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Enumeration of antibody-secreting cells by immunoprinting: sequential readout of different antibody isotypes on individual cell monolayers employing the ELISA plaque assay
Journal of Immunological Methods, 93 (1986) 167-169 Elsevier
167
JIM 04073
Enumeration of antibody-secreting cells by immunoprinting: sequential readout of different antibody isotypes on individual cell monolayers employing the ELISA plaque assay P.G. H o l t * a n d T.M. Plozza Clinical Immunology Research Unit, Princess Margaret Hospital, Thomas Street, Subiaco 6008, Western A ustralia, Australia (Received 6 January 1986, accepted 15 May 1986)
A technique is described for obtaining 'prints' of the antibody output of individual antibody-secreting cells (ASC), by repeated exposure of an immobilized ASC monolayer to coverslips coated with antigen. Zones of bound antibody on the coverslips (each being the 'print' of an individual ASC) are subsequently visualized by ELISA technology. Key words: Antibody-secreting cell; Immunoprinting; ELISA plaque assay
Introduction
We recently described a novel ELISA-based assay system for the detection of antibody-secreting cells (ASC), which in parallel trials appeared more sensitive than conventional haemolytic plaque assays (Sedgwick and Holt, 1983). We now report a modification of this assay, which facilitates repeated probing of individual ASC preparations immobilized in poly-L-lysinetreated microplate wells. In this procedure, sequential 'prints' of the antibody output of ASC are taken by lowering plastic coverslips (coated with appropriate antigen) onto the monolayers. Each print may then be developed for a different antibody isotype employing appropriate anti-Ig * Address for correspondence. Abbreviations: ELISA, enzyme-linked immunosorbent assay; ASC, antibody-secreting cells; Ig, immunoglobulin; AP, alkaline phosphatase; AH, aluminium hydroxide; PLL, polyL-lysine; OA, ovalbumin; BSA, bovine serum albumin; DNP, dinitrophenyl; poly, polymerized antigen; 5-BCIP, 5-bromo-4chloro-3-indolyl-phosphate; i.p., intraperitoneal; FCS, foetal calf serum.
reagents, as in the ELISA plaque assay, and the ASC monolayer may be fixed and subsequently probed by a variety of techniques (e.g., immunohistochemistry, autoradiography etc.). By superimposing the coverslips over the stained cell monolayer in the orientation originally employed whilst immunoprinting, a wide range of information may thus be gained on the ASC responsible for individual ELISA plaques.
Material and methods
Materials Eight-well multiplates (Lux, U.S.A.; well size 26 x 33 mm) were used, together with 24 x 30 mm Thermanox tissue culture coverslips (Lux, U.S.A.). Poly-L-lysine (PLL; 200000), 5-bromo-4-chloro3-indolyl-phosphate(5-BCIP), ovalbumin (OA; Grade V) and bovine serum albumin (BSA) were from Sigma Chemicals (U.S.A.). Antigens Polymerized (poly) OA was prepared via
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
168 glutaraldehyde crosslinking, as described in Holt et al. (1984). Dinitrophenyl conjugates of BSA were prepared as described in Eisen (1975); material with a conjugation ratio of 25:1 was used in this study (see Holt et al., 1984).
Enumeration of A SC by immunoprinting DNP-BSA or poly OA were conjugated to the coverslips as described by Sedgwick and Holt (1983), employing 2.0 m g / m l solutions of the antigen. They were washed three times in PBS0.05% Tween 20 and twice in RPMI plus 10% foetal calf serum (FCS) before use. To the wells of a Lux multiplate were added 1.0 ml volumes of a 1 m g / m l solution of PLL in PBS (determined as optimal in preliminary experiments). After incubation at room temperature overnight or at 37°C for 30-60 rain the plates were washed with PBS/Tween and R P M I / F C S before use. 2.0 ml aliquots of a suspension of OA-immune mouse splenocytes or h y b r i d o m a cells secreting IgE anti-DNP (clone 26-82; Liu et al., 1980) in R P M I / F C S were gently dispensed into PLLcoated multiplate wells, and the plates incubated for 90 min at 37 o C. The attached cell monolayers were then washed gently with R P M I / F C S and 2.0 ml of fresh medium added back to the wells. Coverslips with conjugated antigen (poly OA or DNP-BSA) were then carefully lowered into the wells, and the plates were incubated for 2 h at 37°C (immunoprint 1). This process was repeated up to four times (immunoprints 2-4). Immediately after removal from the wells, each coverslip was washed three times in PBS/Tween. The coverslips were then incubated sequentially with rabbit antimouse IgE or IgG antibody, a sheep anti-rabbit IgG-AP conjugate, and finally an AP substrate (5-BCIP) which yields an insoluble blue product. The reagents and incubation conditions used were exactly as described for the ELISA-plaque assay (Sedgwick and Holt, 1983), except that agarose was not included in the 5-BCIP mixture.
