce cytokines.

1. Warm HISTOPAQUE-1077 to room temperature using a 37°C water bath. Keep HISTOPAQUE-1077 out of direct light.
2. Pipette 15 mL of HISTOPAQUE-1077 into the upper chamber of each Accuspin^TM tube.
3. Centrifuge Accuspin tubes at 800 × g for 30 s to move HISTOPAQUE-1077 into the lower chamber of the Accuspin^TM tube.
4. Gently pipette the whole blood from a tube treated with anticoagulant into the upper chamber of the Accuspin^TM tube (see Note 7).
5. Add sterile 1× PBS into the Accuspin^TM tube up to the 45-mL mark.
6. Gently mix blood and PBS; do not force any blood below the frit.
7. Centrifuge Accuspin^TM tubes at 1,000 × g for 15 min at 25°C with the brake OFF.
8. After centrifugation carefully remove approximately half of the plasma layer using a sterile Pasteur pipette. Do not disturb the white layer (buffy coat) of PBMCs directly above the frit.
9. Using a sterile Pasteur pipette, carefully remove the layer of PBMCs (white hued layer directly above the frit) and transfer it to a 15-mL sterile conical centrifuge tube.
10. Add 1× sterile PBS to PBMCs bringing the volume of liquid in the 15-mL conical centrifuge tube up to the 10-mL mark to wash cells.
11. Resuspend PBMCs by inverting the tube several times.
12. Centrifuge at 500 × g for 10 min at 25°C with brake ON.
13. Remove supernatant without disturbing the cell pellet.
14. Add 5 mL of ACK lysis buffer to the cell pellet. Resuspend cells by pipetting cell suspension up and down.
15. Allow cells to incubate at room temperature for 5 min in the ACK lysis buffer.
16. Add 1× sterile PBS to the cells + ACK lysis buffer, to bring the volume of liquid in the 15-mL conical centrifuge tube up to the 10-mL mark.
17. Centrifuge at 500 × g for 10 min at 25°C with brake ON.
18. Remove supernatant without disturbing the cell pellet then resuspend pellet in 5 mL of 1× sterile PBS.
19. Place a cell strainer on top of a 50-mL conical centrifuge tube. Transfer the cell suspension from the 15-mL conical centrifuge tube to the 50-mL conical centrifuge through the cell strainer (see Note 8).
20. To count the number of live and dead cells, place 200 ML of 1u PBS, 37.5 ML of Trypan blue, and 12.5 ML of cell suspension into a 5-mL falcon tube; mix well then fill a hemacytometer with 10 ML of sample. Count and record the number of unstained (live) cells in the outer four quadrants of the hemocytometer.
21. Total number of cells Number of live cells/4 × 10,000 × 20 (dilution factor [250/12.5]) × total volume of cells (5 mL or pooled total).
22. Centrifuge cell suspension at 500 × g for 10 min at 25°C with brake ON.
23. Adjust cell concentration to 1 u 107 cells/mL with 4°C RPMI freezing medium (see Note 9).
24. Aliquot 1 mL of cell suspension into prelabeled cryogenic freezing tubes.
25. Place cryogenic freezing tubes into a -80°C freezer in a controlled-rate freezing container overnight.
26. Transfer cells to a liquid nitrogen storage tank for long-term storage (see Note 10).

