Journal of Immunological Methods, 29 (1979) 331--342 © Elsevier/North-Holland Biomedical Press
331
TRANSFER PLATE RADIOASSAY USING CELL MONOLAYERS TO DETECT ANTI-CELL SURFACE ANTIBODIES SYNTHESIZED BY LYMPHOCYTE HYBRIDOMAS
MICHAEL D. SCHNEIDER and GEORGE S. EISENBARTH
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Received 2 March 1979, accepted 23 April 1979)
A solid-phase [12sI]Protein A radioassay for anti-cell surface antibodies is described, which employs target cell monolayers cultured on fenestrated polyvinyl chloride 96-well plates ('transfer plates'). The calibrated aperture in the b o t t o m of each well is small enough to retain fluid contents by surface tension during monolayer growth, but also permits fluid to enter the wells when transfer plates are lowered into receptacles containing washing buffer or test sera. To assay for antibodies directed against target cell surface antigens, transfer plates bearing monolayers are inserted into microculture plates with corresponding 96-well geometry, thereby simultaneously sampling 96 wells. This assay allows rapid screening of hundreds of hybrid cell colonies for production of antibodies with desired tissue specificity.
INTRODUCTION
Assays employing either [ 12sI]staphylococcal Protein A ([I:sI]PA; Welsh et al., 1975) or [12SI]F(ab')~ anti-immunoglobulin (Goldstein et al., 1973; Williams, 1977) are widely used to investigate antibodies directed against the cell surface. These assays have recently been successfully applied to the screening of antibody production by somatic cell hybrids (lymphocyte hybridomas) produced by fusion between spleen cells from immunized mice and mouse myeloma cells (KShler and Milstein, 1975a, b; Galfre et al., 1977; Williams et al., 1977). To a considerable extent, the ability to identify tissuespecific antibodies using hybrid cell cultures requires a suitable technology for screening hundreds of culture supernatants both during the initial detection of colonies making the requisite antibodies and during the subsequent cloning of these colonies. In our studies of neural surface-membrane antigens defined by monospecific antibodies produced by hybrid cell lines (Eisenbarth et al., 1978) we have used complement-dependent cytotoxicity (SlCr release) and an [~2sI]PA radioassay to measure antibody binding to target cells in suspension. These assays require mechanical transfer of hundreds of hybridoma supernatants, and even with the use of multichannel pipettes harvesting of supernatants has proven to be a limiting factor. A solid-phase
332 radioassay was therefore developed which employs target cell monolayers cultured on 96-well polyvinyl chloride plates ('transfer plates') with a calibrated orifice at the bottom of each well, allowing the simultaneous sampling of 96 lymphocyte hybridoma colonies simply by insertion of transfer plates bearing monolayers into microculture plates with corresponding 8 by 12 well geometry. MATERIALS AND METHODS Cell lines
The P3-X63Ag8 mouse myeloma cell line (KShler and Milstein, 1975a, b) deficient in hypoxanthine phosphoribosyl transferase (pyrophosphate phosphoribosyl transferase, EC 2.4.2.8) was a gift of Dr. John Minna. Hybrid cell lines F12 A2B5 and F12 B2B2 (Eisenbarth et al., 1978) were produced by the method of Galfre et al. (1977) using polyethylene glycol-induced fusion of P3-X63Ag8 cells with dissociated spleen cells from mice immunized with 8-day chick embryo retina cells. P3-X63Ag8 cells synthesize IgG, (kappa), with 10 pg/ml of immunoglobulin in tissue culture supernatant, determined by radioimmunoassay by a modification of the method of Langone et al. (1977). Hybridomas F12 A2B5 and F12 B2B2 each synthesize a cytotoxic antibody to cell-surface tissue-specific antigens of chick retina. Large quantities of antibody were produced by growing these hybridomas as ascites tumors in BALB/c mice (Potter, 1972). Hybridoma F12 A2B5 ascites fluid contains an antibody concentration of 4 mg/ml. Hybridoma 1.2 ascites (immunoglobulin concentration, 2 mg/ml) was a gift of Dr. Steven Wilson; the antibody produced by this hybridoma does not react with chick retina and was used as a control antibody. Media
