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A novel two colour ELISPOT assay
Journal of Immunological Methods, 115 (1988) 31-37 Elsevier
31
JIM 04952
A novel two colour ELISPOT assay I. Simultaneous detection of distinct types of antibody-secreting cells Cecil Czerkinsky
1,2,3, Zina
Moldoveanu 1, Jiri Mestecky 1, Lars_/~& e Nilsson 2 and Orjan Ouchterlony 2
Department of Microbiology, Universityof Alabama at Birmingham, Birmingham, AL, U.S.A., and Departments of 2 Medical and 3 Oral Microbiology, University of Gothenburg, Gothenburg, Sweden (Received 26 April 1988, revised received 6 June 1988, accepted 29 June 1988)
A novel assay system has been developed which is based on the ELISPOT methodology and employs a combination of two immunoenzyme visualization systems yielding distinct colour products. This variation permits the simultaneous enumeration of two different types of cell secreting antigenically distinct products. Optimal conditions for the concurrent detection of human mononuclear cells secreting IgG or IgA antibodies are described. Key words: ELISPOT, two-color; Antibody-secreting cell
Introduction
Recently, a simple approach based on solidphase immunoenzyme technology has been developed for enumerating antibody-secreting cells (ASC). Since its original description (Czerkinsky et al., 1983; Sedgwick and Holt, 1983), the procedure, termed ELISPOT or ELISA plaque assay, has been employed as an alternative to conventional plaque-forming cell assays (Jerne and Nordin, 1963) to enumerate specific as well as total immunoglobulin-secreting cells and also to detect a variety of cells (lymphoid or nonCorrespondence to: C. Czerkinsky, Departments of Medical and Oral Microbiology, Guldhedsgatan 10, S-413 46 Gothenburg, Sweden. Abbreviations: ASC, antibody-secreting cells; ELISPOT, enzyme-linked immunospot; AP, alkaline phosphatase; HRP, horseradish peroxidase; PBMC, peripheral blood mononuclear cells; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/pnitroblue tetrazolium; AEC/H202, 3-amino-9-ethyl-carbazole/hydrogen peroxide; SFC, spot-forming cell.
lymphoid) secreting antigenic substances (for reviews see Czerkinsky et al., 1988; Sedgwick and Holt, 1988). The original techniques employed either alkaline phosphatase (AP)- (Sedgwick and Holt, 1983) or horseradish peroxidase (HRP)- (Czerkinsky et al., 1983) labelled antibodies and corresponding chromogen substrates as indicator systems for the detection of antibody producing cells. In this communication, we have evaluated a combination of both indicator systems for the concurrent detection of distinct types of ASC. In the procedure which we describe, zones of solid phase-bound IgG and Ig,A antibodies secreted from distinct cells are visualized as blue or red spots corresponding to either of these isotypes. Materials and methods Cells
Four adult human volunteers received a subcutaneous injection of a trivalent influenza virus
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
32 vaccine, types A and B (Wyett Laboratories, PA). Peripheral blood mononuclear cells (PBMC) were collected from 5 to 12 days after immunization, that is at a time when the frequency of antigenspecific spontaneous ASC in the circulation is known to be maximal (Stevens and Saxon, 1978; Stevens et al., 1979; Yarchoan et al., 1981). PBMC were isolated from heparinized venous blood by centrifugation on Ficoll-Hypaque (Bt~yum, 1968). Interface PBMC were washed twice with isotonic phosphate-buffered saline (0.0l M phosphates, 0.15 M NaC1, pH 7.4) (PBS), and resuspended at the appropriate densities in assay culture medium. The latter consisted of RPMI 1640 (Gibco, Glasgow, Scotland) supplemented with 5% foetal bovine serum (FBS) (Irvine Scientific, Santa Ana, CA).