Results and discussion
In principle, the assay system described here employs ELISA technology for the visualization
Fig. 1. ELISA plaques formed by clone 26-82 hybridomacells, incubated over a monolayercomprisingconjugated DNP-BSA. Magnificationx 40.
of zones of bound antibody, which become immobilized on the surface of the antigen-coated coverslips, directly above individual ASC. The methodology employed to develop these zones is identical to that originally described by us as the ELISA plaque assay (Sedgwick and Holt, 1983), in which ASC in suspension are instead layered onto an antigen-conjugated solid phase (surface of a microplate well). The appearance of the resulting ELISA plaques in both systems (Fig. 1) is virtually indistinguishable. The experiments illustrated in Fig. 2 compare the sensitivity of the ELISA plaque assay with that of the immunoprint system in detecting the frequency of cells exhibiting active antibody secretion within a suspension of clone 26-82 hybridoma
169 'U O
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tive to the ELISA plaque assay) falls below 90%, and that a third print still detects in excess of 75%. Comparable data (not shown) have been obtained with OA-immune splenocytes from mice, both for IgE and IgG, and we have confirmed the feasibility of sequential immunoprinting of individual splenocyte monolayers for different antibody isotypes.
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ISlIy Fig. 2. Sequential immunoprinting of individual monolayers of ASC. The frequency of ASC in a suspension of clone 26-82 hybridoma cells was determined by the ELISA plaque assay (Sedgwick and Holt, 1983), and in parallel by repeated 2 h immunoprints, as described in the text. The data are from four separate trims, wherein ASC frequency in the hybridoma preparation was 36-58% of total cells; the variation between replicate immunoprints was < 10%.
cells. The data are presented as the means + SD from four consecutive trials, and are normalized against the figure for the ELISA plaque assay. It can be seen that at least two consecutive immunoprints may be taken from individual ASC monolayers, before the detection efficiency (rela-
This work was supported by the TVW Telethon Foundation, and the Princess Margaret Children's Medical Research Foundation. This is Publication no. 241 from the Clinical Immunology Research Unit of the PMCMRF. We thank Dr. J.D. Sedgwick and Dr. G.A. Stewart for helpful discussions.
References Eisen, H.N., 1984, Methods Med. Res. 10, 94. Holt, P.G., J.D. Sedgwick, G.A. Stewart, C. O'Leary and K. Krska, 1984, J. Immunol. Methods 74, 1. Liu, F.T., J.W. Bohn, E.L. Ferry, H. Yamamoto, C.A. Molinaro, L.A. Sherman, N.R. Klinman and D.H. Katz, 1980, J. Immunol. 124, 2728. Sedgwick, J.D. and P.G. Holt, 1983, J. Immunol. Methods 57, 301.
167
JIM 04073
Enumeration of antibody-secreting cells by immunoprinting: sequential readout of different antibody isotypes on individual cell monolayers employing the ELISA plaque assay P.G. H o l t * a n d T.M. Plozza Clinical Immunology Research Unit, Princess Margaret Hospital, Thomas Street, Subiaco 6008, Western A ustralia, Australia (Received 6 January 1986, accepted 15 May 1986)
A technique is described for obtaining 'prints' of the antibody output of individual antibody-secreting cells (ASC), by repeated exposure of an immobilized ASC monolayer to coverslips coated with antigen. Zones of bound antibody on the coverslips (each being the 'print' of an individual ASC) are subsequently visualized by ELISA technology. Key words: Antibody-secreting cell; Immunoprinting; ELISA plaque assay
Introduction
We recently described a novel ELISA-based assay system for the detection of antibody-secreting cells (ASC), which in parallel trials appeared more sensitive than conventional haemolytic plaque assays (Sedgwick and Holt, 1983). We now report a modification of this assay, which facilitates repeated probing of individual ASC preparations immobilized in poly-L-lysinetreated microplate wells. In this procedure, sequential 'prints' of the antibody output of ASC are taken by lowering plastic coverslips (coated with appropriate antigen) onto the monolayers. Each print may then be developed for a different antibody isotype employing appropriate anti-Ig * Address for correspondence. Abbreviations: ELISA, enzyme-linked immunosorbent assay; ASC, antibody-secreting cells; Ig, immunoglobulin; AP, alkaline phosphatase; AH, aluminium hydroxide; PLL, polyL-lysine; OA, ovalbumin; BSA, bovine serum albumin; DNP, dinitrophenyl; poly, polymerized antigen; 5-BCIP, 5-bromo-4chloro-3-indolyl-phosphate; i.p., intraperitoneal; FCS, foetal calf serum.
reagents, as in the ELISA plaque assay, and the ASC monolayer may be fixed and subsequently probed by a variety of techniques (e.g., immunohistochemistry, autoradiography etc.). By superimposing the coverslips over the stained cell monolayer in the orientation originally employed whilst immunoprinting, a wide range of information may thus be gained on the ASC responsible for individual ELISA plaques.