Eagle's minimal essential medium (MEM) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Grand Island Biological Co. (Gibco). Fetal calf serum (FC8) from North American Biologicals was sterilized by filtration. Dulbecco's phosphate-buffered saline (PB8), pH 7.4 (140 mM NaC1, 2.7 mM KC1, 10 mM Na2HPO4, 1.47 mM KH2PO4, 0.49 mM MgC12, 0.68 mM CaC12), was prepared by the National Institutes of Health Media Unit. P3-X63Ag8 cells were grown in DMEM + 10% FCS. Lymphocyte hybridomas were grown in media containing 10-4M hypoxanthine, 10-6M aminopterin, and 1.6 X 10-SM thymidine (HAT; Littlefield, 1964) formulated in either DMEM + 20% FC8, or in DMEM + 20% FCS supplemented with 2000 pU/ml crystalline bovine insulin (Sigma Chemical Co.), 0.5 mM sodium pyruvate (Gibco), 1 mM oxaloacetic acid (Calbiochem), 1% nonessential amino acids (Gibco), 2 mM glutamine (Gibco), and 10% NCTC 109 lymphocyte growth serum (Microbiological Associates) (Kennett et al., 1978).
333
Preparation of antisera Rabbit anti-chick retina serum (2.5F) was collected from a New Zealand White rabbit immunized with subcutaneous and intraperitoneal injections of 10 s 8 - d a y embryonic chick retina cells, dissociated with 0.02% ethylene bisoxyethylene nitrilotetraacetic acid (Eastman) for 40 min.
Preparation of retina cell monolayers Polyvinyl chloride 96-well V-bottom microtiter plates with 270 pl capacity (catalog number 1-200-25, Dynatech Corporation) were fenestrated by a single passage through the bottom of each well with a 25-gauge, 5/8 in., hypodermic needle (Sherwood Medical Industries), and the supporting plastic rim of each plate was removed with scissors. These plates will subsequently be referred to as 'transfer plates'. The aperture of each well is small enough to retain the fluid contents of the well by surface tension (e.g., during monolayer growth), but also permits fluid to enter when transfer plates are lowered into receptacles containing washing buffer or test sera. Transfer plates were sterilized by immersion in 70% ethanol for 1 min and thoroughly air-dried. Monolayers were also prepared on commercially available tissue culture-treated polyvinyl chloride transfer plates (catalog number 1-22043-1, Dynatech). To prepare target cell monolayers, the neural retinae of four 8-day white Leghorn chick embryos (Truslow Farms) were dissociated in 5 ml of 0.05% trypsin (crystallized X 3; Worthington) in calcium-, magnesium-free PBS for 8 min at 37°C, followed by addition of MEM + 10% FCS (5 ml). The cell suspension was then tritiated X 10 with a 10 ml sterile disposable plastic pipette (catalog number 7551; Falcon Plastics), centrifuged at 1000 Xg (Sorvall GLC-2) for 5 min at room temperature, and the supernatant discarded. The cells were resuspended in 4 ml of MEM + 10% FCS. Approximately 40 X 106 cells/retina were obtained, and greater than 95% were single cells that excluded trypan blue. Suspensions of trypsin-dissociated embryonic chick retina cells were diluted in MEM + 10% FCS (7.9 X 104--3.78 X 107 cells/ml), and 150 pl of cell suspension were distributed to each well of transfer plates by means of an 8- or 12-channel Titertek micropipette (Flow Laboratories) with autoclavable disposable tips. When cell number was not an experimental variable, the retina cell suspensions were added with a sterile 10 ml disposable pipette, inoculating each well with 2 drops of the cell suspension. Plated retina cells were incubated for 6--48 h at 37°C in 5% CO2/ 95% air saturated with H20. During monolayer growth, to ensure sterility transfer plates were suspended by empty 96-well flat-bottomed polystryrene microculture plates and covered with microculture plate lids (catalog numbers 3040, 3041; Falcon Plastics). In addition, commercial transfer plates were elevated by 5 mm X 42 mm strips of filter paper (AP25 042 00; Whatman) along each edge of the microculture plate to prevent contact between the bottom of the microculture plate well and the transfer plate aperture.
334
Dissociated neural retina cells (see above method) were also employed in suspension assays immediately after preparation.
[12si]PA binding assays (1) Cell suspension assay. Retina cells (1 X 106) in 50 pl of MEM + 10% FCS were delivered to each well of a 96-well V-bottom microtiter plate containing 50 #l of hybridoma tissue culture supernatant or 50 pl of diluted ascites from mice with l y m p h o c y t e h y b r i d o m a ascites tumors. Incubation was performed for 30 min at r o o m temperature, followed by centrifugation
E
m B
F
D
H
Fig. 1. Schematic representation of transfer plate radioassay to detect anti-retina antibody using cultured monolayers of embryonic chick retina. A: suspensions of trypsindissociated 8-day embryonic chick retina cells (o) were distributed in 150 pl of MEM + 10% FCS to each well of a transfer plate. B: cultures were incubated at 37°C in 5% CO2/ 95% air with 100% humidity for 6--48 h. C: medium was discarded by sterilely decanting the transfer plate with brisk inversion. D,E: transfer plate bearing retina cell monolayers was inserted into the corresponding wells of an 8 by 12 well microculture plate containing medium conditioned by lymphocyte hybridoma colonies (o, m) (ca. 225 pl/well) or other test sera, permitting inflow of reagent through the aperture in each well of the transfer plate. F: antibody incubation at room temperature (10--180 rain)was followed by 3 washing cycles with PBS-gel. G,H: the presence of cell-bound antibody was quantitated by binding of [12sI]PA ([-*7), incubated for 10---180 min at room temperature and washed 3 times by immersion in PBS-gel to remove unbound radioactivity. Individual wells were then cut from the transfer plate and radioactivity bound was determined with a gamma counter (efficiency = 70%).
335 at 1000 Xg (GLC-2) for 5 min at room temperature. Supernatants were discarded by decanting the plate briskly by inversion onto utility wipes. The pellets were then washed twice by means of a 12-channel micropipette, with 150 pl of PBS containing 0.1% gelatin (PBS-gel; ICN Pharmaceuticals). A 50 pl aliquot of [12sI]PA (0.04 pCi, 30 mCi/mg; Amersham-Searle) in PBS-gel was added to each well and incubated for 45 min at room temperature followed by 3 washing cycles as above. The microtiter plate was cut into individual wells and the bound [12sI]PA determined with a Packard PGD automatic gamma counter (efficiency, 70%). (2) Monolayer assay. (Fig. 1). Transfer plates bearing retina cell monolayers were sterilely decanted by brisk inversion to remove growth medium, and were then inserted into the corresponding wells of flat-bottomed microculture plates (approximately 225 pl/well of antibody-containing medium). Incubation of the monolayers with antibody at room temperature (10--180 min) was terminated by withdrawing the transfer plate from the microculture plate, and 3 washing cycles were performed, each comprising 60-sec immersion in PBS-gel then decanting the plate onto utility wipes. Fifty pl of [12sI]PA (0.04 pCi) in PBS-gel were added to each well and incubated (10-180 min) at room temperature followed by 3 washing cycles as described. Standard assay conditions (subsequent to initial studies) employed retina cell monolayers plated at 1.9 × 106 cells/well, antibody incubation for 30 min, and [~2sI]PA incubation for 45 min. The transfer plates were cut into individual wells with scissors, and [~25I]PA binding was determined as described above.
Complemen t-dependen t cy totoxicity assays Trypsin
331
TRANSFER PLATE RADIOASSAY USING CELL MONOLAYERS TO DETECT ANTI-CELL SURFACE ANTIBODIES SYNTHESIZED BY LYMPHOCYTE HYBRIDOMAS
MICHAEL D. SCHNEIDER and GEORGE S. EISENBARTH
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Received 2 March 1979, accepted 23 April 1979)
A solid-phase [12sI]Protein A radioassay for anti-cell surface antibodies is described, which employs target cell monolayers cultured on fenestrated polyvinyl chloride 96-well plates ('transfer plates'). The calibrated aperture in the b o t t o m of each well is small enough to retain fluid contents by surface tension during monolayer growth, but also permits fluid to enter the wells when transfer plates are lowered into receptacles containing washing buffer or test sera. To assay for antibodies directed against target cell surface antigens, transfer plates bearing monolayers are inserted into microculture plates with corresponding 96-well geometry, thereby simultaneously sampling 96 wells. This assay allows rapid screening of hundreds of hybrid cell colonies for production of antibodies with desired tissue specificity.
INTRODUCTION
Assays employing either [ 12sI]staphylococcal Protein A ([I:sI]PA; Welsh et al., 1975) or [12SI]F(ab')~ anti-immunoglobulin (Goldstein et al., 1973; Williams, 1977) are widely used to investigate antibodies directed against the cell surface. These assays have recently been successfully applied to the screening of antibody production by somatic cell hybrids (lymphocyte hybridomas) produced by fusion between spleen cells from immunized mice and mouse myeloma cells (KShler and Milstein, 1975a, b; Galfre et al., 1977; Williams et al., 1977). To a considerable extent, the ability to identify tissuespecific antibodies using hybrid cell cultures requires a suitable technology for screening hundreds of culture supernatants both during the initial detection of colonies making the requisite antibodies and during the subsequent cloning of these colonies. In our studies of neural surface-membrane antigens defined by monospecific antibodies produced by hybrid cell lines (Eisenbarth et al., 1978) we have used complement-dependent cytotoxicity (SlCr release) and an [~2sI]PA radioassay to measure antibody binding to target cells in suspension. These assays require mechanical transfer of hundreds of hybridoma supernatants, and even with the use of multichannel pipettes harvesting of supernatants has proven to be a limiting factor. A solid-phase
332 radioassay was therefore developed which employs target cell monolayers cultured on 96-well polyvinyl chloride plates ('transfer plates') with a calibrated orifice at the bottom of each well, allowing the simultaneous sampling of 96 lymphocyte hybridoma colonies simply by insertion of transfer plates bearing monolayers into microculture plates with corresponding 8 by 12 well geometry. MATERIALS AND METHODS Cell lines
The P3-X63Ag8 mouse myeloma cell line (KShler and Milstein, 1975a, b) deficient in hypoxanthine phosphoribosyl transferase (pyrophosphate phosphoribosyl transferase, EC 2.4.2.8) was a gift of Dr. John Minna. Hybrid cell lines F12 A2B5 and F12 B2B2 (Eisenbarth et al., 1978) were produced by the method of Galfre et al. (1977) using polyethylene glycol-induced fusion of P3-X63Ag8 cells with dissociated spleen cells from mice immunized with 8-day chick embryo retina cells. P3-X63Ag8 cells synthesize IgG, (kappa), with 10 pg/ml of immunoglobulin in tissue culture supernatant, determined by radioimmunoassay by a modification of the method of Langone et al. (1977). Hybridomas F12 A2B5 and F12 B2B2 each synthesize a cytotoxic antibody to cell-surface tissue-specific antigens of chick retina. Large quantities of antibody were produced by growing these hybridomas as ascites tumors in BALB/c mice (Potter, 1972). Hybridoma F12 A2B5 ascites fluid contains an antibody concentration of 4 mg/ml. Hybridoma 1.2 ascites (immunoglobulin concentration, 2 mg/ml) was a gift of Dr. Steven Wilson; the antibody produced by this hybridoma does not react with chick retina and was used as a control antibody. Media
Eagle's minimal essential medium (MEM) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Grand Island Biological Co. (Gibco). Fetal calf serum (FC8) from North American Biologicals was sterilized by filtration. Dulbecco's phosphate-buffered saline (PB8), pH 7.4 (140 mM NaC1, 2.7 mM KC1, 10 mM Na2HPO4, 1.47 mM KH2PO4, 0.49 mM MgC12, 0.68 mM CaC12), was prepared by the National Institutes of Health Media Unit. P3-X63Ag8 cells were grown in DMEM + 10% FCS. Lymphocyte hybridomas were grown in media containing 10-4M hypoxanthine, 10-6M aminopterin, and 1.6 X 10-SM thymidine (HAT; Littlefield, 1964) formulated in either DMEM + 20% FC8, or in DMEM + 20% FCS supplemented with 2000 pU/ml crystalline bovine insulin (Sigma Chemical Co.), 0.5 mM sodium pyruvate (Gibco), 1 mM oxaloacetic acid (Calbiochem), 1% nonessential amino acids (Gibco), 2 mM glutamine (Gibco), and 10% NCTC 109 lymphocyte growth serum (Microbiological Associates) (Kennett et al., 1978).
333
Preparation of antisera Rabbit anti-chick retina serum (2.5F) was collected from a New Zealand White rabbit immunized with subcutaneous and intraperitoneal injections of 10 s 8 - d a y embryonic chick retina cells, dissociated with 0.02% ethylene bisoxyethylene nitrilotetraacetic acid (Eastman) for 40 min.
Preparation of retina cell monolayers Polyvinyl chloride 96-well V-bottom microtiter plates with 270 pl capacity (catalog number 1-200-25, Dynatech Corporation) were fenestrated by a single passage through the bottom of each well with a 25-gauge, 5/8 in., hypodermic needle (Sherwood Medical Industries), and the supporting plastic rim of each plate was removed with scissors. These plates will subsequently be referred to as 'transfer plates'. The aperture of each well is small enough to retain the fluid contents of the well by surface tension (e.g., during monolayer growth), but also permits fluid to enter when transfer plates are lowered into receptacles containing washing buffer or test sera. Transfer plates were sterilized by immersion in 70% ethanol for 1 min and thoroughly air-dried. Monolayers were also prepared on commercially available tissue culture-treated polyvinyl chloride transfer plates (catalog number 1-22043-1, Dynatech). To prepare target cell monolayers, the neural retinae of four 8-day white Leghorn chick embryos (Truslow Farms) were dissociated in 5 ml of 0.05% trypsin (crystallized X 3; Worthington) in calcium-, magnesium-free PBS for 8 min at 37°C, followed by addition of MEM + 10% FCS (5 ml). The cell suspension was then tritiated X 10 with a 10 ml sterile disposable plastic pipette (catalog number 7551; Falcon Plastics), centrifuged at 1000 Xg (Sorvall GLC-2) for 5 min at room temperature, and the supernatant discarded. The cells were resuspended in 4 ml of MEM + 10% FCS. Approximately 40 X 106 cells/retina were obtained, and greater than 95% were single cells that excluded trypan blue. Suspensions of trypsin-dissociated embryonic chick retina cells were diluted in MEM + 10% FCS (7.9 X 104--3.78 X 107 cells/ml), and 150 pl of cell suspension were distributed to each well of transfer plates by means of an 8- or 12-channel Titertek micropipette (Flow Laboratories) with autoclavable disposable tips. When cell number was not an experimental variable, the retina cell suspensions were added with a sterile 10 ml disposable pipette, inoculating each well with 2 drops of the cell suspension. Plated retina cells were incubated for 6--48 h at 37°C in 5% CO2/ 95% air saturated with H20. During monolayer growth, to ensure sterility transfer plates were suspended by empty 96-well flat-bottomed polystryrene microculture plates and covered with microculture plate lids (catalog numbers 3040, 3041; Falcon Plastics). In addition, commercial transfer plates were elevated by 5 mm X 42 mm strips of filter paper (AP25 042 00; Whatman) along each edge of the microculture plate to prevent contact between the bottom of the microculture plate well and the transfer plate aperture.
334
Dissociated neural retina cells (see above method) were also employed in suspension assays immediately after preparation.
[12si]PA binding assays (1) Cell suspension assay. Retina cells (1 X 106) in 50 pl of MEM + 10% FCS were delivered to each well of a 96-well V-bottom microtiter plate containing 50 #l of hybridoma tissue culture supernatant or 50 pl of diluted ascites from mice with l y m p h o c y t e h y b r i d o m a ascites tumors. Incubation was performed for 30 min at r o o m temperature, followed by centrifugation
E
m B
F
D
H
Fig. 1. Schematic representation of transfer plate radioassay to detect anti-retina antibody using cultured monolayers of embryonic chick retina. A: suspensions of trypsindissociated 8-day embryonic chick retina cells (o) were distributed in 150 pl of MEM + 10% FCS to each well of a transfer plate. B: cultures were incubated at 37°C in 5% CO2/ 95% air with 100% humidity for 6--48 h. C: medium was discarded by sterilely decanting the transfer plate with brisk inversion. D,E: transfer plate bearing retina cell monolayers was inserted into the corresponding wells of an 8 by 12 well microculture plate containing medium conditioned by lymphocyte hybridoma colonies (o, m) (ca. 225 pl/well) or other test sera, permitting inflow of reagent through the aperture in each well of the transfer plate. F: antibody incubation at room temperature (10--180 rain)was followed by 3 washing cycles with PBS-gel. G,H: the presence of cell-bound antibody was quantitated by binding of [12sI]PA ([-*7), incubated for 10---180 min at room temperature and washed 3 times by immersion in PBS-gel to remove unbound radioactivity. Individual wells were then cut from the transfer plate and radioactivity bound was determined with a gamma counter (efficiency = 70%).
335 at 1000 Xg (GLC-2) for 5 min at room temperature. Supernatants were discarded by decanting the plate briskly by inversion onto utility wipes. The pellets were then washed twice by means of a 12-channel micropipette, with 150 pl of PBS containing 0.1% gelatin (PBS-gel; ICN Pharmaceuticals). A 50 pl aliquot of [12sI]PA (0.04 pCi, 30 mCi/mg; Amersham-Searle) in PBS-gel was added to each well and incubated for 45 min at room temperature followed by 3 washing cycles as above. The microtiter plate was cut into individual wells and the bound [12sI]PA determined with a Packard PGD automatic gamma counter (efficiency, 70%). (2) Monolayer assay. (Fig. 1). Transfer plates bearing retina cell monolayers were sterilely decanted by brisk inversion to remove growth medium, and were then inserted into the corresponding wells of flat-bottomed microculture plates (approximately 225 pl/well of antibody-containing medium). Incubation of the monolayers with antibody at room temperature (10--180 min) was terminated by withdrawing the transfer plate from the microculture plate, and 3 washing cycles were performed, each comprising 60-sec immersion in PBS-gel then decanting the plate onto utility wipes. Fifty pl of [12sI]PA (0.04 pCi) in PBS-gel were added to each well and incubated (10-180 min) at room temperature followed by 3 washing cycles as described. Standard assay conditions (subsequent to initial studies) employed retina cell monolayers plated at 1.9 × 106 cells/well, antibody incubation for 30 min, and [~2sI]PA incubation for 45 min. The transfer plates were cut into individual wells with scissors, and [~25I]PA binding was determined as described above.
Complemen t-dependen t cy totoxicity assays Trypsin