Thawing Cryopreserved PBMCs

1. Warm RPMI culture medium supplemented with DNase in a 37°C water bath for a minimum of 15 min.
2. Add 100 μL of RPMI culture medium supplemented with DNase into a 15-mL conical centrifuge for each sample being thawed.
3. Remove one vial of PBMCs (cell concentration 1 × 10⁷) for each sample from liquid nitrogen storage tank.
4. Rapidly thaw PBMCs stored in cryogenic freezing tubes using a 37°C water bath by swirling the vial in the water bath until a small amount of ice remains.
5. Quickly wipe the vial with 70% ethanol and place in a sterile tissue culture hood. 6. Pipette each sample from the cryogenic freezing tube into a 15-mL conical centrifuge tube containing 100 ML of RPMI culture medium supplemented with DNase (prepared in step 2). Do not pipette cells up and down.
6. Mix the cells and medium by gently shaking the 15-mL conical centrifuge tube.
7. Slowly add 500 ML of RPMI culture medium supplemented with DNase while swirling the tube gently to mix the cells and medium together.
8. In 1 min add double the amount (1 mL) of RPMI culture medium supplemented with DNase to the cell suspension in the15-mL conical centrifuge tube.
9. Continue adding double the amount of RPMI culture medium supplemented with DNase every minute until the cell suspension reaches a final volume of 10 mL.
10. Cap each conical tube and invert it five times to mix the cells; do not vortex cell suspension.
11. Centrifuge at 300×g for 7 min at 25°C with brake ON.
12. Remove supernatant then resuspend cells in 10 mL of RPMI culture medium supplemented with DNase.
13. Cap each conical tube and invert it five times to mix the cells; do not vortex cell suspension.
14. Incubate cells at 37°C for 20 min by placing the 15-mL conical centrifuge tubes in a 37°C water bath. Invert tubes once 10 min into the incubation period.
15. After 20-min incubation, place cells on ice for 7 min.
16. Centrifuge cells at 300×g for 7 min at 4°C with brake ON.
17. Carefully remove all supernatant and resuspend cells in 1 mL of RPMI culture medium supplemented with 5% FCS.
18. Place a cell strainer on top of a 50-mL conical centrifuge tube. Transfer the cell suspension from the 15-mL conical centrifuge tube to the 50-mL conical centrifuge through the cell strainer (see Note 11).
19. To count the number of live and dead cells, place 200 μL of 1×PBS, 37.5 ML of Trypan blue, and 12.5 μL of cell suspension into a falcon tube. Mix well and fill a hemacytometer with 10 μL of sample. Count and record the number of unstained (live) cells in the outer four quadrants of the hemocytometer.
20. Total number of cells = Number of live cells/4 × 10,000 × 20 (dilution factor [250/12.5]) u total volume of cells (1 mL or pooled total).
21. Adjust the cell concentration to 2×10⁶ cells/mL by adding RPMI culture medium supplemented with 5% FCS.

Resting PBMCs in the Presence of IL-2

This step is only required if PBMCs were damaged or stressed before or during the isolation procedure, for example, if blood has to be shipped overnight before isolation of PBMCs. IL-2 has been shown to increase T-cell survival and proliferation as well as prevent apoptosis. By resting PBMCs overnight in 50 IU/mL, then recounting and plating for ELISPOT assay, T-cell viability is maintained, thus increasing the precision and reproducibility of the assay. Resting PBMCs in IL-2 is cost and labor intensive and only needs to be performed if the background (negative controls) is above the acceptable thresholds or if PBMCs have low-viability postthawing procedure. If PBMCs were isolated and frozen from a blood sample within 12 h, it is unnecessary to perform an IL-2 resting phase.

1. Add 2-mL of cell suspension from step 22, into each well of a 24-well sterile tissue culture plate (final concentration 4 × 10⁶ cells/well) (see Note 12).
2. Add 1 μL of IL-2 (concentration 1 × 10⁵ IU/mL) per well such that the final concentration of IL-2 in each well is 50 IU/mL.
3. Incubate plate at 37°C in a 5% CO₂ humidified incubator for 18 h.
4. After 18 h, remove medium from wells and pool all medium from one subject into a single 15-mL conical centrifuge tube.
5. Add 0.5 mL of prewarmed (see Note 13) 0.25% Trypsin– EDTA to each well.
6. Place plate back in a 37°C in a 5% CO₂ humidified incubator until cells detach (approximately 10 min). Confirm detachment with a microscope.
7. Remove cells/trypsin suspension from each well and add to the corresponding 15-mL conical centrifuge tube which contains medium harvested from the same wells in step 4.
8. Add another 0.5 mL of prewarmed 0.25% Trypsin–EDTA to each well (see Note 14), and incubate plate for 10 min in a 37°C in a 5% CO₂ humidified incubator.
9. Add 0.5 mL of RPMI culture medium supplemented with 5% FCS to each well and mix by pipetting up and down. Pool suspension from each well with the corresponding 15-mL conical centrifuge tube which contains cells harvested from that well in steps 4 and 7.
10. Bring the volume of each 15-mL conical centrifuge tube up to 10 mL by adding RPMI culture medium supplemented with 5% FCS.
11. Centrifuge cells at 300 × g for 7 min at 4°C with brake ON.
12. Remove supernatant without disturbing the cell pellet then resuspend cell pellet in 0.5 mL of RPMI culture medium supplemented with 5% FCS. Keep cells on ice once resuspended.
13. Count the number of live and dead cells by placing 200 μL of 1× PBS, 37.5μL of Trypan blue, and 12.5μL of cell suspension into a falcon tube. Mix well and fill a hemacytometer with 10μL of sample. Count and record the number of unstained (live) cells in the outer four quadrants of the hemocytometer.
14. Total number of cells Number of live cells/4 u 10,000X20 (dilution factor [250/12.5]) × total volume of cells (0.5 mL).
15. Adjust the cells to the desired concentration by adding RPMI culture medium supplemented with 5% FCS (see Note 15).
16. Keep cells on ice until they are ready to be plated (see Note 16).

