ELISPOT Assay to Detect Cytokine-Secreting Murine ...

ELISPOT Assay to Detect Cytokine-Secreting Murine and Human Cells The filter immunoplaque assay, otherwise called the enzyme-linked immunospot assay (ELISPOT), was initially developed to detect and quantitate individual antibody-secreting B cells (UNIT 7.14). At the time it was developed, the technique provided a rapid and versatile alternative to conventional plaque-forming cell assays. Recent modifications have improved the sensitivity of the ELISPOT assay such that cells producing as few as 100 molecules of specific protein per second can be detected. These assays take advantage of the relatively high concentration of a given protein (such as a cytokine) in the environment immediately surrounding the protein-secreting cell. These cell products are captured and detected using high-affinity antibodies.

UNIT 6.19

BASIC PROTOCOL

The ELISPOT assay utilizes two high-affinity cytokine-specific antibodies directed against different epitopes on the same cytokine molecule: either two monoclonal antibodies or a combination of one monoclonal antibody and one polyvalent antiserum. ELISPOT generates spots based on a colorimetric reaction that detects the cytokine secreted by a single cell. The spot represents a “footprint” of the original cytokine-producing cell. Spots are permanent and can be quantitated visually, microscopically, or electronically. The ELISPOT assay involves five specific steps: (1) coating a purified cytokine-specific antibody to a nitrocellulose-backed microtiter plate; (2) blocking the plate to prevent nonspecific absorption of any other proteins; (3) incubating the cytokine-secreting cells at several different dilutions; (4) adding a labeled second anti-cytokine antibody; and (5) detecting the antibody-cytokine complex (Fig. 6.19.1). CAUTION: When working with human blood cells or infectious agents, biosafety practices must be followed (see Chapter 7). NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used. Materials For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see APPENDIX 2; for suppliers, see APPENDIX 5.

Cytokine-specific primary antibody, purified (Table 6.19.1) Coating buffer (UNIT 7.12) Wash solution: PBS containing 0.25% (v/v) Tween 20 Blocking solution: PBS containing 5% (w/v) BSA or FCS Complete RPMI-10 medium (APPENDIX 2) Cytokine-secreting cells ELISPOT dilution buffer: PBS containing 1% (w/v) BSA Labeled secondary cytokine-specific antibody in ELISPOT dilution buffer (Table 6.19.2) Detecting antibody or protein appropriate for cell type (see recipe) Phosphate-buffered saline (PBS, APPENDIX 2) BCIP/NBT solution (Kirkegaard & Perry; prepare fresh according to manufacturer’s instructions) Aminoethylcarbazole (AEC) solution (see recipe) 96-well nitrocellulose-backed microtiter plates (Millipore) Humidified 37°C, 5% CO2 incubator Dissecting microscope Additional reagents and equipment for stimulating cells (UNITS 3.12, 3.13, 7.10 & 7.19) Contributed by Dennis M. Klinman and Thomas B. Nutman Current Protocols in Immunology (1994) 6.19.1-6.19.8 Copyright © 1994 by John Wiley & Sons, Inc.

Cytokines and Their Cellular Receptors

6.19.1 Supplement 10

coat nitrocellulose-backed microtiter plates with cytokine-specific primary antibody Ab

Ab

block

incubate with cytokine-secreting cells; bind secreted cytokine to primary antibody

C Ab

Ab

wash B

B

Ab

Ab

Ab

Ab

incubate with biotinylated secondary antibody

wash E

B

B

Ab

Ab

Ab

Ab

E

incubate with enzyme-conjugated detection protein

wash add substrate and observe color change; count spots (cytokine-secreting cells) under low magnification

Figure 6.19.1 Schematic of ELISPOT procedure. Abbreviations: Ab, antibody; B, biotin label; C, cytokine-secreting cells; E, enzyme-conjugated detection protein.

