Journal of Immunological Methods, 156 (1992) 191-198 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00

191

JIM06501

A simplification of the enzyme-linked immunospot technique Increased sensitivity for cells secreting IgG antibodies to Haemophilus influenzae type b capsular polysaccharide T o r b e n Barington a

a, Susanne Sparholt b, Lars Juul a and Carsten Heilmann a

Laboratory of Medical Immunology 7544, Medical Department TTA, Rigshospitalet, Copenhagen, Denmark, and bALK Research, Hcrsholm, Denmark (Received 10 February 1992, revised received 4 June 1992, accepted 22 June 1992)

A simplified enzyme-linked immunospot (ELISPOT) technique is described for the detection of cells secreting antibodies to tetanus toxoid (TT), diphtheria toxoid (DT) or Haemophilus influenzae type b capsular polysaccharide (PRP). By combining the cell suspension with the enzyme-linked secondary antibodies in one incubation, the second incubation and washing procedure could be omitted from the original technique. The simplified assay had the same sensitivity for anti-TI" and anti-DT spot-forming cells as the ordinary ELISPOT assay. The IgG anti-PRP spots were, however, improved both in quality and in quantity (median: 40% more spots), while the detection of IgM and IgA anti-PRP spot-forming cells was the same in the two techniques. This simplified technique can probably also be used to save time in other antigen systems and should be considered when designing ELISPOT assays for the detection of polysaccharide-specific antibody-secreting cells. Key words: ELISPOT; Tetanus toxoid; Diphtheria toxoid; Polysaccharide; Affinity; Avidity

Introduction

Since the introduction of the enzyme-linked immunosorbent spot-forming cell (ELISPOT)

Correspondence to: T. Barington, Laboratory of Medical Immunology 7544, Medical Department TTA, Rigshospitalet, Tagensvej 20, DK-2200 Copenhagen N, Denmark. Tel.: +453545-7631; Fax: +45-3139-8766. Abbreviations: ELISPOT, enzyme-linked immunosorbent spot-forming cell; PFC, plaque-forming cell; AbSC, antibodysecreting cell; PRP-D, diphtheria toxoid conjugated PRP; PRP-T, tetanus toxoid conjugated PRP; MNC, mononuclear cell; TT, tetanus toxoid; DT, diphtheria toxoid; PRP, Haemophilus influenzae type b capsular polysaccharide; HRP, horseradish peroxidase; PBS, phosphate-buffered saline.

technique (Czerkinsky et al., 1983; Sedgwick and Holt, 1983) this method has, in many laboratories, gradually replaced the haemolytic plaqueforming cell (PFC) assay (Jerne and Nordin, 1963) for the enumeration of antibody-secreting cells (AbSC). The ELISPOT technique circumvents the need for coating of erythrocytes with antigen and does not depend on a supply of red blood cells and complement, reagents that are difficult to standardize. Furthermore, several groups have reported that the ELISPOT assay has a higher sensitivity than the haemolytic PFC technique, at least for some antigens (Franci et al., 1986). Finally, the ELISPOT technique permits the use of monoclonal antibodies without complementactivating activity as secondary antibodies.

192 To date the ELISPOT assays described have been simple modifications of the ordinary enzyme-linked immunosorbent assays for the detection of antibodies in solution (Engvall and Perlmann, 1972) in which the incubation with serum is replaced by incubation with cells. The current ELISPOT assays thus involve stepwise coating of a solid phase, blocking (sometimes omitted), incubation with cells, incubation with secondary antibody(ies), and addition of substrate - all separated by washings. However, since antibody is not present when the incubation with cells is initiated, but accumulates locally around the AbSC during the assay, there is no obvious reason why the incubation with cells and secondary antibodies should be separated, as long as a surplus of secondary antibody is provided. Recently, we have shown in Jerne-type haemolytic PFC assays that the presence of secondary antibody during the secretion phase increases the sensitivity for B cells secreting IgG directed against capsular polysaccharides (Barington and Heilmann, 1992). Therefore, we wished to determine whether modification of the current ELISPOT technique by combining the incubation with cells and secondary antibody, besides saving time and reagents, might also increase the sensitivity for this type of AbSC. Here we present a comparison of the original ELISPOT technique and a simplified version, with respect to the antigens tetanus toxoid (TT), diphtheria toxoid (DT), and Haemophilus influenzae type b capsular polysaccharide (PRP).

