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Antibodies to cell surface antigens detected by a rapid spot test using ethidium bromide
Journal of Immunological Methods, 49 (1982) 75--82
75
Elsevier Biomedical Press
ANTIBODIES TO CELL SURFACE ANTIGENS DETECTED BY A RAPID SPOT TEST USING ETHIDIUM BROMIDE
MARJORIE L. COHN, HANA GOLDING and MARVIN B. RITTENBERG
Department of Microbiology and Immunology, University of Oregon Health Sciences Center, Portland, OR 97201, U.S.A. (Received 17 July 1981, accepted 24 August 1981)
We describe here a spot test to detect antibodies to non-erythroid cell surface antigens, using ethidium bromide staining to visualize areas of target cell lysis. This assay allows rapid screening of large numbers of samples of culture supernatants, antisera, or ascitic fluids. Antibody titers determined by this spot test are shown to correlate well with titers determined by two other methods, providing a simple method for initial estimation of the a m o u n t of antibody in positive samples.
INTRODUCTION
The hemolytic spot test (Hfibner and Gengozian, 1969) has provided an extremely useful assay for the presence of antibodies in culture supernatants and other fluids. It is limited, however, to use in systems in which responses to erythrocytes or substances easily conjugated to erythrocytes are being examined. Recently, a modified spot test employing trypan blue staining to detect areas of cytolysis has been described (Marshak-Rothstein et al., 1979), allowing detection of antibodies to non-erythroid cell surface determinants. The binding of trypan blue to a variety of proteins (Goding, 1980), however, can result in some lack of specificity in this system. Here we describe a spot test employing ethidium bromide, a compound previously used as a viability stain in other systems (Lee et al., 1975), and demonstrate its usefulness both in screening for the presence of antibodies and in estimating antibody titers. MATERIALS AND METHODS
Mice BALB/c mice were bred in our colony from breeders purchased from the Fred Hutchinson Cancer Research Center, Seattle, WA. AKR/J and AKR X 0022-1759/82/0000--0000/$02.75 © 1982 Elsevier Biomedical Press
76 DBA/2 (AKD2) F1 mice were purchased from Jackson Laboratories, Bar Harbor, ME. In vitro maintenance o f hybridomas and myelomas The IgM anti-Thy 1.2-producing hybridoma, HO-13-4, originally described by Marshak-Rothstein et al. (1979), was obtained from the Cell Distribution Center, the Salk Institute, San Diego, CA. Cells were grown in Dulbecco's modified Eagle's medium (GIBCO) containing 10% fetal calf serum (FCS, Microbiological Associates, Bethesda, MD). When cultures reached a density of 1--2 × 106 cells/ml, cells were removed by centrifugation; supernatants were stored at --20°C. The non-secreting BALB/c myelomas, NS-1 and SP20/Ag-14 (SP-2), were grown in RPMI 1640 medium (GIBCO) containing 15% FCS and were handled as described above. In vivo cell passage AKD2 F1 mice which had been injected with 0.5 ml pristane (Aldrich Chemicals, Milwaukee, WI) 1--8 weeks earlier were injected with 5 × 106 HO13-4 hybridoma cells. One to 2 weeks later, mice were bled and ascitic fluids were collected. TK-4, an IgG anti-TNP producing hybridoma (Foiles and Rittenberg, in preparation), was grown in BALB/c mice to produce ascitic fluid as described above. SP-2 ascitic fluid was similarly obtained from BALB/c mice which had been injected with this myeloma. Tube c y t o t o x i c assay 2 × 10 7 BALB/c thymocytes, suspended in 0.4 ml MEM, were mixed with 0.4 ml of the indicated dilution of HO-13-4 culture supernatant and 0.2 ml of rabbit complement (Low-tox, Accurate Scientific and Chemical Corp., HicksviUe, NY). This mixture was incubated at 37°C for 60 min. Cells were washed once and then resuspended in trypan blue (0.2%) or ethidium bromide (1/~g/ml) for viability determinations, using light and fluorescence microscopy, respectively. Effect o f anti-Thy 1.2 on in vitro PFC response Spleen cells from BALB/c mice which had been primed with TNP-KLH (Golding and Rittenberg, 1980) were treated with HO-13-4 culture supernatant or ascites fluid and complement as previously described (Cohn and Scott, 1979). Cells were then cultured in micro Mishell-Dutton cultures. Seven days later, cultures were harvested, pooling 4--8 wells for assay; 3 such pools were assayed per experimental point. TNP-specific plaque-forming cells (PFC) were assayed (Cunningham and Szenberg, 1968) using TNP-sheep erythrocytes (Rittenberg and Pratt, 1969). Goat anti-mouse IgG antiserum was used to facilitate detection of PFC producing IgG anti-TNP antibodies. IgM PFC were suppressed by inclusion of sheep anti-mouse p chain in the assay mixture (Pierce et al., 1971).