Coat nitrocellulose-backed microtiter plate with cytokine-specific primary antibody 1. Add an optimal amount of purified cytokine-specific primary antibody in coating buffer in a volume of 50 µl to wells of nitrocellulose-backed microtiter plate. Cover plate with lid or plastic wrap. Incubate 2 hr at room temperature or overnight at 4°C. ELISPOT Assay for CytokineSecreting Cells

The optimal concentration of cytokine-specific primary antibody should be determined in advance (see Table 6.19.1). The optimal coating conditions are those with which the most distinct ELISPOTs are obtained. It is generally necessary to use concentrations


Current Protocols in Immunology

Table 6.19.1

Primary Coating Antibodies

Concentration Sourceb (µg/ml)a

Cytokine

Clone

Mouse IL-2 IL-3 IL-4

JES6-1A12 MP2-8F8 BVD4-1D11

10 10 5

Ph E E

IL-5 IL-6 IL-10 IFN-γ

TRFK5 MP5-2053 JES5-2A5 RMMG-1

5 13 20 10

E Ph Ph B

Human IL-4 IL-5

MP4-25D2 JES1-39D10

10 10

Ph Ph

IFN-γ IL-10 GM-CSF

C1-D16 JES3-9D7 BVD2-23B6

5 10 10

C Ph Ph

aFinal working concentration. bAbbreviations: B, Biosource; C, Chromogenix; E, Endogen; LB, Lee

Biomolecular; Ph, Pharmingen. For suppliers’ addresses, see APPENDIX 5.

5- to 10-fold higher than those used in conventional ELISA assays (e.g., 5 to 10 ìg/ml). The antibodies are usually diluted in carbonate buffer (pH 9.6). In some cases, better results are achieved using PBS (pH 7.2 to 7.4) or borate buffer (pH 8.4). Because each antibody used for coating has different binding characteristics, it is necessary to determine the optimal coating conditions for each antibody.

2. Discard coating antibody. Rinse plate three times with wash solution (200 µl per rinse). Shake excess liquid from plate and pat bottom of plate with dry absorbent paper. 3. Add 200 µl blocking solution to each well to saturate remaining binding sites and incubate 30 min at 37°C. 4. Discard blocking solution, wash plate three times with wash solution (200 µl per wash), shake excess liquid from plate, and pat bottom of plate with dry absorbent paper. 5. Add 100 µl complete RPMI-10 and incubate 10 min at room temperature. Discard liquid and pat bottom of plate with dry absorbent paper. The medium dilutes any remaining Tween 20 and prepares the plate for cell incubation. Although this protocol calls for RPMI-10 medium, any culture medium that is appropriate for a given type of cell or cell line can be used (see APPENDIX 2 for recipes).

Incubate cytokine-secreting cells 6. Serially dilute cells with complete RPMI-10 in a separate microtiter plate. Add a suspension of single cells to the coated microtiter plate wells in a final volume of ≤100 µl medium. When volumes >100 ìl are used, leakage through the permeable nitrocellulose bottom plate can occur.


6.19.3 Current Protocols in Immunology

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Table 6.19.2

Secondary Antibodies

Cytokine

Antibody

Label

Concentrationa

Sourceb

Mouse IL-2 IL-3 IL-4 IL-5 IL-6 IL-10 IFN-γ

JES6-5H4 MP2-43D11 BVD6-24G2 TRKF4 MP5-32C11 SXC-1 R46A2

Biotin Biotin Biotin Biotin Biotin Biotin Biotin

2 µg/ml 0.33 µg/ml 0.33 µg/ml 0.25 µg/ml 1 µg/ml 1 µg/ml 0.125 µg/ml

Ph E E E Ph Ph LB

Human IL-4 IL-5 IFN-γ IL-10 GM-CSF

Rabbit anti-IL-4c JES1-5A10 766 JES3-12G8 BVD2-21C11

— Biotin Biotin Biotin Biotin

10 µg/ml 1 µg/ml 3 µg/ml 500 ng/ml 500 ng/ml

G Ph C Ph Ph

aFinal working concentration. bAbbreviations: C, Chromogenix; E, Endogen; G, Genzyme; LB, Lee Biomolecular; Ph,

Pharmingen. For suppliers’ addresses, see APPENDIX 5. cUsed in conjunction with a third detecting antibody—e.g., the alkaline phosphatase–conjugated goat anti-rabbit IgG (Fc-specific; see Reagents and Solutions)

Cells should be plated as soon as they are ready. In the case of adherent cells that must be harvested prior to use, it is not necessary to allow them to rest before use. For peripheral blood cells, a red cell lysis step should be performed (UNIT 3.1). For mouse spleen cells, red cell lysis is not necessary, because they are not present in sufficient quantity to interfere with the formation of a mononuclear cell monolayer. Two- to four-fold cell dilutions starting at 105 or 106 cells per well are appropriate. Typically, ≤106 cells/well is a good starting point for cells that have not been tested. Each experimental condition must be done in triplicate or quadruplicate to ensure well-to-well consistency. If available, a cloned cell line producing the cytokine of interest should also be plated as a positive control.