Reagents AP- or HRP-conjugated affinity-purified goat anti-human IgA and goat anti-human IgG antibodies were purchased commercially (Southern Biotechnology Associate, Birmingham, AL). The specificity of these preparations was further documented by the two-colour ELISPOT assay described below. The AP chromogen substrate solution, consisting of 5-bromo-4-chloro-3-indolyl phosphate toluidine salt and p-nitroblue tetrazolium chloride ( B C I P / N B T ) was prepared according to the manufacturer's instructions (BioRad Laboratories, Richmond, CA). Initially, 15 mg of BCIP reagent and 30 mg of NBT salt were separately dissolved with 1 ml of dimethylformamide (DMF) and then added to 100 ml of a solution of 0.1 M NaHCO3, 1 mM MgC12, pH 9.8. The HRP chromogen substrate solution was prepared by dissolving 25 mg of 3-amino-9-ethyl carbazole (AEC) (Sigma, St. Louis, MO) in 2 ml of DMF, followed by addition of 95 ml of 0.05 M acetate buffer, pH 5.0 ( + 0.2) and 40 /~1 of 30% H202. The above substrate solutions were filtered (0.45 /~m) to remove particulate matter (the AEC/H202 solution becomes colourless after filtration while the B C I P / N B T solution remains pale yellow). Both enzyme substrate solutions could be kept in the dark at 4 ° C for up to 1 week. Two-colour ELISPOT assay The assay consists of five stages: first, a solid-
phase immunoadsorbent is prepared; the second stage, incubation of the cell suspension, is followed by the addition of a mixture of AP- and HRP-conjugated antibodies. The fourth stage comprises the stepwise addition of AP and HRP chromogen substrates which will yield insoluble blue and red spots, respectively. These spots are enumerated in the fifth and final stage. Solid-phase immunoadsorbent. The standard ELISPOT assay was modified by using nitrocellulose membranes as the solid support instead of polystyrene, as described by M/511er and Borrebaeck (1985). Individual wells of nitrocellulose bottomed 96-well Millititer HA plates (Millipore, Bedford, MA) were filled with 0.075-0.1 ml of PBS containing 0.2 ~tg of influenza virus hemagglutinin (Wyett Laboratories). This coating concentration was found optimal in a preliminary checkerboard titration experiment. Plates were allowed to stand overnight at 4 o C. (Coated plates can be stored for several weeks at 4 °C filled with coating solution, or for several months at - 2 0 °C decanted of coating solution.) Unadsorbed proteins were removed by three successive manual washings with PBS (by flicking) and the plates were immersed in this buffer for 5 min. Wells were then emptied of wash buffer and the outer surface of the nitrocellulose membrane was carefully dried with absorbent paper towels. In order to saturate remaining protein binding sites on the solid support individual wells were filled with 0.2 ml of assay culture medium and the plates were incubated at 37°C for at least 30 min in a humidified atmosphere with 7% CO 2. Wells exposed to an irrelevant antigen, tetanus toxoid (TT) (Wyett) (0.1 /~g/well), were prepared in the same way for control purposes. Cell incubation. The content of the wells was replaced with 0.1 ml of cell suspensions containing various numbers of PBMC. Routinely, we used at least three sets of triplicate wells. Each set of wells received 2 x 105, 105 and 5 x 104 PBMC/well. Plates were then incubated undisturbed for 3-4 h at 37 °C in a CO 2 incubator. In one experiment, PBMC were incubated for 5 h at 37°C with various concentrations (5 x 10 - 4 M , 10 - 3 M, 2 × 10-3M) of cycloheximide (Sigma) in assay culture medium, washed, resuspended and plated in cycloheximide containing medium.
33
Immunoenzyme stage. At the completion of the cell incubation period, plates were rinsed three times manually with PBS and three times with PBS containing 0.05% Tween 20 (PBS-T) and were then immersed in PBS-T for 5 min. The dishes were emptied of wash buffer and the outer surface of the plate was blot-dried as described in the first stage. Next, 0.1 ml of PBS-T containing 1% FBS and a mixture of goat anti-human IgG and goat anti-human IgA antibodies conjugated with AP and HRP, respectively or vice versa, was added to each well. Optimal concentrations of AP- and HRP-labeUed antiglobulins were determined in preliminary experiments. Concentrations ranging from 0.5 ~tg/ml to 2.5 ttg/ml were used for both types of enzyme conjugate. Plates were incubated for 3 h at room temperature or overnight at 4°C (whichever was most convenient). Dishes were then rinsed four times with PBS (without detergent) and immersed in 0.05 M Tris buffer saline, pH 8.0, for 5 rain prior to development. Development. Dishes were emptied of wash buffer and blot-dried as above. The wells were then exposed to 0.1 ml of BCIP/NBT substrate solution and examined for the appearance of blue spots. These reactions appeared usually within 5-10 rain. Plates were allowed to develop for an additional period of 5-10 min, after which they were rinsed with PBS and blot-dried. Next, 0.1 ml of AEC/H202 substrate was added to each well, yielding red spots within 1-3 min. Plates were allowed to develop for a variable time (up to 10 rain) and then thoroughly rinsed with tap water. As excessive background staining may result from over-long incubation times, it is important to monitor control wells (not exposed to cells) during the enzyme-substrate reaction. Enumeration of spots. Developed plates were dried and individual wells were examined for the presence of blue and red spots. These reactions were enumerated under low magnification ( x 40 to x to) (a stereomicroscope equipped with a vertical white light source is ideal for this purpose). Under low magnification, positive reactions were defined as circular, well individualized, densely granulated foci contiguous to the background. Their diameter ranged from 0.05 mm to 0.2 mm. Small dense particles could occasionally be observed particularly with over-exposure to either
enzyme substrate. These non-granular dots generally appeared above the plane of the background and could be distinguished from true spots.
Results and discussion
A two-colour immunoenzyme procedure is described for the concurrent detection of two antigenically distinct products secreted by different cells. The procedure follows the general principle of the ELISPOT assay which, in its original descriptions, relied on the use of either alkaline phosphatase (Sedgwick and Holt, 1983) or horseradish peroxidase (Czerkinsky et al., 1983) as indicator enzymes for the detection of antibodysecreting cells. The technique described here now makes use of two antiglobulin conjugates, one labelled with AP, the other with HRP, having distinct immunological specificities. When used in combination with BCIP/NBT and AEC/H202 chromogen substrates, AP and HRP activities can be visualized on the same solid phase as insoluble blue and red products, respectively. In the present study, we have employed this technique to detect simultaneously PBMC secreting IgG- and PBMC-secreting IgA anti-influenza virus antibodies in the circulation of human vaccinees. When plated for a few hours in influenza virus-coated wells, PBMC from recently immunized human volunteers secreted IgA and IgG antibodies. Zones of solid-phase bound IgA and IgG antibodies secreted by distinct cells were visualized by sequential addition of a mixture of anti-IgG and anti-IgA antibody reagents labelled with AP and HRP (or vice versa) and of corresponding chromogen substrates. Within minutes following addition of BCIP/NBT substrate, blue spots appeared on the solid phase at the former location of PBMC-secreting antibodies of a given isotype, e.g., IgA. Subsequent exposure of the solid phase to AEC/H202 substrate yielded red spots corresponding to cells secreting antibodies of a different isotype, e.g., IgG (Fig. 1). Similar numbers of red spots (HRP-AEC/H202 products) were detected in wells that had been previously exposed to both BCIP/NBT and AEC/H202 substrates as compared to wells treated only with AEC/H202 substrate (Table I).