Infecting PBMCs with Vaccinia Virus for IL-10 Secretion

The method below is used for a kit that does not come with precoated microplates. These microplates must be coated with capture antibody (purified antihuman IL-10) before use.

The protocols below do not use HeLa cell lysate as a negative control; rather, culture medium was used as a negative control for ELISPOT assays. Using HeLa cell lysate as a negative control could be beneficial because only one variable is altered (absence of vaccinia virus in the culture). However, the presence of HeLa cell lysate could alter the cytokine secretion patterns of isolated PBMCs and as such it is not a true negative control. In addition, manufacturing and storing HeLa cell lysate costs personnel time, money, and freezer space; thus, using culture medium rather than HeLa cell lysate as a negative control is a scientifically as well as economically attractive option (see Note 17).

1. One day prior to plating PBMCs, coat the PVDF (polyvinylidene difluoride)-backed microplate with capture antibody (purified antihuman IL-10) by adding 100 μL of dilute capture antibody (1:200 in 1× sterile PBS) per well in a sterile culture hood (see Note 18).
2. Cover microplate with the lid and allow it to incubate overnight at 4°C.
3. Two hours before plating cells in the microplate, remove microplate from 4°C refrigerator and discard the contents of the microplate wells inside a sterile culture hood by flicking. Invert plate and blot dry on paper toweling.
4. Wash wells once with RPMI culture medium supplemented with 10% FCS. Discard the contents of the microplate wells inside a sterile culture hood by flicking then invert microplate and blot dry on paper toweling.
5. Block microplate by adding 200 ML/well of RPMI culture medium supplemented with 10% FCS to each well and incubate at room temperature for 2h.
6. Remove and discard all medium from the microplate inside a sterile culture hood by flicking; invert microplate and blot dry on a paper toweling.
7. Remove vaccinia virus aliquots from -80°C freezer and thaw under a cold stream of water.
8. Dilute vaccinia virus stock aliquots to MOI = 0.05 with 4°C RPMI culture medium supplemented with 5% FCS.
9. Add 100 μL of cell suspension (adjusted to 1 × 10⁶ cells/mL) to each well (final concentration 1 x10⁵ cells/well).
10. Add 100 μL of RPMI culture medium supplemented with 5% FCS to columns 1–3 of each row (see Note 19).
11. Add 100 μL of vaccinia virus MOI 0.05 to columns 4–6 of each row.
12. Add 100 μL of PHA-P (concentration 5 μg/mL) to column 7 of each row.
13. Wrap microplate with aluminum foil and incubate for exactly 24 h at 37°C in a 5% CO₂ humidified incubator (see Note 20).

Infecting PBMCs with Vaccinia Virus for IFNγ Secretion

The method below describes the detection and visualization of IFNγ secreting lymphocytes (and specific lymphocyte subsets) using PVDF microplates that are precoated with human anti-IFNγ capture Ab from the manufacturer.