7. Add mitogens, antigens, or other stimulus to the cells. Maintain unstimulated cells (medium alone) to be used to assess background cytokine production and in vivo activation. Stimulation can occur prior to addition of the cells or during incubation on the plate. For standard ELISPOT analyses, the stimuli can be added directly to the cells while in the nitrocellulose-backed microtiter plates. For information about stimulating cells, see UNITS 3.12, 3.13, 7.10 & 7.19. If there is a concern about de novo cytokine production (as opposed to release of preformed cytokine), a protein inhibitor such as cycloheximide (100 ìg/ml) can be used as an additional control. Cycloheximide should be added to 3 or 4 replicate wells, for both unstimulated and stimulated cells, at the initiation of culture.

8. Incubate cells 6 to 24 hr on a level surface in a humidified 37°C, 5% CO2 incubator.

ELISPOT Assay for CytokineSecreting Cells

The length of incubation depends upon the type of cell involved and the nature of the stimulus. For many cytokines, there is little spontaneous production; thus for the majority of cytokines, a stimulus will be necessary to enumerate cells capable of secreting a given cytokine. Each cytokine has its own specific kinetics, and appropriate conditions must be determined for a given set of stimuli. If the incubation is too short, reduced numbers of spots will result. If the incubation is too long, there will be an increase in the assay background.



9. Wash plate extensively (∼10 times) with wash solution (200 µl per wash), shake excess liquid from plate, and pat dry. Removing the cells from the plate is critical. If cells are left behind because of insufficient washing, a high background will result. Distilled water may be used for the final wash, as its hypotonicity will lyse any remaining cells.

Detect spot-forming cells 10. Add 50 µl labeled cytokine-specific secondary antibody in ELISPOT dilution buffer to each well. Incubate 2 hr at room temperature. The appropriate concentration of this antibody must be determined in advance (see suggested concentrations in Table 6.19.2). If the labeled (e.g., biotin-labeled) antibody cannot be obtained commercially, biotinylation can be performed (see UNIT 7.12).

11. Discard the antibody and wash plate six times with wash solution (200 µl per wash). Shake excess fluid from plate and pat dry. 12. Add 50 µl alkaline phosphatase–labeled detecting antibody or protein (e.g., avidin, streptavidin, or goat anti-rabbit IgG) and incubate 2 hr at room temperature. Horseradish peroxidase (HRPO)–labeled detecting antibodies or proteins can also be used. If so, however, a different substrate must be used (see step 14b). The appropriate concentration of the detecting antibody or protein must be determined in advance.

13. Wash plate six times and pat dry as in step 11. Wash the plate once in PBS without Tween 20 and pat dry. 14a. For alkaline-phosphatase–labeled detection: Add 50 µl BCIP/NBT solution to each well and incubate 5 to 30 min at room temperature until color (blue spots) develops. 14b. For HRPO–labeled detection: Add 50 µl AEC solution to each well and incubate 5 to 30 min at room temperature until color (brownish spots) develops. For the positive control, every well plated should yield a spot (100% efficiency).

Fig. 6.19.2 Photograph of an alkaline phosphatase–based ELISPOT for IL-4 shown at 30× magnification. Note the positive spots showing fuzzy borders.



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15. Wash three times with distilled water (200 µl per wash) and air dry. Quantitate spots using a dissecting microscope. The spots can be counted using 10× to 30× magnification any time after they have formed (Fig. 6.19.2). Quantitation can be made more mechanized by image analysis using video camera technology or a simple photograph of each well. A magnifying glass can also be used.

REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2; for suppliers, see APPENDIX 5.

Aminoethylcarbazole (AEC) solution Dissolve 2.5 mg AEC in 200 µl n,n-dimethylformamide (nDF). Add 9 ml of 0.05 M sodium acetate, pH 5.0, and 4 µl of 30% (v/v) H2O2. This solution should be prepared immediately before use.

Detecting antibody or protein The following detecting antibody or proteins may be used: (1) alkaline-phosphatase conjugated Avidin D (for murine assays; Vector); (2) alkaline phosphatase–labeled or horseradish peroxidase–labeled streptavidin (for human assays; Jackson Immunoresearch); or (3) alkaline phosphatase–labeled or horseradish peroxidase–labeled goat anti-rabbit IgG (Fc-specific; Jackson Immunoresearch). The optimal concentrations of the antibody or protein must be determined in advance.