34
35 TABLE I COMPARISON BETWEEN ONEoCOLOUR AND TWOCOLOUR ELISPOT ASSAYS FOR ENUMERATING ANTIGEN SPECIFIC IgG- AND IgA-SECRETINGCELLS Peripheral blood PBMC were obtained from one volunteer 7 days after immunization with influenza virus vaccine. Values are expressed as mean spot-forming cell (SFC) numbers (+ SEM) of quadruplicate assay wells. SFC numbers/106 PBMC in wells developedwith: HRP anti-IgG HRP anti-IgA 5080 (621) a AP anti-IgA 3920 (420) b
3 730 (470)
3445 (376) AP anti-IgG -
4860 (515)
4153 (537)
4540 (420)
3636 (354)
-
Upper values correspond to numbers of red spots developed with HRP-conjugated anti-Ig antibodies and AEC/H202 chromogen substrate. b Lower values indicated in'italics correspond to numbers of blue spots developed with AP-conjugated anti-Ig and BCIP/NBT substrate. a
This observation indicates that A P - B C I P / N B T reactions have no appreciable effects on subsequent development of H R P - A E C / H 2 0 2 reactions. Further, the sensitivity of both enzyme-substrate systems appeared comparable since similar numbers of spot-forming cells (SFC) were detected when using the same anti-immunoglobin preparation labelled with either enzyme (Table I). The quality of the enzyme-antibody conjugates employed in this two-colour ELISPOT assay must be carefully considered as double (indigo) spots may appear with conjugates containing cross-reactive antibodies. Thus, monochromatic (red or blue) spots provide an ideal internal control of the specificity of the assay for detecting antigenically distinct products secreted by different cells. Further, although two enzyme-antibody conjugates may be equally specific, they may exhibit different affinities for binding to cognate immunoglobulins a n d / o r variable enzyme activities, resulting in different staining intensity of the corresponding spots.
In such situations, a correction factor (ratio of number of spots developed with a given antibody conjugated with HRP: number of spots developed with the same antibody preparation but labelled with AP) should be applied to adjust for possible differences in sensitivity. Under optimal conditions PBMC-secreting IgG antibodies and PBMC-secreting IgA antibodies to influenza virus could be detected simultaneously in all four volunteers examined. Virus-specific ASC were detected as early as 5 days after systemic immunization with influenza virus vaccine. Spot forming cell (SFC) numbers reached a maximum on day 7. By day 9-12 the frequency of virusspecific SFC markedly decreased (data not illustrated). Influenza virus specific IgG-SFC and IgA-SFC responses followed a similar kinetic pattern but differed in magnitude. Thus, in three individuals, IgG-SFC predominated (5520 _+ 983 day 7 SFC/106 PBMC versus 140 _+ 69 day 7 IgA SFC/106; n = 3). In the fourth volunteer the magnitude of influenza virus-specific IgA responses on day 7 was very high (3910/106 PBMC + 420 SFC) being almost comparable to that of the IgG responses (5080 _ 621 SFC/106 PBMC). The specificity of the assay for simultaneous demonstration of influenza virus specific-IgA ASC and -IgG ASC was documented by several observations. First, omission of cells, coating antigen, or labelled antibodies prevented subsequent development of spot formation. Second, plating PBMC in wells coated with an irrelevant antigen (tetanus toxoid) resulted in the absence of detectable spots (data not shown). Third, incubation of influenza virus immune cells with graded amounts of influenza virus antigen during cell plating inhibited, in a dose dependent manner, spot formation (Table II). It should be noted that such treatment not only resulted in a reduction of SFC numbers but also in a decrease in the diameter of the remaining spots. In contrast, addition of tetanus toxoid had no effects. Finally, treatment of the cells with cycloheximide prior to and during the cell incubation period markedly inhibited influenza virus specific IgG- and IgA-mediated spot
Fig. 1. Typical appearance of a well followingdevelopmentin the two color ELISPOT assay for simultaneous detection of influenza virus specifichuman IgG antibody-secretingcells (red spots) and IgA antibody-secretingcells (blue spots). A, × 8; B, × 40.