1. Thirty minutes prior to plating, remove a microplate precoated with human anti-IFNγ Ab from 4°C storage.
2. Block microplate by adding 200 μL of RPMI culture medium supplemented with 5% FCS per well and incubate at room temperature for 20 min.
3. Remove and discard medium from all wells inside a sterile culture hood by flicking; invert microplate and blot dry on paper toweling.
4. Remove vaccinia virus aliquots from -80°C freezer and thaw under a cold stream of water.
5. Dilute vaccinia virus stock aliquots to MOI = 5.0 with 4°C RPMI culture medium supplemented with 5% FCS.
6. Add 50 μL of cell suspension (for CD8+ IFNG [cell] 1x10⁷, for total IFNG [cell] 4 x10⁶) to each well (final concentration CD8+ 5 u 105 cells/well; total IFNG = 2 u 105 cells/well).
7. Add 50 μL of RPMI culture medium supplemented with 5% FCS to columns 1–3 of each row (see Note 21).
8. Add 50 μL of vaccinia virus (MOI = 5.0) to columns 4–6 of each row.
9. Add 50 μL of PHA-P (concentration 5 Mg/mL) to column 7 of each row.
10. Cover microplate with aluminum foil and incubate for exactly 24 h for total IFNG or 6 h for CD8+ IFNγ at 37°C in a 5% CO₂ humidified incubator.

Detection of IL-10 Secreting Cells

1. After PVDF microplate has incubated 24 h, discard medium from the microplate into a sink by flicking. Invert microplate and blot dry on paper towels.
2. Wash the microplate two times with 200 μL/well of deionized (DI) water. Allow wells to soak for 3–5 min each wash.
3. After DI wash, wash wells three times with 200 μL/well with wash buffer (1× PBS supplemented with 0.05% Tween 20), discarding wash buffer in the sink after each wash.
4. Dilute detection Ab (biotinylated antihuman IL-10) 1:250 (2 Mg/mL) in 1× PBS supplemented with 10% FCS (see Note 22).
5. Add 100 μL of detection Ab suspension per well and incubate with lid covering the microplate for 2 h at room temperature.
6. After 2-h incubation, flick Ab suspension into a sink, invert microplate, and blot dry on paper towels.
7. Wash wells three times with 200 ML/well with wash buffer (1xPBS supplemented with 0.05% Tween 20). Allow wells to soak 1–2 min each wash.
8. Add 100 ML per well of dilute enzyme conjugate [(Streptavidin HRP) 1:100 in 1× PBS containing 10% FCS] (see Note 23).
9. Cover microplate and incubate it for 1 h at room temperature.
10. After 1 h of incubation, discard dilute enzyme conjugate into the sink by flicking.
11. Wash wells four times with 200 μL/well of wash buffer (1xPBS supplemented with 0.05% Tween 20). Allow wells to soak 1–2 min each wash.
12. Wash wells two times with 200 μL/well of 1x PBS. After final wash invert microplate and blot dry on paper toweling.
13. Add 100 μL of prewarmed to room temperature 3,3c ,5,5c -tetramethylbenzidine (TMB) to each well and incubate microplates in the dark for 30 min (see Note 24).
14. Discard TMB into the sink by flicking.
15. Remove plastic backing from the microplate and discard. Stop substrate reaction by rinsing both the back and front of the microplate in DI water three times.
16. Invert microplate and blot dry on paper toweling; wipe the bottom of the microplate dry with paper toweling.
17. Allow microplate to air-dry overnight.
18. Once the microplate is completely dry, the spots per well can be counted using an automated ELISPOT reader such as an ImmunoSpot® reader or manually using a stereomicroscope.