Mononuclear cells Heparinized blood was drawn on postvaccination days 7 or 8. Mononuclear cells (MNC) were isolated by density gradient centrifugation on metrizoate/Ficoll (Lymphoprep, Nycomed, Oslo, Norway), washed twice in minimal essential medium (Flow, Irvine, UK) supplemented with penicillin (400 IU/ml) and streptomycin (400 /zg/ml) (both from Gibco, Paisley, UK). Finally, the ceils were resuspended in RPMI (Gibco) supplemented with 10% foetal calf serum (Flow). Cells were kept on ice until used in the assays.

Materials and methods

Isolation of surface IgM and IgA positive cells For some experiments 12 X 106 washed MNC were resuspended in 200 /xl RPMI + 5% foetal calf serum containing murine monoclonal antihuman /x chain (Becton Dickinson, Mountain View, CA, USA), a mixture of anti-a I and anti-a 2 chain (Oxoid, Bedford, UK), or anti-CD3 (antiLeu 4, Becton Dickinson) antibodies at a final dilution of 1/5. After incubation for 30 min on ice, the cells were washed three times in ice-cold medium and resuspended in 600 txl medium with 60 N 106 washed magnetizable beads coated with sheep anti-mouse IgG antibodies (Dynabeads M450, Dynal, Oslo, Norway) and rotated end-overend for 30 min at 4°C. The cells were washed three times on a magnet to remove MNC not binding to beads, and the bead-binding fraction was resuspended in R P M I + 10% foetal calf serum and tested directly in the ELISPOT assays. Samples treated with antibody to the T cell marker, CD3, which is not present on B cells, served as a control for non-specific binding of AbSC to the beads.

Vaccines and vaccinees Healthy adult volunteers were, after informed consent, vaccinated subcutaneously with one dose of one of the following vaccines: AI(OH) 3 adsorbed TI" (40 IU) or DT (5 IU) (State Serum Institute, Copenhagen, Denmark), 25 Ixg of PRP conjugated to 18 Izg DT (PRP-D) (Prohibit, Connaught Laboratories, Swiftwater, PA, USA), 10 /zg of PRP coupled to 24 /zg of TT (PRP-T) (Pasteur M6rieux, Lyon, France). The protocol was approved by the regional ethical committee.

Ordinary ELISPOT assays These assays were based on the principles originally described by Czerkinsky et al. (1983) and Sedgwick and Holt (1983) and modified as described elsewhere (Sparholt et al., 1991). All reagents were added in 100 Izl aliquots and unless otherwise stated, incubations were performed at room temperature. For the assays detecting PRP AbSC, microtiter plates (Immulon 2, Dynatech, Chantilly, VA, USA) were incubated overnight with PRP coupled to poly-L-lysine (MW

193 55,000) (kindly supplied by Connaught Laboratories) by a modification of the method of Gray (1979) at a final concentration of 10 /zg PRP coupled to 2.5 /~g poly-L-lysine/ml. For assays detecting TT and DT AbSC, Nunc-Immunoplate Maxisorp microtiter plates (Nunc, Roskilde, Denmark) were coated with 10 /~g/ml purified TT and DT (both from the State Serum Institute), respectively, diluted in sodium bicarbonate buffer, pH 9.6. After three washings with 0.5 M sodium chloride containing 0.044% (v/v) Tween 20 (Merck, Darmstadt, Germany) and once with phosphate-buffered saline (PBS, pH 7.4) (to remove detergent), MNC were added in supplemented RPMI and the plates were incubated for 3.5 h at 37°C in a humidified atmosphere containing 5% CO 2. After three washings the plates were incubated overnight with rabbit anti-human immunoglobulin antibodies coupled to horseradish peroxidase (HRP) (Dako, Glostrup, Denmark) diluted 1/1000 in PBS containing 1% bovine serum albumin (Sigma, St. Louis, MO, USA). Substrate was prepared by adding 3 ml of 96% (v/v) ethanol containing 24 mg of dioctyl sulfosuccinate (Sigma) and 100/xl DMSO (Merck) containing 7.2 mg of tetramethylbenzidine (Sigma) to 15 ml of 0.75% melted agarose (Litex, Denmark) in citrate/phosphate buffer pH 5.0. All the reagents were kept on a waterbath at 60°C. Finally, 6/zl of 35% (v/v) H202 were added and the mixture was immediately transferred to the wells and allowed to harden on a level surface for a few minutes. Spots were formed within 20 min. The plates were sealed with an adhesive plastic film and kept at 4°C until the spots were enumerated using a dark-field stereo microscope at low magnification. Optimal concentrations of antigen and secondary antibodies were determined by log-2 checkerboard titrations performed separately for each isotype.