COMMENTARY Background Information


The ELISPOT assay for detecting individual cytokine-secreting cells is based on the use of highly specific monoclonal and polyclonal antibodies to human and murine cytokines. The procedure is based on the ELISPOT assay, initially described to detect individual B cells secreting antibody, for use in polystyrene plates with alkaline phosphatase (Sedgwick and Holt, 1983) or horseradish peroxidase (Czerkinsky et al., 1983) and later modified for use on nitrocellulose plates (Moller and Borrebaeck, 1985; Czerkinsky et al., 1988b). Although two-color analysis has been used successfully to detect the production of two different immunoglobulin isotypes on the same plate (Czerkinsky et al., 1988a), two-color staining for cells secreting more than one cytokine has been less successful, presumably because of relatively lower local protein concentration. However, a “sandwich” ELISPOT assay, in which the primary antibody is directed to one cytokine and the labeled secondary antibody is directed to a different cytokine, can be used to detect single cells producing two different cytokines simultaneously (Shirai et al., in press). The ELISPOT technique can be utilized in almost all systems where ELISA measure-

ments can be made, although the information obtained from the ELISPOT assay differs from that obtained in ELISAs. First, the ELISPOT technique is 20- to 200-fold more sensitive than ELISA (Shirai et al., 1993) in that cells secreting a particular cytokine can be detected in situations in which cytokine protein cannot be measured in cell-free supernatants (King et al., 1993). Moreover, this technique gives additional information about the frequency of cytokine-secreting cell populations that is not obtainable from conventional ELISAs or biosassays. When used in parallel with ELISAs, the ELISPOT technique can provide an estimate of the amount of cytokine protein produced on a per-cell basis (King et al., 1991). When coupled with cell separation techniques, the frequency of leukocyte subpopulations secreting a given cytokine can also be determined. Although this has been used most successfully for T cell subpopulations obtained from peripheral blood mononuclear cells (PBMCs; Mahanty et al., 1992), it has also been applied to purified monocyte and granulocyte populations. Using cloned cell lines (e.g., CHO) stably transfected with specific cytokine genes, this technique has an efficiency that reaches 100%—i.e., every cell plated can be detected.



Nevertheless, it is likely that, even when stimulated maximally, some cells purified from peripheral blood, spleen, tonsils, or lymph nodes may produce cytokines in concentrations too low to be detected by ELISPOT. Ongoing studies are directed toward determining the frequency of cytokine-producing cells in normal and disease states (Shirai, 1993). These frequencies depend upon the cytokine being studied, the cell subpopulation being analyzed, and the presence or absence of exogenous stimuli.

Critical Parameters The appearance of spots for any given cytokine must be carefully defined in preliminary experiments. For the human system, finding cell lines that constitutively express a high level of a given cytokine makes optimization experiments much easier. In the absence of such a defined cell line, making or obtaining cell lines stably transfected with a given cytokine gene is certainly possible (UNITS 10.15 & 10.16). This makes it possible to take advantage of the fact that every cell placed on a microtiter plate will be producing the given cytokine; thus, there should be a one-to-one correlation between the number of cells placed on the plate and the number of spots that are seen after the assay is optimized. The major factor in successful frequency analysis for cytokines using ELISPOT is the availability of high-affinity purified anti-cytokine antibodies that are directed against different epitopes on the cytokine molecule. Because the ELISPOT assay requires a labeled secondary anti-cytokine antibody (which occasionally must be labeled by the investigator), using antibody that is affinity-purified, purified from nude mouse ascites, or in serum-free medium allows for the highest signal-to-noise ratio. Because many cytokines are not constitutively expressed, it is important to determine the optimal type, strength, and duration of stimulus that induces each cytokine. Nevertheless, with every set of stimuli, a control set of replicate unstimulated cultures should be used to identify spontaneous cytokine production.

Troubleshooting When few or no ELISPOTs are detected among cells that have been appropriately stimulated, the problem usually lies in the selection of anti-cytokine antibodies (most commonly the coating antibody). Occasionally, the substrate solutions or the enzyme-labeled de-