36 TABLE II SPECIFICITY OF TWO-COLOUR ELISPOT ASSAY FOR SIMULTANEOUS DETECTION OF INFLUENZA VIRUS SPECIFIC IgA-SECRETING PBMC AND IgG-SECRETING PBMC Peripheral blood PBMC were obtained from one donor on day 7 following immunization with influenza virus vaccine. PBMC were assayed by two-colour ELISPOT assay for numbers of virus-specific ASC. IgG SFC and IgA SFC were developed with AP-conjugated anti-lgG and HRP-conjugated anti-IgA, respectively followed by BCIP/NBT (blue) and AEC/H202 (red) enzyme substrates. Values represent mean SFC numbers of quadruplicate assay wells/106 PBMC. Data in parentheses indicate percentages of inhibition. Inhibitor added per assay well
SFC numbers/106 PBMC IgG (blue)
IgA (red)
6560
220
Influenza virus 15/zg 3/~g 0.6/~g 0.12/~g
120 (98%) 1 400 (79%) 2720 (59%) 3 840 (42%)
0 (100%) 45 (80%) 96 (57%) 240 ( - 11%)
Tetanus toxoid 100 #g
6120 (6.7%)
197
(10.4%)
Cycloheximide a 2X10 -3 M 10 -3 M 5X10 - 4 M
720 (89%) 2000 (69.5%) 2960(54.8%)
20 70 100
(91%) (68.2%) (54.5%)
Treatment with cycloheximide (see section Materials and Methods) did not affect cell viability (assessed by trypan blue dye exclusion) at all 3 concentrations of drug tested. a
f o r m a t i o n ( T a b l e II). T h a t ' d o u b l e ' ( b i o c h r o m a t i c ) spots were never o b s e r v e d c o n f i r m e d the high degree of specificity of the e n z y m e l a b e l l e d antib o d y p r e p a r a t i o n s e m p l o y e d as class specific reagents in this assay. In p r e l i m i n a r y c o m p a r a t i v e experiments, the p r o c e d u r e d e s c r i b e d here a p p e a r e d at least five times as sensitive as the original E L I S P O T techniques p e r f o r m e d o n plastic surfaces a n d develo p e d with either p a r a p h e n y l e n e d i a m i n e / H 2 0 2 in a g a r ( H R P system) ( C z e r k i n s k y et al., 1983) or B C I P ( A P system) (Sedgwick a n d Holt, 1983) a m p l i f i e d with N B T ( F r a n c i et al., 1986). T h e high b i n d i n g a n d r e t e n t i o n p r o p e r t i e s of nitrocellulose s u p p o r t s for p r o t e i n antigens m a y a c c o u n t for such an increase in sensitivity. T h e use of nitrocellulose m e m b r a n e s in the E L I S P O T assay, r e c e n t l y
i n t r o d u c e d b y MOiler a n d B o r r e b a e c k (1985) also m i n i m i z e s s u b s t a n t i a l l y the r e q u i r e m e n t s of the original techniques for relatively large a m o u n t s of c o a t i n g material. In a d d i t i o n , this m o d i f i c a t i o n a p p e a r s s i m p l e r since it does n o t rely on the use of a gel overlay, as is the case with certain p e r o x i d a s e c h r o m o g e n s on plastic surfaces. W e have c o n f i r m e d the suitability of this twoc o l o u r E L I S P O T assay for d e t e c t i n g simultan e o u s l y cells secreting o t h e r Ig i s o t y p e s (IgM, I g G subclasses) in b o t h h u m a n a n d m u r i n e systems. W o r k is in p r o g r e s s to use this t w o - c o l o u r assay system for the d e t e c t i o n of distinct l y m p h o k i n e secreting cells. A d d i t i o n a l visualization systems, e.g., silver i m m u n o g o l d staining ( W a l k e r a n d Dawe, 1987) are also u n d e r e v a l u a t i o n for the c o n c u r r e n t d e t e c t i o n of m u l t i p l e i m m u n o r e a c t i v e substances. These d e v e l o p m e n t s should m a k e it p o s s i b l e to investigate w h e t h e r two or m o r e antigenically distinct p r o d u c t s originate f r o m the same or two different cells.
Acknowledgements This s t u d y was s u p p o r t e d b y U.S. Public H e a l t h Service G r a n t AI-10702 a n d b y G r a n t 08320 f r o m the Swedish M e d i c a l R e s e a r c h Council. T h e technical assistance of Miss Shirley Prince a n d Mrs. A n n e t t e Pitts is g r a t e f u l l y a c k n o w l e d g e d .
References B6yum, A. (1968) Isolation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21, 97. Czerkinsky, C., Nilsson, L.-~,., Nygren, H., Ouchterlony, O, and Tarkowski, A. (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65, 109. Czerkinsky, C., Tarkowski, A., Nilsson, L.-A., Koopman, W.J., Mestecky, J. and Ouchterlony, 0. (1988) The enzyme-linked immunospot assay for detection of specific antibodysecreting cells: methodology and applicability. In: D.M. Kemeny and S.J. Challacombe (Eds.), Theoretical and Technical Aspects of ELISA and Other Solid Phase Immunoassays. John Wiley, Oxford, p. 217. Franci, C., Ingles, J., Castro, R. and Vidal, J. (1986) Further studies on the ELISA-spot technique. Its application to particulate antigens and potential improvement in sensitivity. J. Immunol. Methods 88, 225. Jerne, N.K. and Nordin, A.A. (1963) Plaque formation in agar by single antibody-producing cells. Science 140, 405.