Simplified ELISPOT assays These were performed exactly as the ordinary assays described above, except that the enzymeantibody conjugates were added to the cell suspensions at the initiation of culture in a final dilution of 1/1000 (Table I). After incubation for 3.5 h the plates were washed and the wells left with PBS + 1% bovine serum albumin overnight.

TABLE I THE THREE VERSIONS OF THE ELISPOT TECHNIQUE USED IN THIS REPORT Assay version Ordinary

Present in the wells First incubation Secondincubation 3.5 h overnight MNC HRP-anti-Ig

Simplified

MNC+ HRP-anti-Ig

Buffer a

Facilitated

MNC+ F(ab')2 antilight chain

HRP-anti-Ig

a

This step can be omitted (see results section).

Optimal concentrations of secondary antibodies were determined by log 2 titration experiments.

Preincubation with anti-immunoglobulin For some experiments MNC were incubated in a 24-well culture dish (Nunclon, Nunc, 2 ml/well, 1 × 106/ml in supplemented medium) for 2 h at 37°C in a humidified atmosphere containing 5% CO 2 with or without HRP-conjugated rabbit anti-human immunoglobulin antibodies (pooled anti-IgM, -IgG, and -IgA, all at a final dilution of 1/1000). The cells were carefully harvested, washed three times, resuspended, and tested in the ordinary PRP ELISPOT assays as described above. The cells used for these experiments were obtained from three PRP-T and one PRP-D vaccinated donors as described above. Immediately after separation, however, they had been frozen in RPMI + 15% foetal calf serum (FCS) + 10% DMSO and stored in liquid nitrogen until the day of the experiment.

Facilitation of spot formation by F(ab') 2 anti-light chain antibodies In some experiments the ordinary assays detecting PRP AbSC were modified by the addition of F(ab') 2 anti-K light chain antibodies and F(ab') 2 anti-A light chain antibodies (Dako) to the cell suspension at the initiation of culture (Table I); a final dilution of 1/500 was employed (determined by log 2 titration experiments). After incubation, the plates were treated as in the ordinary assays described above.

194

+

PRP

+

DT

+

TT

Control

Fig. 1. Inhibition of spot formation in the simplified TT-specific ELISPOT assay (IgG) by the addition of free antigen to the cell suspension. The following concentrations were used: PRP, 1000 /zg/ml; DT and TT, 200 p~g/ml. The right-hand well was an antigen-free control.

Statistics The numbers of spots obtained in the different versions of the technique were compared by the Wilcoxon matched-pairs test (paired data) and the Mann-Whitney U test (unpaired data) using a significance level of p = 0.05.

Results

The antigen specificity of the simplified ELISPOT assays was studied by inhibition experiments with free antigen present in the culture medium during antibody secretion. For the PRPand DT-specific assays spot formation was inhibited in a dose-dependent way ending with 0-2% (mean, n = 3) visible spots when using the highest concentration of the relevant antigen (PRP: 1000

~zg/ml; and DT: 200 txg/ml, respectively) as compared with an uninhibited control. The number of Tr-specific AbSC was not significantly reduced by 200/zg/ml of TT. Qualitatively, however, the remaining spots in the wells with TT were clearly reduced in size (Fig. 1), indicating that a specific interaction between antigen and secreted antibody had indeed occurred. No inhibition (qualitative or quantitative) was seen when the irrelevant antigens were added at the same concentrations. Antigen specificity of all the assays was further established by the fact that no spots were formed if MNC obtained from donors vaccinated with an irrelevant antigen were used (in the case of the PRP-specific assays: DT; Trspecific assays: PRP-D; DT-specific assays: PRPT) (data not shown). The simplified ELISPOT assay was found to