Detection of IFNγ Secreting Cells

1. After PVDF microplate has incubated 24 h, discard medium from the microplate into a sink by flicking. Invert microplate and blot dry on paper towels.
2. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot until dry. To make wash buffer, add 50 mL of wash buffer concentrate (provided in the kit) to 450 mL of H2O.
3. Immediately prior to use, prepare detection antibody (biotinylated antihuman IFNγ) by mixing 100 μL of detection Ab concentrate with Dilution Buffer 1. Mix thoroughly, then add 100 μL of Ab suspension per well.
4. Incubate microplate overnight at 4°C.
5. The following day, discard detection Ab suspension from the microplate into a sink by flicking; invert microplate and blot dry.
6. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot dry.
7. Directly prior to use, prepare Streptavidin-AP by adding 100 μL of Streptavidin-AP concentrate A to bottle of Dilution Buffer 2. Mix thoroughly, then add 100 μL of Streptavidin-AP suspension per well.
8. Incubate microplate for 2 h at room temperature.
9. Prewarm BCIP/NBT Chromogen substrate to room temperature using a 37°C water bath.
10. After the microplate has incubated for 2 h, discard Streptavidin-AP solution from wells into a sink by flicking.
11. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot dry.
12. Add 100 μL of BCIP/NBT Chromogen substrate per well.
13. Incubate microplate in the dark for 30 min at room temperature.
14. After the microplate has incubated for 30 min in the dark, discard BCIP/NBT Chromogen substrate from wells into a sink by flicking; invert microplate and blot dry.
15. Remove plastic backing from the microplate and discard. Stop substrate reaction by rinsing both the back and front of the microplate in DI water three times.
16. Invert microplate and blot dry on paper toweling; wipe the bottom of the microplate dry with paper toweling.
17. Allow microplate to air-dry overnight.
18. Once the microplate is completely dried, the spots per well can be counted using an automated ELISPOT reader such as an ImmunoSpot® reader from CTL or manually using a stereomicroscope.

Detection of IFNγ CD8+ Secreting T Cells

1. After the microplate has incubated for 6 h in 37°C in a 5% CO₂ humidified incubator, discard the contents of the microplate wells into a container containing bleach within a sterile environment by flicking. Invert microplate and blot dry on paper toweling.
2. Remove any unbound cells by washing microplate with 250 μL of 1× sterile PBS three times. After each wash, invert microplate and blot dry.
3. After three washes, add 100 μL of RPMI culture medium supplemented with 5% FCS to each well.
4. Wrap microplate in aluminum foil and incubate microplate for 18 additional hours (24 h total) in a 5% CO2 humidified incubator.
5. After the microplate has incubated for 18 h, discard medium from wells into a sink by flicking.
6. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot dry. To make wash buffer, add 50 mL of wash buffer concentrate (provided in the kit) to 450 mL of H₂O.
7. Immediately prior to use, prepare detection antibody (biotinylated antihuman IFNγ) by mixing 100 μL of detection Ab concentrate with Dilution Buffer 1. Mix thoroughly, then add 100 μL of Ab suspension per well.
8. Incubate microplate overnight at 4°C.
9. The following day, discard the content of the microplate wells into a sink by flicking; invert microplate and blot dry.
10. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot dry.
11. Directly prior to use, prepare Streptavidin-AP by adding 100 μL of Streptavidin-AP concentrate A to bottle of Dilution Buffer 2. Mix thoroughly, then add 100 μL of Streptavidin-AP suspension per well.
12. Incubate microplate for 2 h at room temperature.
13. Prewarm BCIP/NBT Chromogen substrate to room temperature using a 37°C water bath.
14. After the microplate has incubated for 2 h, discard Streptavidin-AP solution from wells into a sink by flicking.
15. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot dry.
16. Add 100 ML of BCIP/NBT Chromogen substrate per well.
17. Incubate microplate in the dark for 30 min at room temperature.
18. After the microplate has incubated for 30 min in the dark, discard BCIP/NBT Chromogen substrate from wells into a sink by flicking; invert microplate and blot dry.
19. Remove plastic backing from the microplate and discard. Stop substrate reaction by rinsing both the back and front of the microplate in DI water three times.
20. Invert microplate and blot dry on paper toweling; wipe the bottom of the microplate dry with paper toweling.
21. Allow microplate to air-dry overnight.
22. Once the microplate is completely dry, the spots per well can be counted using an automated ELISPOT reader such as an ImmunoSpot® reader from CTL or manually using a stereomicroscope.

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