Material and methods
Materials Eight-well multiplates (Lux, U.S.A.; well size 26 x 33 mm) were used, together with 24 x 30 mm Thermanox tissue culture coverslips (Lux, U.S.A.). Poly-L-lysine (PLL; 200000), 5-bromo-4-chloro3-indolyl-phosphate(5-BCIP), ovalbumin (OA; Grade V) and bovine serum albumin (BSA) were from Sigma Chemicals (U.S.A.). Antigens Polymerized (poly) OA was prepared via
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
168 glutaraldehyde crosslinking, as described in Holt et al. (1984). Dinitrophenyl conjugates of BSA were prepared as described in Eisen (1975); material with a conjugation ratio of 25:1 was used in this study (see Holt et al., 1984).
Enumeration of A SC by immunoprinting DNP-BSA or poly OA were conjugated to the coverslips as described by Sedgwick and Holt (1983), employing 2.0 m g / m l solutions of the antigen. They were washed three times in PBS0.05% Tween 20 and twice in RPMI plus 10% foetal calf serum (FCS) before use. To the wells of a Lux multiplate were added 1.0 ml volumes of a 1 m g / m l solution of PLL in PBS (determined as optimal in preliminary experiments). After incubation at room temperature overnight or at 37°C for 30-60 rain the plates were washed with PBS/Tween and R P M I / F C S before use. 2.0 ml aliquots of a suspension of OA-immune mouse splenocytes or h y b r i d o m a cells secreting IgE anti-DNP (clone 26-82; Liu et al., 1980) in R P M I / F C S were gently dispensed into PLLcoated multiplate wells, and the plates incubated for 90 min at 37 o C. The attached cell monolayers were then washed gently with R P M I / F C S and 2.0 ml of fresh medium added back to the wells. Coverslips with conjugated antigen (poly OA or DNP-BSA) were then carefully lowered into the wells, and the plates were incubated for 2 h at 37°C (immunoprint 1). This process was repeated up to four times (immunoprints 2-4). Immediately after removal from the wells, each coverslip was washed three times in PBS/Tween. The coverslips were then incubated sequentially with rabbit antimouse IgE or IgG antibody, a sheep anti-rabbit IgG-AP conjugate, and finally an AP substrate (5-BCIP) which yields an insoluble blue product. The reagents and incubation conditions used were exactly as described for the ELISA-plaque assay (Sedgwick and Holt, 1983), except that agarose was not included in the 5-BCIP mixture.
Results and discussion
In principle, the assay system described here employs ELISA technology for the visualization
Fig. 1. ELISA plaques formed by clone 26-82 hybridomacells, incubated over a monolayercomprisingconjugated DNP-BSA. Magnificationx 40.
of zones of bound antibody, which become immobilized on the surface of the antigen-coated coverslips, directly above individual ASC. The methodology employed to develop these zones is identical to that originally described by us as the ELISA plaque assay (Sedgwick and Holt, 1983), in which ASC in suspension are instead layered onto an antigen-conjugated solid phase (surface of a microplate well). The appearance of the resulting ELISA plaques in both systems (Fig. 1) is virtually indistinguishable. The experiments illustrated in Fig. 2 compare the sensitivity of the ELISA plaque assay with that of the immunoprint system in detecting the frequency of cells exhibiting active antibody secretion within a suspension of clone 26-82 hybridoma
169 'U O
1.0'
tive to the ELISA plaque assay) falls below 90%, and that a third print still detects in excess of 75%. Comparable data (not shown) have been obtained with OA-immune splenocytes from mice, both for IgE and IgG, and we have confirmed the feasibility of sequential immunoprinting of individual splenocyte monolayers for different antibody isotypes.
--'~
4)
"o @ u) ,<
0.8'
0.6 O i.
o .Q
E
~J C
0.4
Acknowledgements
0.2
E
i
Ellea
plaque
i
i
I
i
1
2
3
4
ISlIy Fig. 2. Sequential immunoprinting of individual monolayers of ASC. The frequency of ASC in a suspension of clone 26-82 hybridoma cells was determined by the ELISA plaque assay (Sedgwick and Holt, 1983), and in parallel by repeated 2 h immunoprints, as described in the text. The data are from four separate trims, wherein ASC frequency in the hybridoma preparation was 36-58% of total cells; the variation between replicate immunoprints was < 10%.
cells. The data are presented as the means + SD from four consecutive trials, and are normalized against the figure for the ELISA plaque assay. It can be seen that at least two consecutive immunoprints may be taken from individual ASC monolayers, before the detection efficiency (rela-
This work was supported by the TVW Telethon Foundation, and the Princess Margaret Children's Medical Research Foundation. This is Publication no. 241 from the Clinical Immunology Research Unit of the PMCMRF. We thank Dr. J.D. Sedgwick and Dr. G.A. Stewart for helpful discussions.
References Eisen, H.N., 1984, Methods Med. Res. 10, 94. Holt, P.G., J.D. Sedgwick, G.A. Stewart, C. O'Leary and K. Krska, 1984, J. Immunol. Methods 74, 1. Liu, F.T., J.W. Bohn, E.L. Ferry, H. Yamamoto, C.A. Molinaro, L.A. Sherman, N.R. Klinman and D.H. Katz, 1980, J. Immunol. 124, 2728. Sedgwick, J.D. and P.G. Holt, 1983, J. Immunol. Methods 57, 301.