TABLE II COMPARISON OF SIMPLIFIED TT- AND DT-SPECIFIC ELISPOT ASSAYS AND THE CORRESPONDING ORDINARY ELISPOT ASSAYS The number of spots per 10 6 MNC in the simplified assay is given as a percentage of that obtained in the ordinary assay. Assay

Isotype

Vaccine

specificity TT

DT

Simplified assay result as a % of ordinary ELISPOT assay Median

(quartiles)

(n)

IgG

TT PRP-T

102 105

(98-113) (98-111)

(15) (8)

IgA

TT PRP-T

93 87

(80-108) (83-109)

(16) (6)

IgG

DT PRP-D

101 108

(88-118) (103-119)

(12) (10)

IgA

DT PRP-D

89 85

(81-100) (82- 96)

(7) (6)

195 be reliable and easy to perform. In the comparative experiments outlined below the wells in the simplified assay were left overnight with incubation buffer in order to allow them to be present on the same plate and developed along with the wells of the ordinary and F ( a b ' ) 2 anti-light chain-facilitated assays. The overnight incubation could, however, be omitted and the substrate added directly without changing the results (data not shown). In the latter case it was necessary to soak the wells with washing buffer for 5 min during the two last washes in order to eliminate background staining. Due to the omission of one incubation, the assay could be performed in six hours including the isolation of MNC. As shown in Table II, the sensitivity of the simplified assay for IgA and I g G T-F and D T AbSC did not differ significantly from that of the original E L I S P O T technique ( p > 0.16). This was true both when the M N C were derived from donors immunized with the protein antigen itself and when protein-polysaccharide conjugates were used for immunization. Qualitatively, no significant difference in spot size, colour density or background was observed between the two E L I S P O T techniques. The simplified assay had a higher sensitivity for I g G anti-PRP AbSC (median 40% more spots detected) than the ordinary technique (table III), while similar sensitivities for IgM and I g A P R P AbSC were observed in the two techniques (Table III, p > 0.2). Qualitatively, no difference was observed in the IgM- and IgA-specific assays but, a s illustrated in Fig. 2, I g G anti-PRP spots were larger and more colour intensive when the simpli-

Ordinary

Simplified

assay

assay

Fig. 2. The quality of IgG anti-PRP spots in the ordinary ELISPOT assay and the simplified ELISPOT assay. lied technique was used. Selective facilitation of the detection of I g G anti-PRP AbSC was also observed when F ( a b ' ) 2 anti-light chain antibodies were added to the cell suspension at the initiation of culture followed by washing and incubation with enzyme-linked antibodies as in the original assay (Table III). In fact, the results obtained in the F ( a b ' ) anti-light chain antibody facilitated assays did not differ significantly from those obtained in the simplified assays ( p > 0.2). To ascertain whether the larger number of spots detected w h e n using the simplified E L I S P O T assay for I g G P R P AbSC was possibly due to co-detection of AbSC secreting anti-PRP antibody of other isotypes rather than IgG, the isotype specificity of the assays was evaluated. To this end, we purified surface IgM- and IgA-positive cells from P R P - T or P R P - D vaccinated donors and tested them in the simplified anti-PRP

TABLE III THE RELATIVE PERFORMANCE OF THE THREE VERSIONS OF THE PRP-SPECIFIC ELISPOT TECHNIQUE The number of spots per ordinary assays Isotype

IgM IgG IgA

10 6

MNC both the simplified and the facilitated assays are given as a percentage of that obtained in the

Simplified assay result as % of ordinary ELISPOT assay Median 96 140 a 97

Facilitated assay result as % of ordinary ELISPOT assay

(quartiles)

(n)

Median

(quartiles)

(n)

(74-102) (126-170) (93-109)

(14) (12) (17)

98 150 a 103

(95-111) (123-183) (93-112)

(14) (12) (17)

a Significantly higher than the spot numbers obtained in the ordinary assay (p < 0.004).