veloping antibody lose activity and need to be replaced. One useful approach to solving the technical problems associated with cytokine ELISPOT assays is to include positive control cells on each plate—i.e., cells that constitutively produce a given cytokine (or set of cytokines). Once identified or constructed, these cells can be divided into aliquots, cryopreserved (APPENDIX 3), thawed, and included in each experiment as a positive control. Two types of background problems can be seen in an ELISPOT assay. The first is a diffuse darkening of the nitrocellulose membrane. This is most commonly observed when large numbers of cytokine-secreting cells per well are studied, and reflects the accumulation of secreted cytokine in the culture supernatant and its subsequent binding to the membrane. This diffuse background can be reduced by decreasing either the number of cells incubated in each well or the duration of the cell incubation. The second type of background problem is the appearance of very small, very dark spots that do not reflect secreted cytokine. The trained observer can usually discriminate between real ELISPOTs and these artifacts; real spots have dark centers and light rims (the latter formed by the diffusion of cytokine outward from the secreting cell). Artifactual spots are smaller and of uniform intensity. They may appear when the anti-cytokine antibodies used in the ELISPOT assay aggregate, but more frequently are caused by inadequate removal of cells from the plate. Some cells have endogenous phosphatase activity, or nonspecifically bind biotin-labeled antibodies, leading to the formation of these artifacts. This problem can generally be prevented by washing the plates at least once in distilled water.

Anticipated Results The spots appear blue when alkaline phosphatase (with BCIP/NBT) is used and brownish when horseradish peroxidase (with AEC) is used. They are granular in appearance and tend to be darker at the center than at the periphery. This method should have 100% efficiency for the positive control cells—every plated cell should result in a spot. The efficiency of experimental preparations will vary depending on the cell type and experimental conditions.

Time Considerations The procedures vary with the cytokine to be measured. The major time commitment in-



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volves incubating cells with their appropriate stimulus. In general, between 5 and 20 hr are required to optimize ELISPOT formation. Although washing the plates takes only a few minutes, plates should be treated with antibody ≥1 hr at 37°C or ≥2 hr at room temperature. Once the assay is finished, the spots can be counted at the convenience of the investigator. Although spots can develop in a few minutes, they become more distinct after 24 hr, and do not fade over time.

Literature Cited Czerkinsky, C.C., Nilsson, L.A., Nygren, H., Ouchterlony, O., and Tarkowski, A. 1983. A solidphase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65:109-121. Czerkinsky, C., Andersson, G., Ekre, H.P., Nilsson, L.A., Klareskog, L., and Ouchterlony, O. 1988a. Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gammainterferon-secreting cells. J. Immunol. Methods 110:29-36. Czerkinsky, C., Moldoveanu, Z., Mestecky, J., Nilsson, L.A., and Ouchterlony, O. 1988b. A novel two colour ELISPOT assay. I. Simultaneous detection of distinct types of antibody-secreting cells. J. Immunol. Methods 115:31-37. Czerkinsky, C., Anderson, G., Ferrua, B., Nordstrom, I., Quiding, M., Eriksson, K., Larson, L., Hellstrand, K., and Ekre, H. 1991. Detection of human cytokine-secreting cells in distinct anatomical compartments. Immunol. Rev. 119:5-22. King, C.L., Poindexter, R.W., Ragunathan, J., Fleisher, T.A., Ottesen, E.A., and Nutman, T.B. 1991. Frequency analysis of IgE-secreting B lymphocytes in persons with normal or elevated serum IgE levels. J. Immunol. 146:1478-1483.

King, C.L., Low, C.C., and Nutman, T.B. 1993. IgE production in human helminth infection. Reciprocal interrelationship between IL-4 and IFNgamma. J. Immunol. 150:1873-1880. Mahanty, S., Abrams, J.S., King, C.L., Limaye, A.P., and Nutman, T.B. 1992. Parallel regulation of IL-4 and IL-5 in human helminth infections. J. Immunol. 148:3567-3571. Moller, S.A. and Borrebaeck, C.A. 1985. A filter immuno-plaque assay for the detection of antibody-secreting cells in vitro. J. Immunol. Methods 79:195-204. Sedgwick, J.D. and Holt, P.G. 1983. A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J. Immunol. Methods 57:301-309. Shirai, A., Holmes, K., and Klinman, D.M. 1993. Detection and quantitation of cells secreting IL-6 under physiologic conditions in BALB/c mice. J. Immunol. 150:793-799. Shirai, A., Sierra, V., Kelly, C.I., and Klinman, D.M. Individual cells simultaneously produce both IL4 and IL-6 in vivo. Cytokine. In press.

Key Reference Czerkinsky et al., 1991. See above. Provides useful overview of available cytokine ELISPOT assays.

Contributed by Dennis M. Klinman Center for Biologics Evaluation & Research Food and Drug Administration Bethesda, Maryland Thomas B. Nutman National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland


