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Reverse enzyme-linked immunospot assay (RELISPOT) for the detection of cells secreting immunoreactive substances
Journal of Immunological Methods.
72 (1984) 489-496
489
Elsevier JIM03181
Reverse Enzyme-Linked Immunospot Assay (RELISPOT) for the Detection of Cells Secreting Immunoreactive Substances C.C. Czerkinsky 1, A. Tarkowski 1,3, L.-A. Nilsson 1,., O. Ouchterlony 1, H. Nygren 2 and C. Gretzer i Departments of ~ Medical Microbiology, ~"Histology and -~ Rheumatologv. Universi(v of G~teborg, S- 413 46 G~teborg, Sweden
(Received 23 January 1984, accepted 2 May 1984)
A reverse modificationof the recently described enzyme-linkedimmunospotassay (ELISPOT), based on localized enzyme-substrate reactions in gel, is described for the enumeration of antigen-secreting cells using petri dishes coated with specific antibodies. As a model the detection of mouse and human immunoglobulin-secreting cells has been evaluated. Simple and sensitive, this new method, termed RELISPOT, can be adapted for the quantitation of secreted antigen thus providing additional information on the metabolic state of the population of cells tested. Key words: antigen - immunoglobulin - E L I S P O T - single cells
Introduction The secretion of antigenic macromolecules can be assessed at the individual cell level by the 'reverse' modifications of the original hemolytic plaque assay (Jerne and Nordin, 1963) using indicator erythrocytes coated with either purified antibodies (Molinaro and Dray, 1974) or staphylococcal protein A (Gronowicz et al., 1976) in the presence of an appropriate developer antiserum. It has thus been possible to study the secretion of immunoglobulins (Ig) from single mouse and human B cells (Molinaro and Dray, 1974; Gronowicz et al., 1976; Bird and Britton, 1979) as well as the secretion of other molecules such as albumin (Molinaro et al., 1975; Smith and Hammarstri3m, 1979) and lymphokines (Primi et al., 1979) by individual mouse hepatocytes and T lymphocytes respectively. Although both procedures possess considerable biological potential, their reproducibility remains questionable (Merchant and Inman, 1977; Truffa-Bachi and Bordenave, 1980; Burns and Pyke, 1981) because of batch-to-batch variations in the stability of the conjugated red * Correspondence to: Dr. L.-,k Nilsson, Department of Medical Microbiology,University of G6teborg, Guldhedsgatan 10, S-413 46 G6teborg, Sweden. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
490
blood cells and their susceptibility to complement-mediated lysis. In addition, conjugation of red blood cells is usually by the chromium chloride method, a procedure which is difficult to standardize (Kofler and Wick, 1977). Moreover, hemolytic plaque assays are not suited to the quantitation of secreted products, i.e., antibodies or antigen. Such analyses would be useful for the evaluation of the metabolic state of the cell population tested. In the present report, a 'reverse' modification of the recently described enzymelinked immunospot assay (ELISPOT) (Czerkinsky et al., 1983), based on localized enzyme-substrate reactions in gel, has been elaborated for the enumeration of antigen-secreting cells and the simultaneous quantitation of secreted antigens. As a model, the secretion of immunoglobulin, irrespective of its antigenic specificity, by mouse splenocytes and Epstein-Barr virus (EBV)-stimulated human peripheral blood lymphocytes is herein described.
Materials and Methods
Cells Erythrocyte-free, single spleen cell suspensions were prepared from B A L B / c mice (age and sex matched) as described elsewhere (Czerkinsky et al., 1983). H u m a n mononuclear cells ( H M N C ) were isolated from heparinized venous blood of healthy adult donors by centrifugation on Lymphoprep TM (Nyegaard, Oslo) (B6yum, 1968). Interface M N C were washed twice with phosphate-buffered saline (0.01 M phosphate buffer in 0.15 M NaC1, p H 7.4; PBS), resuspended, counted and adjusted at the appropriate density in culture medium. The human M N C were cultured in 16 m m well diameter cluster tissue culture plates (Nunc, Roskilde). A total of 2 x 106 H M N C in a final volume of 2 ml was dispensed in each well. The standard culture medium consisted of bicarbonate-buffered Iscove's medium supplemented with L-glutamine (2 raM), sodium pyruvate (1 raM), 5 x 1 0 -s M mercaptoethanol and 10% Nu T M Serum (Collaborative Research, Lexington, MA), a semi-defined growth supplement used as an alternative to fetal bovine serum (FBS). Preliminary experiments unequivocally established that this growth supplement, in contrast to several batches of FBS tested, was able to support Ig production as well as specific antibody secretion (Czerkinsky et al., manuscript submitted for publication) without detectable mitogenic activity. Cultures received 0.1 ml of a 10-fold diluted EBV stock prepared from the EBV transformed marmoset lymphoid cell line B95,/8 or 0.1 ml of culture medium (control cultures). After incubation for 6 days at 37°C in a humid atmosphere with 10% CO 2, the cultures were harvested and centrifuged at 400 x g for 10 rain. The supernatants were further clarified by centrifugation at 600 x g for 5 rain and stored at - 2 0 ° C until assayed. The M N C pellets were washed 3 times, counted and adjusted to 106 trypan blue excluding M N C / m l in culture medium and immediately assayed for numbers of immunoglobulin-secreting cells (ISC).
Antibody reagents Affinity-purified rabbit anti-mouse IgG (heavy and light chains specific) anti-
491 bodies (RaM Ig) were obtained from Cappel Laboratories (Cochranville, PA) and used at a concentration of 10 # g / m l for the coating of petri dishes. A mixture of F(ab')2 fragments of rabbit antibodies to human IgA, IgG and IgM ( R a i l Ig) (Cappel Laboratories) at concentrations of 20 /~g/ml, 10 /Lg/ml and 20 /~g/ml respectively were prepared for coating petri dishes and ELISA microtiter plates. All the above reagents were diluted in PBS containing 0.02% N a N 3. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig ( H R P RaM Ig) and rabbit anti-human Ig ( H R P R a i l Ig), both purchased from Dakopatts (Copenhagen), were used at a dilution of 1 : 200 in PBS containing 0.05% Tween 20 (PBS-Tween), found optimal for both reagents in preliminary checkerboard titration experiments. Rabbit anti-mouse albumin was purchased from Nordic Immunological Laboratories (Tilburg) and specific antibodies were prepared by chromatography on Sepharose 4B (Pharmacia, Uppsala) to which purified mouse albumin (Nordic) had been coupled. The immunoglobulin fraction of normal rabbit serum was prepared according to Steinbuch and Audran (1969).
Reverse enzyme-linked immunospot assay (RELISPOT) and in situ ELISA The assays consist of 4 stages, first preparing a solid-phase immunoadsorbent, then the cell incubation stage followed by the enzyme-labeled antiglobulin stage. The fourth and last stage consists of adding a chromogen substrate either in a gelling form (RELISPOT) or in free solution (in situ ELISA). The procedures used in all experiments described were as follows: (1) Preparation of the solid-phase. Five centimeter diameter polystyrene petri dishes (Nunc, Roskilde) were filled with 2.5 ml of RaM Ig or R a i l Ig and allowed to stand overnight at room temperature in humid chambers. Unadsorbed antibodies could be recovered and re-used on 2 successive occasions. The dishes were rinsed 3 times with PBS, then exposed to 3 ml of PBS containing 5% FBS for 1 - 2 h at 37°C. Dishes coated with an irrelevant antibody preparation a n d / o r FBS were prepared for control purposes. (2) Cell incubation stage. Five hundred microliters of cell suspensions containing putative ISC (mouse splenocytes or cultured human MNC) were dispensed into the pertinent antiglobulin-coated dishes and incubated on a levelled surface for 4 h at 37°C in a humid atmosphere containing 10% CO 2. The cell layer was then removed with PBS and the dish incubated for 10-15 min with 5 ml of PBS containing 50 mM EDTA. (3) Enzyme-labelled antiglobulin stage. The dishes were washed 4 additional times with PBS-Tween and filled with 2.5 ml of the appropriate HRP-antiglobulin preparation ( H R P RaM Ig of H R P R a i l Ig) diluted in PBS-Tween. After 3 h at room temperature or overnight at 4°C, unreacted H R P antiglobulins were removed by 4 washings with PBS and the plates allowed to stand with the last wash prior to being developed. (4) Enzyme-substrate reaction stage. A molten solution of PBS containing 1% Noble agar (Difco, Detroit, MI) was cooled to 48°C. The chromogen substrate, paraphenylenediamine (PPD) was dissolved in methanol at a concentration of 5 m g / m l , mixed with H202 (final concentration 0.3%) and immediately added to 20
492
vols. of agar solution. The dishes were carefully decanted, poured with the agar-substrate mixture and decanted again immediately in order to leave a thin layer of gel. After a few minutes the plates were examined for the appearance of dark-brown spots which were enumerated at 30 min with the naked eye or under low magnification (× 6). Alternatively, contact photographs of the developed dishes were made allowing the count to be performed at leisure on the print. In situ ELISA measurement of the Ig secreted was performed in parallel by exposing replicate dishes to orthophenylenediamine (OPD) (10 mg/ml) in citrate buffer 0.1 M, pH 4.5, containing 0.01% H202. After incubation for 10 min at room temperature the contents of the dishes were transferrred into microELISA plates and color development of enzyme-substrate reactions was monitored spectrophotometrically (492 nm) using a semi-automated device.
Cycloheximide treatments Requirements for de novo protein synthesis were assessed in the RELISPOT assay by adding cycloheximide (Sigma, St. Louis, MO) (25 ~tg/ml) to some of the HMNC cultures 16 h before harvesting the cells and at the time of plating. Mouse splenocytes were preincubated with the drug for 4 h at 37°C and during the cell incubation step. Analysis of lg synthesized in cultures of human MNC by conventional ELISA Supernates from EBV infected and non-infected human MNC cultures were assayed for Ig content by conventional solid-phase ELISA in 96-well Nunc polystyrene plates. Wells were filled with 100 /~1 of Rail Ig mixture; coating and blocking conditions were as described above for the petri dishes. One hundred microliters of appropriately diluted cell culture supernates were dispensed into triplicate wells and incubated for 3 h at 37°C in a CO 2 incubator. After 4 washings with PBS-Tween, the wells were exposed to 100/~1 of diluted HRP RaHIg conjugate for 3 h at room temperature, washed thoroughly with PBS and developed with OPD-substrate (200/~l/well) as above. Hemolytic plaque-forming cell (PFC) assay Mouse spleen cells were assayed for ISC numbers by the protein A plaque assay (Gronowicz et al., 1976) following the detailed procedure of Bird and Britton (1979). Staphylococcus Cowan 1 protein A (Pharmacia, Uppsala) was coupled to sheep erythrocytes by the chromium chloride method according to Gronowicz et al. (1976). The Ig fraction of rabbit anti-mouse Ig antiserum (Dakopatts) was used as developer in the presence of sheep red blood cell-absorbed guinea pig serum as source of complement. Plaques were counted after 4 h incubation at 37°C. Results
Detection and enumeration of immunoglobulin-secreting mouse splenocytes by the RELISPOT test Mouse splenocytes ( 1 0 4 - 1 0 6 ) were plated for 4 h in antiglobulin-coated assay
493 TABLE I SPECIFICITY OF THE RELISPOT TEST FOR THE ENUMERATION OF IMMUNOGLOBULINSECRETING MOUSE SPLENOCYTES Culture medium with
SFC/2 x 105 splenocytes cultured in dishes coated with
-
125
8
2
Rabbit anti-mouse Ig Rabbit anti-mouse albumin Cycloheximide
17 105 28
ND a 2 ND
ND ND ND
Rabbit antimouse Ig
Rabbit antimouse albumin
FBS
a Not done. culture dishes. A f t e r stepwise a d d i t i o n of e n z y m e - l a b e l e d a n t i - m o u s e Ig a n t i b o d i e s a n d agar c h r o m o g e n - s u b s t r a t e d a r k - b r o w n i s h spots a p p e a r e d within minutes, indic a t i n g the former l o c a t i o n of i m m u n o g l o b u l i n secreting cells. O c c a s i o n a l artefacts d u e to cell debris a n d / o r aggregates were easily distinguished f r o m the singular p a t t e r n of true reactions, i.e., i n d i v i d u a l i z e d a n d h o m o g e n e o u s l y g r a n u l a t e d foci (Fig. 1). Spots d i d not develop in dishes that h a d been c o a t e d with an irrelevant a n t i b o d y p r e p a r a t i o n ( r a b b i t a n t i - m o u s e a l b u m i n ) or in dishes e x p o s e d to F B S o n l y (Table I). A d d i t i o n of R a M Ig to the cells d u r i n g p l a t i n g i n h i b i t e d the f o r m a t i o n of spots whereas a d d i t i o n of n o r m a l r a b b i t Ig h a d no effect. P r e i n c u b a t i o n of the cells with
Fig. 1. Typical appearance of reactions (spots) generated by mouse immunoglobulin-secreting cells and developed by means of the RELISPOT test. Artefacts may appear occasionally and are indicated here by an arrow. The indicated scale corresponds to 1 mm.
494
the inhibitor of protein synthesis cycloheximide (25 # g / m l ) for 4 h and during plating reduced the number of spots by almost 80%. As inferred from the above observations, the generation of spots by mouse splenocytes appeared to be due to the specific binding of Ig molecules secreted by metabolically active cells.
In situ ELISA measurement of Ig secreted by mouse splenocytes Replicate dishes were exposed to OPD-substrate solution instead of agar-PPD. Color development of enzyme-bound activity was recorded allowing estimation of the total amount of Ig secreted and bound within the cell population tested. This enabled the detection of immunoglobulin secreted from as few as 5 × 104 mouse splenocytes (Fig. 2). This corresponded to approximately 50 spot-forming cells (SFC). Comparison of R E L I S P O T test and protein A plaque-forming cell assay jor the enumeration of immunog[obulin secreting mouse ~p[enoc),tes Normal mouse splenocytes were assayed in parallel for ISC numbers by means of the protein A plaque assay and by the RELISPOT test. The results obtained by both procedures were comparable (1650 + 210 PFC and 1480 + 130 SFC per 106 splenocytes respectively). However, increasing the cell incubation period from 4 to 6 h resulted in a substantial increase of ISC detected as spots by the RELISPOT test (approximately 30%) whereas hemolysis started to occur spontaneously in the corresponding PFC assay (data not illustrated). Detection and enumeration of immunoglobulin-secreting cells (ISC) generated in cultures of human mononuclear cells exposed to EB V Human peripheral blood MNC were assayed by the RELISPOT test for ISC A492 0.5
0,4
0.3
0.2
T+--I
0 0.1
0 2 5 0.5
1
2
4 xlO s cells plated
Fig. 2. In situ E L I S A m e a s u r e m e n t of Ig secreted for various n u m b e r s of mouse splenocytes cultured for 4 h in R a M Ig coated dishes ( e ) a n d in FBS-coated control dishes ( O - - - - O). Results are expressed as m e a n a b s o r b a n c e values _+ SEM of triplicate determinations.
495 T A B L E II A N A L Y S I S OF I M M U N O G L O B U L I N SECRETIO N BY H U M A N L Y M P H O C Y T E S S T I M U L A T E D WITH EPSTEIN-BARR VIRUS
PERIPHERAL
Donor
EBV
ELISA
1
+ -
0.85 0.06
110000 220
2
+ -
0.70 0.12
62000 1 500
a
RELISPOT
BLOOD
a
Results are expressed as mean absorbance values (A492) and SFC numbers for 2 × 106 cells cultured.
numbers in Rail Ig-coated dishes following in vitro exposure to EBV for 6 days. The results shown in Table II indicate that human ISC could be detected and enumerated by means of the RELISPOT test. It should be pointed out that there was no difference in the macroscopical appearance of spots generated by HMNC and mouse ISC. In addition the cell culture supernatants were assayed for total Ig contents by a conventional ELISA technique. Table II shows that the RELISPOT test appeared more discriminative (ratio of stimulated cultures to unstimulated cultures) than the ELISA procedure.
Discussion
The 'reverse' enzyme-linked immunospot test (RELISPOT) described here provides a tool whereby the secretion of antigenic macromolecules, e.g., immunoglobulins, can be assessed at the individual cell level. Results obtained by this method are comparable to those obtained with the protein A-plaque assay (Gronowicz et al., 1976) for enumeration of mouse spleen cells secreting immunoglobulins. However, the immunoenzyme procedure offers advantages. Unlike conjugated red blood cells whose preparation and storage are complicated (Merchant and Inman, 1977), plastic immunoadsorbents can be prepared very simply without any cumbersome coupling step and are generally stable over long periods of storage. These properties are shared with those of the recently described ELISPOT assay (Czerkinsky et al., 1983) for the detection of specific antibody-secreting cells and should ease the standardization of the present method from one laboratory to another. The stability of the immunoadsorbent during the cell incubation is another advantage. It should be particularly important when studying the secretion of antigenic molecules by cells displaying a relatively low rate of synthesis and thus requiring longer incubation periods in order to generate detectable reactions (spots).
496
Unlike hemolytic plaque assays, the RELISPOT test can easily be adapted for the quantitation of secreted antigen using the same cell culture device, thus providing information concerning the average rate of antigen secretion for a given population of cells. When compared to recently described culture well enzyme-immunoassay systems (Weetman et al., 1981; Boerrigter et al., 1983) for the quantitation of secreted immunoglobulin, the RELISPOT test offers a major advantage by permitting the determination of the frequency of Ig-secreting cells. We have successfully applied the RELISPOT test for the detection of immunoglobulin-secreting cells generated upon in vitro exposure of human peripheral blood mononuclear cells to Epstein-Barr virus. It may be mentioned that the use of F(ab')2 fragments of anti-human antiglobulin reagents, although not necessary in the present study, is required to enable isotype-specific determination of immunoglobulin secreting cells by avoiding interference due to rheumatoid factors secreted by human lymphocytes stimulated in vitro with Epstein-Barr virus (Fong et al.. 1981). The RELISPOT assay design may be a general tool for detecting antigen-secreting cells as spot-forming cells provided the corresponding specific antibodies are available. The assay should provide a useful alternative to reverse hemolytic plaqueforming cell assays for studies assessing the secretion of immunoreactive molecules of individual cells.
References Bird, G.A. and S. Brinon, 1979, Immunol. Rev. 45, 41. Boerrigter, G.H., A. Vos and R.J. Scheper, 1983, J. Immunol. Methods 61, 377. B6yum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (suppl. 97), 77. Burns, G.F. and B.L. Pyke, 1981, J. Immunol. Methods 41,269. Czerkinsky, C.C., L.A. Nilsson, H. Nygren, ~). Ouchterlony and A. Tarkowski, 1983, J. Immunol. Methods 65, 109. Fong, S., C.D. Tsoukas and L.A. Frincke, 1981, J. Immunol. 126, 910. Gronowicz, E.A., A. Coutinho and F. Melchers, 1976, Eur. J. Immunol. 6, 588. Jerne, N.K. and A.A. Nordin, 1963, Science 140, 405. Kofler, R. and G. Wick, 1977, J. lmmunol. Methods 16, 201. Merchant, B. and J.K. Inman, 1977, J. Exp. Med. 145, 372. Molinaro, G.A. and S. Dray, 1974, Nature (London) 248, 515. Molinaro, G.A., E. Maron, W.C. Eby and S. Dray, 1975, Eur. J. Immunol. 5, 771. Primi, D., G.K. Lewis and J.W. Goodman, 1979, J. Exp. Med. 150, 987. Smith, C.I.E. and L. Hammarstr6m, 1979, Eur. J. Immunol. 9, 570. Steinbuch, M. and R. Audran, 1969, Arch. Biochem. 134, 279. Truffa-Bachi, P. and G.R. Bordenave, 1980, Cell. Immunol. 50, 261. Weetman, A.P., A.M.M. MacGregor, J.H. Lazarus and R. Hall, 1981, Clin. Exp. Immunol. 48, 196.
72 (1984) 489-496
489
Elsevier JIM03181
Reverse Enzyme-Linked Immunospot Assay (RELISPOT) for the Detection of Cells Secreting Immunoreactive Substances C.C. Czerkinsky 1, A. Tarkowski 1,3, L.-A. Nilsson 1,., O. Ouchterlony 1, H. Nygren 2 and C. Gretzer i Departments of ~ Medical Microbiology, ~"Histology and -~ Rheumatologv. Universi(v of G~teborg, S- 413 46 G~teborg, Sweden
(Received 23 January 1984, accepted 2 May 1984)
A reverse modificationof the recently described enzyme-linkedimmunospotassay (ELISPOT), based on localized enzyme-substrate reactions in gel, is described for the enumeration of antigen-secreting cells using petri dishes coated with specific antibodies. As a model the detection of mouse and human immunoglobulin-secreting cells has been evaluated. Simple and sensitive, this new method, termed RELISPOT, can be adapted for the quantitation of secreted antigen thus providing additional information on the metabolic state of the population of cells tested. Key words: antigen - immunoglobulin - E L I S P O T - single cells
Introduction The secretion of antigenic macromolecules can be assessed at the individual cell level by the 'reverse' modifications of the original hemolytic plaque assay (Jerne and Nordin, 1963) using indicator erythrocytes coated with either purified antibodies (Molinaro and Dray, 1974) or staphylococcal protein A (Gronowicz et al., 1976) in the presence of an appropriate developer antiserum. It has thus been possible to study the secretion of immunoglobulins (Ig) from single mouse and human B cells (Molinaro and Dray, 1974; Gronowicz et al., 1976; Bird and Britton, 1979) as well as the secretion of other molecules such as albumin (Molinaro et al., 1975; Smith and Hammarstri3m, 1979) and lymphokines (Primi et al., 1979) by individual mouse hepatocytes and T lymphocytes respectively. Although both procedures possess considerable biological potential, their reproducibility remains questionable (Merchant and Inman, 1977; Truffa-Bachi and Bordenave, 1980; Burns and Pyke, 1981) because of batch-to-batch variations in the stability of the conjugated red * Correspondence to: Dr. L.-,k Nilsson, Department of Medical Microbiology,University of G6teborg, Guldhedsgatan 10, S-413 46 G6teborg, Sweden. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
490
blood cells and their susceptibility to complement-mediated lysis. In addition, conjugation of red blood cells is usually by the chromium chloride method, a procedure which is difficult to standardize (Kofler and Wick, 1977). Moreover, hemolytic plaque assays are not suited to the quantitation of secreted products, i.e., antibodies or antigen. Such analyses would be useful for the evaluation of the metabolic state of the cell population tested. In the present report, a 'reverse' modification of the recently described enzymelinked immunospot assay (ELISPOT) (Czerkinsky et al., 1983), based on localized enzyme-substrate reactions in gel, has been elaborated for the enumeration of antigen-secreting cells and the simultaneous quantitation of secreted antigens. As a model, the secretion of immunoglobulin, irrespective of its antigenic specificity, by mouse splenocytes and Epstein-Barr virus (EBV)-stimulated human peripheral blood lymphocytes is herein described.
Materials and Methods
Cells Erythrocyte-free, single spleen cell suspensions were prepared from B A L B / c mice (age and sex matched) as described elsewhere (Czerkinsky et al., 1983). H u m a n mononuclear cells ( H M N C ) were isolated from heparinized venous blood of healthy adult donors by centrifugation on Lymphoprep TM (Nyegaard, Oslo) (B6yum, 1968). Interface M N C were washed twice with phosphate-buffered saline (0.01 M phosphate buffer in 0.15 M NaC1, p H 7.4; PBS), resuspended, counted and adjusted at the appropriate density in culture medium. The human M N C were cultured in 16 m m well diameter cluster tissue culture plates (Nunc, Roskilde). A total of 2 x 106 H M N C in a final volume of 2 ml was dispensed in each well. The standard culture medium consisted of bicarbonate-buffered Iscove's medium supplemented with L-glutamine (2 raM), sodium pyruvate (1 raM), 5 x 1 0 -s M mercaptoethanol and 10% Nu T M Serum (Collaborative Research, Lexington, MA), a semi-defined growth supplement used as an alternative to fetal bovine serum (FBS). Preliminary experiments unequivocally established that this growth supplement, in contrast to several batches of FBS tested, was able to support Ig production as well as specific antibody secretion (Czerkinsky et al., manuscript submitted for publication) without detectable mitogenic activity. Cultures received 0.1 ml of a 10-fold diluted EBV stock prepared from the EBV transformed marmoset lymphoid cell line B95,/8 or 0.1 ml of culture medium (control cultures). After incubation for 6 days at 37°C in a humid atmosphere with 10% CO 2, the cultures were harvested and centrifuged at 400 x g for 10 rain. The supernatants were further clarified by centrifugation at 600 x g for 5 rain and stored at - 2 0 ° C until assayed. The M N C pellets were washed 3 times, counted and adjusted to 106 trypan blue excluding M N C / m l in culture medium and immediately assayed for numbers of immunoglobulin-secreting cells (ISC).
Antibody reagents Affinity-purified rabbit anti-mouse IgG (heavy and light chains specific) anti-
491 bodies (RaM Ig) were obtained from Cappel Laboratories (Cochranville, PA) and used at a concentration of 10 # g / m l for the coating of petri dishes. A mixture of F(ab')2 fragments of rabbit antibodies to human IgA, IgG and IgM ( R a i l Ig) (Cappel Laboratories) at concentrations of 20 /~g/ml, 10 /Lg/ml and 20 /~g/ml respectively were prepared for coating petri dishes and ELISA microtiter plates. All the above reagents were diluted in PBS containing 0.02% N a N 3. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig ( H R P RaM Ig) and rabbit anti-human Ig ( H R P R a i l Ig), both purchased from Dakopatts (Copenhagen), were used at a dilution of 1 : 200 in PBS containing 0.05% Tween 20 (PBS-Tween), found optimal for both reagents in preliminary checkerboard titration experiments. Rabbit anti-mouse albumin was purchased from Nordic Immunological Laboratories (Tilburg) and specific antibodies were prepared by chromatography on Sepharose 4B (Pharmacia, Uppsala) to which purified mouse albumin (Nordic) had been coupled. The immunoglobulin fraction of normal rabbit serum was prepared according to Steinbuch and Audran (1969).
Reverse enzyme-linked immunospot assay (RELISPOT) and in situ ELISA The assays consist of 4 stages, first preparing a solid-phase immunoadsorbent, then the cell incubation stage followed by the enzyme-labeled antiglobulin stage. The fourth and last stage consists of adding a chromogen substrate either in a gelling form (RELISPOT) or in free solution (in situ ELISA). The procedures used in all experiments described were as follows: (1) Preparation of the solid-phase. Five centimeter diameter polystyrene petri dishes (Nunc, Roskilde) were filled with 2.5 ml of RaM Ig or R a i l Ig and allowed to stand overnight at room temperature in humid chambers. Unadsorbed antibodies could be recovered and re-used on 2 successive occasions. The dishes were rinsed 3 times with PBS, then exposed to 3 ml of PBS containing 5% FBS for 1 - 2 h at 37°C. Dishes coated with an irrelevant antibody preparation a n d / o r FBS were prepared for control purposes. (2) Cell incubation stage. Five hundred microliters of cell suspensions containing putative ISC (mouse splenocytes or cultured human MNC) were dispensed into the pertinent antiglobulin-coated dishes and incubated on a levelled surface for 4 h at 37°C in a humid atmosphere containing 10% CO 2. The cell layer was then removed with PBS and the dish incubated for 10-15 min with 5 ml of PBS containing 50 mM EDTA. (3) Enzyme-labelled antiglobulin stage. The dishes were washed 4 additional times with PBS-Tween and filled with 2.5 ml of the appropriate HRP-antiglobulin preparation ( H R P RaM Ig of H R P R a i l Ig) diluted in PBS-Tween. After 3 h at room temperature or overnight at 4°C, unreacted H R P antiglobulins were removed by 4 washings with PBS and the plates allowed to stand with the last wash prior to being developed. (4) Enzyme-substrate reaction stage. A molten solution of PBS containing 1% Noble agar (Difco, Detroit, MI) was cooled to 48°C. The chromogen substrate, paraphenylenediamine (PPD) was dissolved in methanol at a concentration of 5 m g / m l , mixed with H202 (final concentration 0.3%) and immediately added to 20
492
vols. of agar solution. The dishes were carefully decanted, poured with the agar-substrate mixture and decanted again immediately in order to leave a thin layer of gel. After a few minutes the plates were examined for the appearance of dark-brown spots which were enumerated at 30 min with the naked eye or under low magnification (× 6). Alternatively, contact photographs of the developed dishes were made allowing the count to be performed at leisure on the print. In situ ELISA measurement of the Ig secreted was performed in parallel by exposing replicate dishes to orthophenylenediamine (OPD) (10 mg/ml) in citrate buffer 0.1 M, pH 4.5, containing 0.01% H202. After incubation for 10 min at room temperature the contents of the dishes were transferrred into microELISA plates and color development of enzyme-substrate reactions was monitored spectrophotometrically (492 nm) using a semi-automated device.
Cycloheximide treatments Requirements for de novo protein synthesis were assessed in the RELISPOT assay by adding cycloheximide (Sigma, St. Louis, MO) (25 ~tg/ml) to some of the HMNC cultures 16 h before harvesting the cells and at the time of plating. Mouse splenocytes were preincubated with the drug for 4 h at 37°C and during the cell incubation step. Analysis of lg synthesized in cultures of human MNC by conventional ELISA Supernates from EBV infected and non-infected human MNC cultures were assayed for Ig content by conventional solid-phase ELISA in 96-well Nunc polystyrene plates. Wells were filled with 100 /~1 of Rail Ig mixture; coating and blocking conditions were as described above for the petri dishes. One hundred microliters of appropriately diluted cell culture supernates were dispensed into triplicate wells and incubated for 3 h at 37°C in a CO 2 incubator. After 4 washings with PBS-Tween, the wells were exposed to 100/~1 of diluted HRP RaHIg conjugate for 3 h at room temperature, washed thoroughly with PBS and developed with OPD-substrate (200/~l/well) as above. Hemolytic plaque-forming cell (PFC) assay Mouse spleen cells were assayed for ISC numbers by the protein A plaque assay (Gronowicz et al., 1976) following the detailed procedure of Bird and Britton (1979). Staphylococcus Cowan 1 protein A (Pharmacia, Uppsala) was coupled to sheep erythrocytes by the chromium chloride method according to Gronowicz et al. (1976). The Ig fraction of rabbit anti-mouse Ig antiserum (Dakopatts) was used as developer in the presence of sheep red blood cell-absorbed guinea pig serum as source of complement. Plaques were counted after 4 h incubation at 37°C. Results
Detection and enumeration of immunoglobulin-secreting mouse splenocytes by the RELISPOT test Mouse splenocytes ( 1 0 4 - 1 0 6 ) were plated for 4 h in antiglobulin-coated assay
493 TABLE I SPECIFICITY OF THE RELISPOT TEST FOR THE ENUMERATION OF IMMUNOGLOBULINSECRETING MOUSE SPLENOCYTES Culture medium with
SFC/2 x 105 splenocytes cultured in dishes coated with
-
125
8
2
Rabbit anti-mouse Ig Rabbit anti-mouse albumin Cycloheximide
17 105 28
ND a 2 ND
ND ND ND
Rabbit antimouse Ig
Rabbit antimouse albumin
FBS
a Not done. culture dishes. A f t e r stepwise a d d i t i o n of e n z y m e - l a b e l e d a n t i - m o u s e Ig a n t i b o d i e s a n d agar c h r o m o g e n - s u b s t r a t e d a r k - b r o w n i s h spots a p p e a r e d within minutes, indic a t i n g the former l o c a t i o n of i m m u n o g l o b u l i n secreting cells. O c c a s i o n a l artefacts d u e to cell debris a n d / o r aggregates were easily distinguished f r o m the singular p a t t e r n of true reactions, i.e., i n d i v i d u a l i z e d a n d h o m o g e n e o u s l y g r a n u l a t e d foci (Fig. 1). Spots d i d not develop in dishes that h a d been c o a t e d with an irrelevant a n t i b o d y p r e p a r a t i o n ( r a b b i t a n t i - m o u s e a l b u m i n ) or in dishes e x p o s e d to F B S o n l y (Table I). A d d i t i o n of R a M Ig to the cells d u r i n g p l a t i n g i n h i b i t e d the f o r m a t i o n of spots whereas a d d i t i o n of n o r m a l r a b b i t Ig h a d no effect. P r e i n c u b a t i o n of the cells with
Fig. 1. Typical appearance of reactions (spots) generated by mouse immunoglobulin-secreting cells and developed by means of the RELISPOT test. Artefacts may appear occasionally and are indicated here by an arrow. The indicated scale corresponds to 1 mm.
494
the inhibitor of protein synthesis cycloheximide (25 # g / m l ) for 4 h and during plating reduced the number of spots by almost 80%. As inferred from the above observations, the generation of spots by mouse splenocytes appeared to be due to the specific binding of Ig molecules secreted by metabolically active cells.
In situ ELISA measurement of Ig secreted by mouse splenocytes Replicate dishes were exposed to OPD-substrate solution instead of agar-PPD. Color development of enzyme-bound activity was recorded allowing estimation of the total amount of Ig secreted and bound within the cell population tested. This enabled the detection of immunoglobulin secreted from as few as 5 × 104 mouse splenocytes (Fig. 2). This corresponded to approximately 50 spot-forming cells (SFC). Comparison of R E L I S P O T test and protein A plaque-forming cell assay jor the enumeration of immunog[obulin secreting mouse ~p[enoc),tes Normal mouse splenocytes were assayed in parallel for ISC numbers by means of the protein A plaque assay and by the RELISPOT test. The results obtained by both procedures were comparable (1650 + 210 PFC and 1480 + 130 SFC per 106 splenocytes respectively). However, increasing the cell incubation period from 4 to 6 h resulted in a substantial increase of ISC detected as spots by the RELISPOT test (approximately 30%) whereas hemolysis started to occur spontaneously in the corresponding PFC assay (data not illustrated). Detection and enumeration of immunoglobulin-secreting cells (ISC) generated in cultures of human mononuclear cells exposed to EB V Human peripheral blood MNC were assayed by the RELISPOT test for ISC A492 0.5
0,4
0.3
0.2
T+--I
0 0.1
0 2 5 0.5
1
2
4 xlO s cells plated
Fig. 2. In situ E L I S A m e a s u r e m e n t of Ig secreted for various n u m b e r s of mouse splenocytes cultured for 4 h in R a M Ig coated dishes ( e ) a n d in FBS-coated control dishes ( O - - - - O). Results are expressed as m e a n a b s o r b a n c e values _+ SEM of triplicate determinations.
495 T A B L E II A N A L Y S I S OF I M M U N O G L O B U L I N SECRETIO N BY H U M A N L Y M P H O C Y T E S S T I M U L A T E D WITH EPSTEIN-BARR VIRUS
PERIPHERAL
Donor
EBV
ELISA
1
+ -
0.85 0.06
110000 220
2
+ -
0.70 0.12
62000 1 500
a
RELISPOT
BLOOD
a
Results are expressed as mean absorbance values (A492) and SFC numbers for 2 × 106 cells cultured.
numbers in Rail Ig-coated dishes following in vitro exposure to EBV for 6 days. The results shown in Table II indicate that human ISC could be detected and enumerated by means of the RELISPOT test. It should be pointed out that there was no difference in the macroscopical appearance of spots generated by HMNC and mouse ISC. In addition the cell culture supernatants were assayed for total Ig contents by a conventional ELISA technique. Table II shows that the RELISPOT test appeared more discriminative (ratio of stimulated cultures to unstimulated cultures) than the ELISA procedure.
Discussion
The 'reverse' enzyme-linked immunospot test (RELISPOT) described here provides a tool whereby the secretion of antigenic macromolecules, e.g., immunoglobulins, can be assessed at the individual cell level. Results obtained by this method are comparable to those obtained with the protein A-plaque assay (Gronowicz et al., 1976) for enumeration of mouse spleen cells secreting immunoglobulins. However, the immunoenzyme procedure offers advantages. Unlike conjugated red blood cells whose preparation and storage are complicated (Merchant and Inman, 1977), plastic immunoadsorbents can be prepared very simply without any cumbersome coupling step and are generally stable over long periods of storage. These properties are shared with those of the recently described ELISPOT assay (Czerkinsky et al., 1983) for the detection of specific antibody-secreting cells and should ease the standardization of the present method from one laboratory to another. The stability of the immunoadsorbent during the cell incubation is another advantage. It should be particularly important when studying the secretion of antigenic molecules by cells displaying a relatively low rate of synthesis and thus requiring longer incubation periods in order to generate detectable reactions (spots).
496
Unlike hemolytic plaque assays, the RELISPOT test can easily be adapted for the quantitation of secreted antigen using the same cell culture device, thus providing information concerning the average rate of antigen secretion for a given population of cells. When compared to recently described culture well enzyme-immunoassay systems (Weetman et al., 1981; Boerrigter et al., 1983) for the quantitation of secreted immunoglobulin, the RELISPOT test offers a major advantage by permitting the determination of the frequency of Ig-secreting cells. We have successfully applied the RELISPOT test for the detection of immunoglobulin-secreting cells generated upon in vitro exposure of human peripheral blood mononuclear cells to Epstein-Barr virus. It may be mentioned that the use of F(ab')2 fragments of anti-human antiglobulin reagents, although not necessary in the present study, is required to enable isotype-specific determination of immunoglobulin secreting cells by avoiding interference due to rheumatoid factors secreted by human lymphocytes stimulated in vitro with Epstein-Barr virus (Fong et al.. 1981). The RELISPOT assay design may be a general tool for detecting antigen-secreting cells as spot-forming cells provided the corresponding specific antibodies are available. The assay should provide a useful alternative to reverse hemolytic plaqueforming cell assays for studies assessing the secretion of immunoreactive molecules of individual cells.
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