196

ELISPOT assays. Due to non-specific binding of some AbSC to the beads, it was necessary to include a control suspension prepared in the presence of an irrelevant antibody (i.e., anti-CD3 antibody). Fig. 3 shows that purified surface IgMand IgA-positive cells did not form more spots in the assays of other isotype specificities than the numbers found in the control. The same was found in experiments in which a mixture of surface IgM- or surface-IgA-positive cells and unseparated MNC from donors vaccinated with DT was used in the PRP-specific simplified ELISPOT assays (data not shown). The latter experiments were performed to exclude any spot formation resulting from an interaction between the secondary antibody, PRP-specific IgM or IgA AbSC, and IgG-secreting cells of other antigen specificities present in the same wells. One possible mechanism behind the increased sensitivity of the simplified assay for IgG PRP AbSC and the better quality of these spots as compared with the ordinary assay was studied. The presence of the secondary antibody during the incubation with the cells in the simplified

TABLE IV EFFECT OF PREINCUBATING MNC FOR 2 h WITH HRP-ANTI-IgM, -IgG, AND -IgA ON THE NUMBER OF SPOTS DETECTED IN THE ORDINARY PRP ELISPOT ASSAYS Data are given in percent of the spot numbers obtained with cells preincubated without anti-immunoglobulin. Isotype

Spots after preincubation with anti-lg in% of control (median and range, n = 4)

IgM IgG IgA

103 (96-126) 104 (98-113) 105 (91-113)

assay might be beneficial because it increased the rate of antibody secretion by cross-linking the antigen receptor present on at least some of the AbSC. This possibility was tested by preincubating the cells for 2 h with anti-immunoglobulin before testing them in the ordinary assays. This treatment did not increase the number of PRP AbSC detected (Table IV) and did not affect the quality of the spots.

Simplified PRP ELIspof assay Number of spofs (~) SIgM-pos cells

Discussion SIgA-pos cells

lOO

50

o IgM IgG IgA

IgM IgG IgA

Fig. 3. Spots formed in the three isotype-specific simplified PRP ELISPOT assays by purified surface IgM- and surface IgA-positive cells (closed bars) and by a control suspension similarly separated using an irrelevant antibody directed against the T cell surface molecule, CD3, not present on B cells (open bars). The latter served as a control for non-specific binding of AbSC to the beads used for purification. For the surface IgM- and IgA-positive cell suspensions, the numbers of spots are expressed as a percentage of the number detected in the IgM and IgA assays, respectively (mean and SE, n = 4).

In the enzyme-linked immunosorbent assays detecting antibodies in solution specific for antigen fixed to the solid phase, separation of the incubation with test sample from the incubation with secondary antibody is crucial. Without the intervening washing procedure there would not be a simple relationship between the amount of antibody in the test sample and the amount of secondary antibody bound. When Czerkinsky et al. (1983) and Sedgwick and Holt (1983) independently introduced the ELISPOT technique this scheme was adopted and has to our knowledge been used in all subsequently published modifications of the technique. However, there is no obvious reason why this separation of incubations is required in the ELISPOT technique as long as the secondary antibody does not inhibit the binding of the primary antibody to the antigen, and as long as the number of cells secreting immunoglobulin of the relevant isotype in the well

197

is not so high that the amount of secondary antibody fixed to the individual spots is below the limit of detection. The data presented in this report demonstrate that a time-saving combination of the two incubations is indeed possible and results in reliable assays with equal (or higher) sensitivity for the individual AbSC without losing antigen specificity. The simplified assays have low background and good quality of the individual spots is maintained. The fact that it was not necessary to increase the total amount of secondary antibody present per well in the simplified assay compared with the ordinary assay indicates that the amount of secondary antibody neutralized by antigennon-specific immunoglobulin is negligible. Theoretically, however, there must exist an upper limit for the number of cells secreting the relevant isotype that may be present in the well without interfering with spot formation. In this study, potentially the most critical aspect was the measurement of IgA DT and IgA q-T AbSC after vaccination with a conjugate. In these situations the cell suspension often contained more than 100 times as many IgA PRP AbSC as IgA DT or IgA TI" AbSC. Despite the fact that 200-500 IgA PRP AbSC were often present in the wells when measuring these spots, no sign of inhibition was seen. However, one should be aware of this possibility when using simplified ELISPOT assays for the detection of rare types of AbSC in the presence of high numbers of antigen-nonspecific cells secreting the same isotype. The demonstration of higher sensitivity of the simplified assay for cells secreting IgG (but not IgM or IgA) to PRP was not surprising to us. We have recently shown that special problems exist when measuring polysaccharide-specific AbSC of the IgG isotype in haemolytic PFC assays, since they are underestimated if an IgG-aggregating principle is not present in the assay during the phase of antibody secretion (Barington and Heilmann, 1992). Thus, it was found that the addition of secondary antibodies or F(ab') 2 anti-light chain antibodies from the beginning of the assay greatly enhanced detection of IgG anti-PRP and antipneumococcal polysaccharide AbSC without influencing the detection of IgM or IgA AbSC or IgG AbSC with specificity for DT or TI'. Based