37 MSller, S.A. and Borrebaeck, C.A.K. (1985) A filter immunoplaque assay for the detection of antibody-secreting cells in vitro. J. Immunol. Methods 79, 195. 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. Sedgwick, J.D. and Holt, P.G. (1988) ELISA-plaque assay for the detection of single antibody-secreting cells. In: D.M. Kemeny and S.Y ChaUacombe (Eds.), Theoretical and Technical Aspects of ELISA and Other Solid Phase Immunoassays. John Wiley, Oxford, p. 241. Stevens, R.H. and Saxon, A. (1978) Immunoregulation in humans. Control of antitetanus antibody production after booster immunization. J. Clin. Invest. 62, 1154.
Stevens, R.H., Macy, E., Morrow, C. and Saxon, A. (1979) Characterization of a circulating subpopulation of spontaneous antitetanus antibody producing B cells following in vivo booster immunization. J. Immunol. 122, 2498. Walker, A.G. and Dawe, K.I. (1987) The rapid and sensitive enumeration of antibody-secreting cells using immunogold/silver staining. J. Immunol. Methods 104, 281. Yarchoan, R., Murphy, B.R., Strober, W, Clements, M.L. and Nelson, D.L. (1981) In vitro production of anti-influenza virus antibody after intranasal inoculation with coldadapted influenza virus. J. Immunol. 127, 1958.
31
JIM 04952
A novel two colour ELISPOT assay I. Simultaneous detection of distinct types of antibody-secreting cells Cecil Czerkinsky
1,2,3, Zina
Moldoveanu 1, Jiri Mestecky 1, Lars_/~& e Nilsson 2 and Orjan Ouchterlony 2
Department of Microbiology, Universityof Alabama at Birmingham, Birmingham, AL, U.S.A., and Departments of 2 Medical and 3 Oral Microbiology, University of Gothenburg, Gothenburg, Sweden (Received 26 April 1988, revised received 6 June 1988, accepted 29 June 1988)
A novel assay system has been developed which is based on the ELISPOT methodology and employs a combination of two immunoenzyme visualization systems yielding distinct colour products. This variation permits the simultaneous enumeration of two different types of cell secreting antigenically distinct products. Optimal conditions for the concurrent detection of human mononuclear cells secreting IgG or IgA antibodies are described. Key words: ELISPOT, two-color; Antibody-secreting cell
Introduction
Recently, a simple approach based on solidphase immunoenzyme technology has been developed for enumerating antibody-secreting cells (ASC). Since its original description (Czerkinsky et al., 1983; Sedgwick and Holt, 1983), the procedure, termed ELISPOT or ELISA plaque assay, has been employed as an alternative to conventional plaque-forming cell assays (Jerne and Nordin, 1963) to enumerate specific as well as total immunoglobulin-secreting cells and also to detect a variety of cells (lymphoid or nonCorrespondence to: C. Czerkinsky, Departments of Medical and Oral Microbiology, Guldhedsgatan 10, S-413 46 Gothenburg, Sweden. Abbreviations: ASC, antibody-secreting cells; ELISPOT, enzyme-linked immunospot; AP, alkaline phosphatase; HRP, horseradish peroxidase; PBMC, peripheral blood mononuclear cells; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/pnitroblue tetrazolium; AEC/H202, 3-amino-9-ethyl-carbazole/hydrogen peroxide; SFC, spot-forming cell.
lymphoid) secreting antigenic substances (for reviews see Czerkinsky et al., 1988; Sedgwick and Holt, 1988). The original techniques employed either alkaline phosphatase (AP)- (Sedgwick and Holt, 1983) or horseradish peroxidase (HRP)- (Czerkinsky et al., 1983) labelled antibodies and corresponding chromogen substrates as indicator systems for the detection of antibody producing cells. In this communication, we have evaluated a combination of both indicator systems for the concurrent detection of distinct types of ASC. In the procedure which we describe, zones of solid phase-bound IgG and Ig,A antibodies secreted from distinct cells are visualized as blue or red spots corresponding to either of these isotypes. Materials and methods Cells
Four adult human volunteers received a subcutaneous injection of a trivalent influenza virus
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
32 vaccine, types A and B (Wyett Laboratories, PA). Peripheral blood mononuclear cells (PBMC) were collected from 5 to 12 days after immunization, that is at a time when the frequency of antigenspecific spontaneous ASC in the circulation is known to be maximal (Stevens and Saxon, 1978; Stevens et al., 1979; Yarchoan et al., 1981). PBMC were isolated from heparinized venous blood by centrifugation on Ficoll-Hypaque (Bt~yum, 1968). Interface PBMC were washed twice with isotonic phosphate-buffered saline (0.0l M phosphates, 0.15 M NaC1, pH 7.4) (PBS), and resuspended at the appropriate densities in assay culture medium. The latter consisted of RPMI 1640 (Gibco, Glasgow, Scotland) supplemented with 5% foetal bovine serum (FBS) (Irvine Scientific, Santa Ana, CA).
Reagents AP- or HRP-conjugated affinity-purified goat anti-human IgA and goat anti-human IgG antibodies were purchased commercially (Southern Biotechnology Associate, Birmingham, AL). The specificity of these preparations was further documented by the two-colour ELISPOT assay described below. The AP chromogen substrate solution, consisting of 5-bromo-4-chloro-3-indolyl phosphate toluidine salt and p-nitroblue tetrazolium chloride ( B C I P / N B T ) was prepared according to the manufacturer's instructions (BioRad Laboratories, Richmond, CA). Initially, 15 mg of BCIP reagent and 30 mg of NBT salt were separately dissolved with 1 ml of dimethylformamide (DMF) and then added to 100 ml of a solution of 0.1 M NaHCO3, 1 mM MgC12, pH 9.8. The HRP chromogen substrate solution was prepared by dissolving 25 mg of 3-amino-9-ethyl carbazole (AEC) (Sigma, St. Louis, MO) in 2 ml of DMF, followed by addition of 95 ml of 0.05 M acetate buffer, pH 5.0 ( + 0.2) and 40 /~1 of 30% H202. The above substrate solutions were filtered (0.45 /~m) to remove particulate matter (the AEC/H202 solution becomes colourless after filtration while the B C I P / N B T solution remains pale yellow). Both enzyme substrate solutions could be kept in the dark at 4 ° C for up to 1 week. Two-colour ELISPOT assay The assay consists of five stages: first, a solid-
phase immunoadsorbent is prepared; the second stage, incubation of the cell suspension, is followed by the addition of a mixture of AP- and HRP-conjugated antibodies. The fourth stage comprises the stepwise addition of AP and HRP chromogen substrates which will yield insoluble blue and red spots, respectively. These spots are enumerated in the fifth and final stage. Solid-phase immunoadsorbent. The standard ELISPOT assay was modified by using nitrocellulose membranes as the solid support instead of polystyrene, as described by M/511er and Borrebaeck (1985). Individual wells of nitrocellulose bottomed 96-well Millititer HA plates (Millipore, Bedford, MA) were filled with 0.075-0.1 ml of PBS containing 0.2 ~tg of influenza virus hemagglutinin (Wyett Laboratories). This coating concentration was found optimal in a preliminary checkerboard titration experiment. Plates were allowed to stand overnight at 4 o C. (Coated plates can be stored for several weeks at 4 °C filled with coating solution, or for several months at - 2 0 °C decanted of coating solution.) Unadsorbed proteins were removed by three successive manual washings with PBS (by flicking) and the plates were immersed in this buffer for 5 min. Wells were then emptied of wash buffer and the outer surface of the nitrocellulose membrane was carefully dried with absorbent paper towels. In order to saturate remaining protein binding sites on the solid support individual wells were filled with 0.2 ml of assay culture medium and the plates were incubated at 37°C for at least 30 min in a humidified atmosphere with 7% CO 2. Wells exposed to an irrelevant antigen, tetanus toxoid (TT) (Wyett) (0.1 /~g/well), were prepared in the same way for control purposes. Cell incubation. The content of the wells was replaced with 0.1 ml of cell suspensions containing various numbers of PBMC. Routinely, we used at least three sets of triplicate wells. Each set of wells received 2 x 105, 105 and 5 x 104 PBMC/well. Plates were then incubated undisturbed for 3-4 h at 37 °C in a CO 2 incubator. In one experiment, PBMC were incubated for 5 h at 37°C with various concentrations (5 x 10 - 4 M , 10 - 3 M, 2 × 10-3M) of cycloheximide (Sigma) in assay culture medium, washed, resuspended and plated in cycloheximide containing medium.
33
Immunoenzyme stage. At the completion of the cell incubation period, plates were rinsed three times manually with PBS and three times with PBS containing 0.05% Tween 20 (PBS-T) and were then immersed in PBS-T for 5 min. The dishes were emptied of wash buffer and the outer surface of the plate was blot-dried as described in the first stage. Next, 0.1 ml of PBS-T containing 1% FBS and a mixture of goat anti-human IgG and goat anti-human IgA antibodies conjugated with AP and HRP, respectively or vice versa, was added to each well. Optimal concentrations of AP- and HRP-labeUed antiglobulins were determined in preliminary experiments. Concentrations ranging from 0.5 ~tg/ml to 2.5 ttg/ml were used for both types of enzyme conjugate. Plates were incubated for 3 h at room temperature or overnight at 4°C (whichever was most convenient). Dishes were then rinsed four times with PBS (without detergent) and immersed in 0.05 M Tris buffer saline, pH 8.0, for 5 rain prior to development. Development. Dishes were emptied of wash buffer and blot-dried as above. The wells were then exposed to 0.1 ml of BCIP/NBT substrate solution and examined for the appearance of blue spots. These reactions appeared usually within 5-10 rain. Plates were allowed to develop for an additional period of 5-10 min, after which they were rinsed with PBS and blot-dried. Next, 0.1 ml of AEC/H202 substrate was added to each well, yielding red spots within 1-3 min. Plates were allowed to develop for a variable time (up to 10 rain) and then thoroughly rinsed with tap water. As excessive background staining may result from over-long incubation times, it is important to monitor control wells (not exposed to cells) during the enzyme-substrate reaction. Enumeration of spots. Developed plates were dried and individual wells were examined for the presence of blue and red spots. These reactions were enumerated under low magnification ( x 40 to x to) (a stereomicroscope equipped with a vertical white light source is ideal for this purpose). Under low magnification, positive reactions were defined as circular, well individualized, densely granulated foci contiguous to the background. Their diameter ranged from 0.05 mm to 0.2 mm. Small dense particles could occasionally be observed particularly with over-exposure to either
enzyme substrate. These non-granular dots generally appeared above the plane of the background and could be distinguished from true spots.
Results and discussion
A two-colour immunoenzyme procedure is described for the concurrent detection of two antigenically distinct products secreted by different cells. The procedure follows the general principle of the ELISPOT assay which, in its original descriptions, relied on the use of either alkaline phosphatase (Sedgwick and Holt, 1983) or horseradish peroxidase (Czerkinsky et al., 1983) as indicator enzymes for the detection of antibodysecreting cells. The technique described here now makes use of two antiglobulin conjugates, one labelled with AP, the other with HRP, having distinct immunological specificities. When used in combination with BCIP/NBT and AEC/H202 chromogen substrates, AP and HRP activities can be visualized on the same solid phase as insoluble blue and red products, respectively. In the present study, we have employed this technique to detect simultaneously PBMC secreting IgG- and PBMC-secreting IgA anti-influenza virus antibodies in the circulation of human vaccinees. When plated for a few hours in influenza virus-coated wells, PBMC from recently immunized human volunteers secreted IgA and IgG antibodies. Zones of solid-phase bound IgA and IgG antibodies secreted by distinct cells were visualized by sequential addition of a mixture of anti-IgG and anti-IgA antibody reagents labelled with AP and HRP (or vice versa) and of corresponding chromogen substrates. Within minutes following addition of BCIP/NBT substrate, blue spots appeared on the solid phase at the former location of PBMC-secreting antibodies of a given isotype, e.g., IgA. Subsequent exposure of the solid phase to AEC/H202 substrate yielded red spots corresponding to cells secreting antibodies of a different isotype, e.g., IgG (Fig. 1). Similar numbers of red spots (HRP-AEC/H202 products) were detected in wells that had been previously exposed to both BCIP/NBT and AEC/H202 substrates as compared to wells treated only with AEC/H202 substrate (Table I).
34
35 TABLE I COMPARISON BETWEEN ONEoCOLOUR AND TWOCOLOUR ELISPOT ASSAYS FOR ENUMERATING ANTIGEN SPECIFIC IgG- AND IgA-SECRETINGCELLS Peripheral blood PBMC were obtained from one volunteer 7 days after immunization with influenza virus vaccine. Values are expressed as mean spot-forming cell (SFC) numbers (+ SEM) of quadruplicate assay wells. SFC numbers/106 PBMC in wells developedwith: HRP anti-IgG HRP anti-IgA 5080 (621) a AP anti-IgA 3920 (420) b
3 730 (470)
3445 (376) AP anti-IgG -
4860 (515)
4153 (537)
4540 (420)
3636 (354)
-
Upper values correspond to numbers of red spots developed with HRP-conjugated anti-Ig antibodies and AEC/H202 chromogen substrate. b Lower values indicated in'italics correspond to numbers of blue spots developed with AP-conjugated anti-Ig and BCIP/NBT substrate. a
This observation indicates that A P - B C I P / N B T reactions have no appreciable effects on subsequent development of H R P - A E C / H 2 0 2 reactions. Further, the sensitivity of both enzyme-substrate systems appeared comparable since similar numbers of spot-forming cells (SFC) were detected when using the same anti-immunoglobin preparation labelled with either enzyme (Table I). The quality of the enzyme-antibody conjugates employed in this two-colour ELISPOT assay must be carefully considered as double (indigo) spots may appear with conjugates containing cross-reactive antibodies. Thus, monochromatic (red or blue) spots provide an ideal internal control of the specificity of the assay for detecting antigenically distinct products secreted by different cells. Further, although two enzyme-antibody conjugates may be equally specific, they may exhibit different affinities for binding to cognate immunoglobulins a n d / o r variable enzyme activities, resulting in different staining intensity of the corresponding spots.
In such situations, a correction factor (ratio of number of spots developed with a given antibody conjugated with HRP: number of spots developed with the same antibody preparation but labelled with AP) should be applied to adjust for possible differences in sensitivity. Under optimal conditions PBMC-secreting IgG antibodies and PBMC-secreting IgA antibodies to influenza virus could be detected simultaneously in all four volunteers examined. Virus-specific ASC were detected as early as 5 days after systemic immunization with influenza virus vaccine. Spot forming cell (SFC) numbers reached a maximum on day 7. By day 9-12 the frequency of virusspecific SFC markedly decreased (data not illustrated). Influenza virus specific IgG-SFC and IgA-SFC responses followed a similar kinetic pattern but differed in magnitude. Thus, in three individuals, IgG-SFC predominated (5520 _+ 983 day 7 SFC/106 PBMC versus 140 _+ 69 day 7 IgA SFC/106; n = 3). In the fourth volunteer the magnitude of influenza virus-specific IgA responses on day 7 was very high (3910/106 PBMC + 420 SFC) being almost comparable to that of the IgG responses (5080 _ 621 SFC/106 PBMC). The specificity of the assay for simultaneous demonstration of influenza virus specific-IgA ASC and -IgG ASC was documented by several observations. First, omission of cells, coating antigen, or labelled antibodies prevented subsequent development of spot formation. Second, plating PBMC in wells coated with an irrelevant antigen (tetanus toxoid) resulted in the absence of detectable spots (data not shown). Third, incubation of influenza virus immune cells with graded amounts of influenza virus antigen during cell plating inhibited, in a dose dependent manner, spot formation (Table II). It should be noted that such treatment not only resulted in a reduction of SFC numbers but also in a decrease in the diameter of the remaining spots. In contrast, addition of tetanus toxoid had no effects. Finally, treatment of the cells with cycloheximide prior to and during the cell incubation period markedly inhibited influenza virus specific IgG- and IgA-mediated spot
Fig. 1. Typical appearance of a well followingdevelopmentin the two color ELISPOT assay for simultaneous detection of influenza virus specifichuman IgG antibody-secretingcells (red spots) and IgA antibody-secretingcells (blue spots). A, × 8; B, × 40.
36 TABLE II SPECIFICITY OF TWO-COLOUR ELISPOT ASSAY FOR SIMULTANEOUS DETECTION OF INFLUENZA VIRUS SPECIFIC IgA-SECRETING PBMC AND IgG-SECRETING PBMC Peripheral blood PBMC were obtained from one donor on day 7 following immunization with influenza virus vaccine. PBMC were assayed by two-colour ELISPOT assay for numbers of virus-specific ASC. IgG SFC and IgA SFC were developed with AP-conjugated anti-lgG and HRP-conjugated anti-IgA, respectively followed by BCIP/NBT (blue) and AEC/H202 (red) enzyme substrates. Values represent mean SFC numbers of quadruplicate assay wells/106 PBMC. Data in parentheses indicate percentages of inhibition. Inhibitor added per assay well
SFC numbers/106 PBMC IgG (blue)
IgA (red)
6560
220
Influenza virus 15/zg 3/~g 0.6/~g 0.12/~g
120 (98%) 1 400 (79%) 2720 (59%) 3 840 (42%)
0 (100%) 45 (80%) 96 (57%) 240 ( - 11%)
Tetanus toxoid 100 #g
6120 (6.7%)
197
(10.4%)
Cycloheximide a 2X10 -3 M 10 -3 M 5X10 - 4 M
720 (89%) 2000 (69.5%) 2960(54.8%)
20 70 100
(91%) (68.2%) (54.5%)
Treatment with cycloheximide (see section Materials and Methods) did not affect cell viability (assessed by trypan blue dye exclusion) at all 3 concentrations of drug tested. a
f o r m a t i o n ( T a b l e II). T h a t ' d o u b l e ' ( b i o c h r o m a t i c ) spots were never o b s e r v e d c o n f i r m e d the high degree of specificity of the e n z y m e l a b e l l e d antib o d y p r e p a r a t i o n s e m p l o y e d as class specific reagents in this assay. In p r e l i m i n a r y c o m p a r a t i v e experiments, the p r o c e d u r e d e s c r i b e d here a p p e a r e d at least five times as sensitive as the original E L I S P O T techniques p e r f o r m e d o n plastic surfaces a n d develo p e d with either p a r a p h e n y l e n e d i a m i n e / H 2 0 2 in a g a r ( H R P system) ( C z e r k i n s k y et al., 1983) or B C I P ( A P system) (Sedgwick a n d Holt, 1983) a m p l i f i e d with N B T ( F r a n c i et al., 1986). T h e high b i n d i n g a n d r e t e n t i o n p r o p e r t i e s of nitrocellulose s u p p o r t s for p r o t e i n antigens m a y a c c o u n t for such an increase in sensitivity. T h e use of nitrocellulose m e m b r a n e s in the E L I S P O T assay, r e c e n t l y
i n t r o d u c e d b y MOiler a n d B o r r e b a e c k (1985) also m i n i m i z e s s u b s t a n t i a l l y the r e q u i r e m e n t s of the original techniques for relatively large a m o u n t s of c o a t i n g material. In a d d i t i o n , this m o d i f i c a t i o n a p p e a r s s i m p l e r since it does n o t rely on the use of a gel overlay, as is the case with certain p e r o x i d a s e c h r o m o g e n s on plastic surfaces. W e have c o n f i r m e d the suitability of this twoc o l o u r E L I S P O T assay for d e t e c t i n g simultan e o u s l y cells secreting o t h e r Ig i s o t y p e s (IgM, I g G subclasses) in b o t h h u m a n a n d m u r i n e systems. W o r k is in p r o g r e s s to use this t w o - c o l o u r assay system for the d e t e c t i o n of distinct l y m p h o k i n e secreting cells. A d d i t i o n a l visualization systems, e.g., silver i m m u n o g o l d staining ( W a l k e r a n d Dawe, 1987) are also u n d e r e v a l u a t i o n for the c o n c u r r e n t d e t e c t i o n of m u l t i p l e i m m u n o r e a c t i v e substances. These d e v e l o p m e n t s should m a k e it p o s s i b l e to investigate w h e t h e r two or m o r e antigenically distinct p r o d u c t s originate f r o m the same or two different cells.
Acknowledgements This s t u d y was s u p p o r t e d b y U.S. Public H e a l t h Service G r a n t AI-10702 a n d b y G r a n t 08320 f r o m the Swedish M e d i c a l R e s e a r c h Council. T h e technical assistance of Miss Shirley Prince a n d Mrs. A n n e t t e Pitts is g r a t e f u l l y a c k n o w l e d g e d .
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