77 PROCEDURE • TriP"
2 % Thymocyles,
BOTTOM: 14% Agorose in PBS .5 F, somples --10-]5' 37° (uncovered)
Rot)bit complement (1:10) --60'
--,5'
L0
37 °
Ethidiurn bromide ( 2 p g / m l ) Room letup
aveen
U V source
Fig. 1. Spot test procedure.
Spot test Spot tests were carried o u t using a modification of the m e t h o d of Marshak-Rothstein et al. (1979) (Fig. 1). Five ml o f 1.4% agarose (L'Industrie Biologique Fran~aise) in PBS were poured into a 100 m m diameter plastic dish (Falcon No. 1029). After the first layer hardened, it was overlaid with 5 ml of 0.7% agarose in MEM containing 2% BALB/c t h y m o c y t e s . Samples (5/~1 each) were spotted onto the plate and allowed to soak in. Two ml of rabbit C, diluted 1/10 in PBS, were added and the plates were incubated at 37°C for 60--90 rain. After removal of C, plates were stained for 5 min with either 2 ml o f 0.2% trypan blue in PBS or 2 ml of 2 pg/ml ethidium bromide (Calbiochem) in PBS. Plates stained with trypan blue were rinsed with PBS until areas o f lysis could be detected; those stained with ethidium bromide were examined immediately over a low wavelength ultraviolet light source (Fig. 2). RESULTS
Comparison of ethidium bromide and trypan blue staining of spot plates Samples o f culture supernatants, ascites fluids and sera were assayed on layers o f agarose containing BALB/c t h y m o c y t e s ; the results of such an assay are shown in Table 1. Ethidium bromide staining of test plates detected spots produced by HO-13-4 anti Thy 1.2 which were strongly positive and uniformly c o m p l e m e n t dependent; similar results were seen whether the monoclonal a n t i b o d y was contained in culture supernatant, ascitic fluid, or serum. These anti-Thy 1.2 reagents produced no ethidium bromide stained
78
Fig. 2. An ethidium bromide stained spot test plate. Samples of ascitic fluid (upper row) or culture supernatant (lower row) from the anti-Thy 1.2 producing HO-13-4 hybridoma (left) or the non-secreting SP-2 myeloma (right) were assayed on a layer of BALB/c (Thy 1.2) thymocytes.
79 TABLE 1 Comparison of ethidium bromide and trypan blue staining of BALB/c thymocyte spot plates. Stain
Ethidium bromide
Trypan blue
RC
+a
-- a
+
+++
k
÷÷÷
Cult. sup. HO-13-4 (~-Thy 1.2) b SP-2 (--) NS-1 (--) Ascitic fluid HO-13-4 (~-Thy 1.2) SP-2 (--) TK-4 (~-TNP) Serum HO-13-4 (~-Tby 1.2) Normal BALB/c (--)
D
+++
++
--
D
÷÷÷
÷÷÷
m
÷÷÷
÷÷÷
m
÷÷÷
÷÷÷
q-÷
÷÷
-b-I-
÷÷
a Plates were incubated with rabbit complement as described (÷) or with MEM (--) before staining with the indicated dye. b Antibody specificity of each sample is indicated in parentheses.
spots, however, when assayed on an A K R ( Thy 1.1) t h y m o c y t e layer (data n o t shown). In contrast, under similar conditions c o m p l e m e n t d e p e n d e n c e o f spots d e t e c t e d by t r y p a n blue staining could only be shown with culture supernatant
Titration o f cytotoxic activity o f H 0 - 1 3 - 4 culture supernatant Serial dilutions o f a sample o f HO-13-4 culture supernatant were assayed for their ability to p r o d u c e cytolysis in a spot test; these same dilutions were also used in a tu b e assay t o d e t e r m i n e c y t o t o x i c titers. The data from such an e x p e r i m e n t are shown in Fig. 3. At high c o n c e n t r a t i o n s o f culture supernatant, greater than 95% c y t o t o x i c i t y occurred; these c o n c e n t r a t i o n s of culture s u p er n atan t also gave very intense spots in the spot test. At increasing
80 ÷+
++
+÷
I 30
~ 3i
J 32
++
+
+
+I-
I 34
I 35
I 36
I00
8O
60
40
20
J 3~,
I 37
L 3e
I 39
p 310
I/dilution Fig. 3. Titration of cytotoxm activity of HO-13-4 culture supernatant. BALB/c thymocytes were treated with the indicated dilutions of HO-13-4 culture supernatant and rabbit complement, and stained with ethidium bromide for viability counts. Treatment with complement alone resulted in 33% dead cells; a minimum of 300 cells was counted at each point. Results of a spot test conducted simultaneously are shown above.
dilutions o f supernatant (1 in 34 or 3s), c y t o t o x i c i t y decreased to 80--85%, and th e corresponding spots were somewhat less intense. As dilution of the supernatant c o n t i n u e d , c y t o t o x i c i t y declined dramatically, accompanied by negative results in t he spot test. Similar results were obtained when samples f r o m tu b e assays were stained with t r y p a n blue rather than ethidium b ro m id e (data n o t shown). Thus it appears t hat c y t o t o x i c titers obtained by t u b e d eter min atio n s o f cell viability correlate well with spot test titrations.
Correlation o f spot test results with functional assay o f H0-13-4 activity To examine the ef f ect o f HO-13-4 antibodies on helper T cell function, spleen cells f r o m TNP-KLH primed BALB/c mice were treated with dilutions o f 'HO-13-4 culture supernatant (Fig. 4) or ascitic fluid (Fig. 5) plus rabbit C prior to c u l t u r e with TNP-KLH. These cells were assayed after 7 days of culture f o r IgG anti-TNP PFC. As can be seen in Fig. 4, a 1/10 dilution of HO-13-4 culture supernatant eliminated m o r e than 90% of the secondary TNP-KLH response, while a 1/30 dilution had little effect on the response, correlating well with the results of a spot test c o n d u c t e d on the same culture supernatant d i l u t i o n s . A similar correlation was seen when HO-13-4 ascitic fluid was assayed (Fig. 5). A 1/25 dilution o f ascitic fluid eliminated greater than 99% o f th e anti-TNP response o f u n t r e a t e d or c o m p l e m e n t treated cells, a 1/125 dilution, which gave a positive bu t less intense spot, eliminated a p p r o x i m a t e l y 80% of the TNpoKLH response, and a 1/625 dilution, negative in the spot test, resulted in less than 40% decrease in the t h y m u s d e p e n d e n t PFC response. In these experiments, no significant effect of HO13-4 t r e a t m e n t was seen when treated cells were cultured with the t h y m u s i n d e p e n d e n t antigens TNP-Brucella abortus or DNP-dextran (146% and
81 25
I/dilution N spot test
C
I0
30
++
+/-
90
I/dilution spot test
N
C
oi 25 ++
125 .625 +
Fig. 4. E f f e c t o f HO-13-4 c u l t u r e s u p e r n a t a n t o n h e l p e r T cell f u n c t i o n . S p l e e n cells f r o m T N P - K L H p r i m e d mice were t r e a t e d w i t h t h e i n d i c a t e d d i l u t i o n s o f HO-13-4 c u l t u r e s u p e r n a t a n t a n d c o m p l e m e n t b e f o r e c u l t u r e w i t h T N P - K L H t o g e n e r a t e a n IgG a n t i - T N P P F C r e s p o n s e . C o n t r o l r e s p o n s e s o f cells t r e a t e d w i t h c o m p l e m e n t a l o n e (C) a n d o f u n t r e a t e d cells ( N ) are also s h o w n , as are results o f a s p o t t e s t c o n d u c t e d o n t h e d a y o f culture initiation. Fig. 5. E f f e c t o f HO-13-4 ascitic fluid o n h e l p e r T cell f u n c t i o n . Ascitic fluid d i l u t i o n s were used in a n assay o t h e r w i s e i d e n t i c a l t o t h a t o f Fig. 4.
144% of control respectively at 1/10 dilution of culture supernatant, 75% and 110% of control respectively at 1/25 dilution of ascitic fluid). DISCUSSION
In this paper, we have described a rapid m e t h o d for screening various fluids for the presence of antibodies specific for cell surface antigens. While the studies here have used t h y m o c y t e layers to assay Thy 1.2 specific antibodies, antibodies specific for any antigen widely distributed in a given tissue could be similarly assayed. The use of ethidium bromide as a viability stain in this spot test eliminates the lack of specificity frequently seen with sera and ascitic fluids when trypan blue staining is used; the necessity of rinsing plates extensively to remove excess d y e when trypan blue staining is used is also eliminated here. We have also shown that antibody titers as determined by spot tests correlated well with titers determined by t w o other methods. Antibodies of classes other than IgM can be assessed b y including an additional incubation step using an appropriate anti-immunoglobulin reagent prior to incubation with complement. The m e t h o d described here can also be used in conjunction with the replica plating system described b y Bankert e t a l . (1980) thereby providing a simple, rapid m e t h o d of screening hybridomas for production of antibodies to cell surface antigens and for examining their specificity by assaying supernatants on a variety of cell types.
82 ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y G r a n t A I 1 4 9 8 5 f r o m t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , M a r c h o f D i m e s Basic R e s e a r c h G r a n t 1-633, a n d A m e r i c a n C a n c e r S o c i e t y G r a n t I M - 1 7 2 . M.L.C. a n d H . G . are F e l l o w s o f t h e N . L . T a r t a r Foundation. REFERENCES
Bankert, R.B., D. DesSoye and L. Powers, 1980, J. Immunol. Methods 35, 23. Cohn, M.L. and D.W. Scott, 1979, J. Immunol. 123, 2083. Cunningham, A. and A. Szenberg, 1968, Immunology 14,599. Goding, J.W., 1980, J. Immunol. Methods 39, 289. Golding, H. and M.B. Rittenberg, 1980, J. Immunol. 124, 1284. Hiibner, K.R. and N. Gengozian, 1969, J. Immunol. 102, 155. Lee, S.-K., J. Singh and R.B. Taylor, 1975, Eur. J. Immunol. 5, 259. Marshak-Rothstein, A., P. Fink, T. Gridley, D.H. Raulet, M.J. Bevan and M.L. Gefter, 1979, J. Immunol. 122, 2491. Pierce, C.W., B.M. Johnson, H.E. Gershon and R. Asofsky, 1971, J. Exp. Med. 134, 395. Rittenberg, M.B. and K.L. Pratt, 1969, Proc. Soc. Exp. Biol. Med. 132, 575.
75
Elsevier Biomedical Press
ANTIBODIES TO CELL SURFACE ANTIGENS DETECTED BY A RAPID SPOT TEST USING ETHIDIUM BROMIDE
MARJORIE L. COHN, HANA GOLDING and MARVIN B. RITTENBERG
Department of Microbiology and Immunology, University of Oregon Health Sciences Center, Portland, OR 97201, U.S.A. (Received 17 July 1981, accepted 24 August 1981)
We describe here a spot test to detect antibodies to non-erythroid cell surface antigens, using ethidium bromide staining to visualize areas of target cell lysis. This assay allows rapid screening of large numbers of samples of culture supernatants, antisera, or ascitic fluids. Antibody titers determined by this spot test are shown to correlate well with titers determined by two other methods, providing a simple method for initial estimation of the a m o u n t of antibody in positive samples.
INTRODUCTION
The hemolytic spot test (Hfibner and Gengozian, 1969) has provided an extremely useful assay for the presence of antibodies in culture supernatants and other fluids. It is limited, however, to use in systems in which responses to erythrocytes or substances easily conjugated to erythrocytes are being examined. Recently, a modified spot test employing trypan blue staining to detect areas of cytolysis has been described (Marshak-Rothstein et al., 1979), allowing detection of antibodies to non-erythroid cell surface determinants. The binding of trypan blue to a variety of proteins (Goding, 1980), however, can result in some lack of specificity in this system. Here we describe a spot test employing ethidium bromide, a compound previously used as a viability stain in other systems (Lee et al., 1975), and demonstrate its usefulness both in screening for the presence of antibodies and in estimating antibody titers. MATERIALS AND METHODS
Mice BALB/c mice were bred in our colony from breeders purchased from the Fred Hutchinson Cancer Research Center, Seattle, WA. AKR/J and AKR X 0022-1759/82/0000--0000/$02.75 © 1982 Elsevier Biomedical Press
76 DBA/2 (AKD2) F1 mice were purchased from Jackson Laboratories, Bar Harbor, ME. In vitro maintenance o f hybridomas and myelomas The IgM anti-Thy 1.2-producing hybridoma, HO-13-4, originally described by Marshak-Rothstein et al. (1979), was obtained from the Cell Distribution Center, the Salk Institute, San Diego, CA. Cells were grown in Dulbecco's modified Eagle's medium (GIBCO) containing 10% fetal calf serum (FCS, Microbiological Associates, Bethesda, MD). When cultures reached a density of 1--2 × 106 cells/ml, cells were removed by centrifugation; supernatants were stored at --20°C. The non-secreting BALB/c myelomas, NS-1 and SP20/Ag-14 (SP-2), were grown in RPMI 1640 medium (GIBCO) containing 15% FCS and were handled as described above. In vivo cell passage AKD2 F1 mice which had been injected with 0.5 ml pristane (Aldrich Chemicals, Milwaukee, WI) 1--8 weeks earlier were injected with 5 × 106 HO13-4 hybridoma cells. One to 2 weeks later, mice were bled and ascitic fluids were collected. TK-4, an IgG anti-TNP producing hybridoma (Foiles and Rittenberg, in preparation), was grown in BALB/c mice to produce ascitic fluid as described above. SP-2 ascitic fluid was similarly obtained from BALB/c mice which had been injected with this myeloma. Tube c y t o t o x i c assay 2 × 10 7 BALB/c thymocytes, suspended in 0.4 ml MEM, were mixed with 0.4 ml of the indicated dilution of HO-13-4 culture supernatant and 0.2 ml of rabbit complement (Low-tox, Accurate Scientific and Chemical Corp., HicksviUe, NY). This mixture was incubated at 37°C for 60 min. Cells were washed once and then resuspended in trypan blue (0.2%) or ethidium bromide (1/~g/ml) for viability determinations, using light and fluorescence microscopy, respectively. Effect o f anti-Thy 1.2 on in vitro PFC response Spleen cells from BALB/c mice which had been primed with TNP-KLH (Golding and Rittenberg, 1980) were treated with HO-13-4 culture supernatant or ascites fluid and complement as previously described (Cohn and Scott, 1979). Cells were then cultured in micro Mishell-Dutton cultures. Seven days later, cultures were harvested, pooling 4--8 wells for assay; 3 such pools were assayed per experimental point. TNP-specific plaque-forming cells (PFC) were assayed (Cunningham and Szenberg, 1968) using TNP-sheep erythrocytes (Rittenberg and Pratt, 1969). Goat anti-mouse IgG antiserum was used to facilitate detection of PFC producing IgG anti-TNP antibodies. IgM PFC were suppressed by inclusion of sheep anti-mouse p chain in the assay mixture (Pierce et al., 1971).
77 PROCEDURE • TriP"
2 % Thymocyles,
BOTTOM: 14% Agorose in PBS .5 F, somples --10-]5' 37° (uncovered)
Rot)bit complement (1:10) --60'
--,5'
L0
37 °
Ethidiurn bromide ( 2 p g / m l ) Room letup
aveen
U V source
Fig. 1. Spot test procedure.
Spot test Spot tests were carried o u t using a modification of the m e t h o d of Marshak-Rothstein et al. (1979) (Fig. 1). Five ml o f 1.4% agarose (L'Industrie Biologique Fran~aise) in PBS were poured into a 100 m m diameter plastic dish (Falcon No. 1029). After the first layer hardened, it was overlaid with 5 ml of 0.7% agarose in MEM containing 2% BALB/c t h y m o c y t e s . Samples (5/~1 each) were spotted onto the plate and allowed to soak in. Two ml of rabbit C, diluted 1/10 in PBS, were added and the plates were incubated at 37°C for 60--90 rain. After removal of C, plates were stained for 5 min with either 2 ml o f 0.2% trypan blue in PBS or 2 ml of 2 pg/ml ethidium bromide (Calbiochem) in PBS. Plates stained with trypan blue were rinsed with PBS until areas o f lysis could be detected; those stained with ethidium bromide were examined immediately over a low wavelength ultraviolet light source (Fig. 2). RESULTS
Comparison of ethidium bromide and trypan blue staining of spot plates Samples o f culture supernatants, ascites fluids and sera were assayed on layers o f agarose containing BALB/c t h y m o c y t e s ; the results of such an assay are shown in Table 1. Ethidium bromide staining of test plates detected spots produced by HO-13-4 anti Thy 1.2 which were strongly positive and uniformly c o m p l e m e n t dependent; similar results were seen whether the monoclonal a n t i b o d y was contained in culture supernatant, ascitic fluid, or serum. These anti-Thy 1.2 reagents produced no ethidium bromide stained
78
Fig. 2. An ethidium bromide stained spot test plate. Samples of ascitic fluid (upper row) or culture supernatant (lower row) from the anti-Thy 1.2 producing HO-13-4 hybridoma (left) or the non-secreting SP-2 myeloma (right) were assayed on a layer of BALB/c (Thy 1.2) thymocytes.
79 TABLE 1 Comparison of ethidium bromide and trypan blue staining of BALB/c thymocyte spot plates. Stain
Ethidium bromide
Trypan blue
RC
+a
-- a
+
+++
k
÷÷÷
Cult. sup. HO-13-4 (~-Thy 1.2) b SP-2 (--) NS-1 (--) Ascitic fluid HO-13-4 (~-Thy 1.2) SP-2 (--) TK-4 (~-TNP) Serum HO-13-4 (~-Tby 1.2) Normal BALB/c (--)
D
+++
++
--
D
÷÷÷
÷÷÷
m
÷÷÷
÷÷÷
m
÷÷÷
÷÷÷
q-÷
÷÷
-b-I-
÷÷
a Plates were incubated with rabbit complement as described (÷) or with MEM (--) before staining with the indicated dye. b Antibody specificity of each sample is indicated in parentheses.
spots, however, when assayed on an A K R ( Thy 1.1) t h y m o c y t e layer (data n o t shown). In contrast, under similar conditions c o m p l e m e n t d e p e n d e n c e o f spots d e t e c t e d by t r y p a n blue staining could only be shown with culture supernatant
Titration o f cytotoxic activity o f H 0 - 1 3 - 4 culture supernatant Serial dilutions o f a sample o f HO-13-4 culture supernatant were assayed for their ability to p r o d u c e cytolysis in a spot test; these same dilutions were also used in a tu b e assay t o d e t e r m i n e c y t o t o x i c titers. The data from such an e x p e r i m e n t are shown in Fig. 3. At high c o n c e n t r a t i o n s o f culture supernatant, greater than 95% c y t o t o x i c i t y occurred; these c o n c e n t r a t i o n s of culture s u p er n atan t also gave very intense spots in the spot test. At increasing
80 ÷+
++
+÷
I 30
~ 3i
J 32
++
+
+
+I-
I 34
I 35
I 36
I00
8O
60
40
20
J 3~,
I 37
L 3e
I 39
p 310
I/dilution Fig. 3. Titration of cytotoxm activity of HO-13-4 culture supernatant. BALB/c thymocytes were treated with the indicated dilutions of HO-13-4 culture supernatant and rabbit complement, and stained with ethidium bromide for viability counts. Treatment with complement alone resulted in 33% dead cells; a minimum of 300 cells was counted at each point. Results of a spot test conducted simultaneously are shown above.
dilutions o f supernatant (1 in 34 or 3s), c y t o t o x i c i t y decreased to 80--85%, and th e corresponding spots were somewhat less intense. As dilution of the supernatant c o n t i n u e d , c y t o t o x i c i t y declined dramatically, accompanied by negative results in t he spot test. Similar results were obtained when samples f r o m tu b e assays were stained with t r y p a n blue rather than ethidium b ro m id e (data n o t shown). Thus it appears t hat c y t o t o x i c titers obtained by t u b e d eter min atio n s o f cell viability correlate well with spot test titrations.
Correlation o f spot test results with functional assay o f H0-13-4 activity To examine the ef f ect o f HO-13-4 antibodies on helper T cell function, spleen cells f r o m TNP-KLH primed BALB/c mice were treated with dilutions o f 'HO-13-4 culture supernatant (Fig. 4) or ascitic fluid (Fig. 5) plus rabbit C prior to c u l t u r e with TNP-KLH. These cells were assayed after 7 days of culture f o r IgG anti-TNP PFC. As can be seen in Fig. 4, a 1/10 dilution of HO-13-4 culture supernatant eliminated m o r e than 90% of the secondary TNP-KLH response, while a 1/30 dilution had little effect on the response, correlating well with the results of a spot test c o n d u c t e d on the same culture supernatant d i l u t i o n s . A similar correlation was seen when HO-13-4 ascitic fluid was assayed (Fig. 5). A 1/25 dilution o f ascitic fluid eliminated greater than 99% o f th e anti-TNP response o f u n t r e a t e d or c o m p l e m e n t treated cells, a 1/125 dilution, which gave a positive bu t less intense spot, eliminated a p p r o x i m a t e l y 80% of the TNpoKLH response, and a 1/625 dilution, negative in the spot test, resulted in less than 40% decrease in the t h y m u s d e p e n d e n t PFC response. In these experiments, no significant effect of HO13-4 t r e a t m e n t was seen when treated cells were cultured with the t h y m u s i n d e p e n d e n t antigens TNP-Brucella abortus or DNP-dextran (146% and
81 25
I/dilution N spot test
C
I0
30
++
+/-
90
I/dilution spot test
N
C
oi 25 ++
125 .625 +
Fig. 4. E f f e c t o f HO-13-4 c u l t u r e s u p e r n a t a n t o n h e l p e r T cell f u n c t i o n . S p l e e n cells f r o m T N P - K L H p r i m e d mice were t r e a t e d w i t h t h e i n d i c a t e d d i l u t i o n s o f HO-13-4 c u l t u r e s u p e r n a t a n t a n d c o m p l e m e n t b e f o r e c u l t u r e w i t h T N P - K L H t o g e n e r a t e a n IgG a n t i - T N P P F C r e s p o n s e . C o n t r o l r e s p o n s e s o f cells t r e a t e d w i t h c o m p l e m e n t a l o n e (C) a n d o f u n t r e a t e d cells ( N ) are also s h o w n , as are results o f a s p o t t e s t c o n d u c t e d o n t h e d a y o f culture initiation. Fig. 5. E f f e c t o f HO-13-4 ascitic fluid o n h e l p e r T cell f u n c t i o n . Ascitic fluid d i l u t i o n s were used in a n assay o t h e r w i s e i d e n t i c a l t o t h a t o f Fig. 4.
144% of control respectively at 1/10 dilution of culture supernatant, 75% and 110% of control respectively at 1/25 dilution of ascitic fluid). DISCUSSION
In this paper, we have described a rapid m e t h o d for screening various fluids for the presence of antibodies specific for cell surface antigens. While the studies here have used t h y m o c y t e layers to assay Thy 1.2 specific antibodies, antibodies specific for any antigen widely distributed in a given tissue could be similarly assayed. The use of ethidium bromide as a viability stain in this spot test eliminates the lack of specificity frequently seen with sera and ascitic fluids when trypan blue staining is used; the necessity of rinsing plates extensively to remove excess d y e when trypan blue staining is used is also eliminated here. We have also shown that antibody titers as determined by spot tests correlated well with titers determined by t w o other methods. Antibodies of classes other than IgM can be assessed b y including an additional incubation step using an appropriate anti-immunoglobulin reagent prior to incubation with complement. The m e t h o d described here can also be used in conjunction with the replica plating system described b y Bankert e t a l . (1980) thereby providing a simple, rapid m e t h o d of screening hybridomas for production of antibodies to cell surface antigens and for examining their specificity by assaying supernatants on a variety of cell types.
82 ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y G r a n t A I 1 4 9 8 5 f r o m t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , M a r c h o f D i m e s Basic R e s e a r c h G r a n t 1-633, a n d A m e r i c a n C a n c e r S o c i e t y G r a n t I M - 1 7 2 . M.L.C. a n d H . G . are F e l l o w s o f t h e N . L . T a r t a r Foundation. REFERENCES
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