on the low intrinsic affinity of anti-PRP antibody (Hetherington, 1988), we proposed that the mechanism behind this phenomenon in the haemolytic PFC assay was that aggregation of the secreted IgG by increasing the avidity, reduced the tendency for detachment of antibody bound to the target erythrocytes. Detachment and subsequent diffusion away from the AbSC before the addition of complement would reduce the size of the plaques and ultimately interfere with their detection. The lack of effect of Ig-aggregating molecules on the number of IgM and IgA antipolysaccharide AbSC and on the number of AbSC with specificity for protein antigens was explained by the polymeric nature of the molecules (and therefore higher avidity) and higher intrinsic affinity, respectively. However, one might argue that the effect of Ig-aggregating molecules in the haemolytic plaque assays might be due to the formation of more lytic immune complexes rather than to an increase in the avidity of the secreted antibodies. The finding in this study of a similar effect of aggregation of IgG in the ELISPOT technique (not depending on activation of complement) strongly supports our hypothesis and indicates that it might be a general problem associated with quantitation of cells secreting monomeric low-affinity antibody. It is, however, possible that the beneficial effect of IgG-aggregating molecules in the PRP-specific assays could be the result of a stimulatory effect of cross-linking of the antigen receptor on B cells rather than the effect of cross-linking of secreted antibody. However, we find this possibility unlikely for several reasons. Firstly, aggregation of the antigen receptor is expected to occur even in the ordinary assays due to antigen on the target cells or on the solid phase. Secondly, the lack of an effect of preincubating the cells with secondary antibodies before they are added to the ordinary IgG PRP ELISPOT assay (Table IV) argues against this hypothesis. It is concluded that the simplified ELISPOT assays are fast, reliable, specific and in the case of IgG anti-PRP AbSC, more sensitive than ordinary ELISPOT assays. This technique can probably be used with other antigens and should clearly be considered when measuring AbSC with specificity for polysaccharides.

198

Acknowledgements Marianne Pedersen is thanked for excellent technical assistance. This study was supported by the Danish Medical Research Council (grant no. 12-8419 and 120251-1), the Lundbeck Foundation, Institut MErieux Pasteur, and Connaught Laboratories, Inc.

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immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65, 109. Engvall, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109, 129. Francl, C., Ingl6s, J., Castro, R. and Vidal, J. (1986) Further studies on the ELISA-spot technique. Its application to particulate antigens and a potential improvement in sensitivity. J. Immunol. Methods 88, 225. Gray, B.M. (1979) ELISA methodology for polysaccharide antigens: Protein coupling of polysaccharides for adsorption to plastic tubes. J. Immunol. Methods 28, 187. Hetherington, S.V. (1988) The intrinsic affinity constant (K) of anticapsular antibody to oligosaccharides of Haemophilus influenzae type b. J. Immunol. 140, 3966. Jerne, N.K. and Nordin, A.A. (1963) Plaque formation in agar by single antibody-producing cells. Science 140, 405. Sedgwick, J.D. and Holt, P.G. (1983) A solid-phase immnnoenzymatic technique for the enumeration of specific antibody-secreting cells. J. Immunol. Methods 57, 301. Sparholt, S.H., Barington, T. and Schou, C. (1991) Detection of B-lymphocytes secreting antibodies to Dermatophagoides antigens. Clin. Exp. Allergy 21, 85.