A microtiter plate assay for detecting IgE-binding molecules and cells bearing Fc receptors for IgE

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Journal oflmmunologicalMethods, 84(1985) 11-24 Elsevier

JIM03666

A Microtiter Plate Assay for Detecting IgE-Binding Molecules and Cells Bearing Fc Receptors for IgE Susan A. H u d a k a n d M a r i l y n R. K e h r y DNA X Research Institute for Molecular and Cellular Biology, Inc., 90l California Avenue, Palo Alto, CA 94304, U.S.A.

(Received 1 April 1985, accepted 2 July 1985)

A microtiter plate assay is described for detecting cells bearing Fc receptors for IgE (Fc~ receptors) and for assaying IgE-binding molecules. Cells are bound specifically to IgE-coated wells of microtiter plates, and the bound cells are enumerated in a quantitative colorimetric assay. IgE-binding molecules and monoclonal antibodies are assayed as inhibitors of the IgE-dependent cell binding. Major advantages of the plate assay compared to rosetting assays are its ability to accommodate many test samples for replicates and titrations and the ease with which results are read out. The versatility of the assay is discussed with respect to detecting other immunoglobulin Fc receptor-bearing cells. Key words: IgE receptor assay - Fc receptor assay antibodies

IgE-binding factors - Fc receptors

monoclonal

Introduction

The interaction of cell surface receptors for the Fc portion of IgE (Fg receptor) with IgE plus antigen initiates several important regulatory processes in the immune system. One class of Fc~ receptors has a high affinity for IgE, is found on the surface of basophils/mast cells and has been extensively characterized biochemically (Metzger et al., 1982). Cells bearing these Fg receptors play a critical role in immediate hypersensitivity reactions (Ishizaka and Ishizaka, 1978). Low-affinity Fg receptors ( - 100-fold lower affinity than those on basophils/mast cells) have been demonstrated on subpopulations of T cells, B cells and monocytes (Spiegelberg, 1981). Eosinophils mediate an IgE-dependent cytotoxic response against parasites via cell surface Fg receptors (Capron et al., 1981) and T cells bearing Fg receptors Abbreviations: PBS, phosphate-buffered saline that contains 0.132 M NaCl, 0.01 M Na2HPO 4, 0.003 M KHzPO 4, pH 7.2; FGG, chicken gamma globulin; KLH, keyhole limpet hemocyanin; FCS, fetal bovine serum; TNP, trinitrophenyl; HBSS, Hanks' balanced salt solution; Hepes, N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Fc~ receptor, Fc receptor for IgE.

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© 1985 Elsevier Science Publishers B.V. (Biomedical Division)

12 are thought to be involved in regulation of IgE production (Yodoi et al., 1980; Spiegelberg, 1981). The number of Fc~ receptor-positive lymphoid cells is usually determined by enumerating the cells that form rosettes with IgE-coated erythrocytes (Spiegelberg, 1981). Soluble IgE-binding molecules produced by Fc~ receptor-positive T cells have been reported to be mediators for regulating IgE production by B cells (Hirashima et al., 1980; Yodoi et al., 1980, 1981; Suemura et al., 1981; Marcelletti and Katz, 1984). The majority of the regulatory IgE-binding molecules are produced in very small quantities and exhibit a low affinity for IgE. IgE-binding ability is typically assayed as an inhibition of the formation of IgE-specific rosettes with lymphocytes bearing low-affinity Fc, receptors (Yodoi and Ishizaka, 1980; Spiegelberg, 1981). Although the rosette inhibition assay is less time-consuming than biological assays for B cell regulation, it is laborious, has a subjective readout, and accommodates large numbers of samples only with difficulty. We have developed an alternative to the rosetting assay that accommodates large numbers of samples and is versatile in its applicability to detecting cells bearing various kinds of receptors. We demonstrate that both lymphocytes and basophils/ mast cells bind to IgE-coated wells of a microtiter plate in an IgE-specific fashion. Bound cells are enumerated colorimetrically. IgE-binding molecules (including monoclonal antibodies) inhibit the interaction of cells with the IgE-coated plate and can be titrated in the assay.

Materials and Methods

Animals BALB/cByJ mice (8 to 10 weeks old) were purchased from the Jackson Laboratory, Bar Harbour, ME. Rats of the L O U / M N strain were obtained from Stanford University and maintained as a breeding colony at DNAX.

Immunoglobulins and protein coupling Monoclonal rat IgE was purified from the ascites of L O U / M N rats injected with the IR162 immunocytoma (Bazin et al., 1974) as described (Isersky et al., 1974; Vander-Mallie et al., 1982). Monoclonal mouse IgE specific for the trinitrophenyl (TNP) hapten was purified from the ascites of B A L B / c mice injected with the IGEL a2 hybridoma (Rudolph et al., 1981). Mouse IgE was isolated by the same method as used for the isolation of rat IgE. Purified mouse IgG1 (35-20) specific for digitalis (Hunter et al., 1982) was the kind gift of Dr. J.S. Abrams. Rat monoclonal antibodies specific for mouse IgE were the generous gift of Dr. Z. Eshhar. The antibodies (EM-126.6, IgG2b; EM-95.3, IgG2a; and EM-51.3, IgG1) were used as culture supernatants (Baniyash and Eshhar, 1984). Purified IgE and bovine albumin (fatty acid-free, Sigma Chemical Co., St. Louis, MO) were covalently coupled to Affi-Gel 10 (Bio-Rad Laboratories, Richmond, CA) according to the manufacturer's instructions.

13 Chicken gamma globulin (FGG, Cappel Laboratories, Cochranville, PA) was derivatized with T N P groups as follows. A stock solution of 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma) was prepared in 0.1 M Na2CO 3 and added at various concentrations (10 ~g T N B S / m g F G G for coupling - 2 5 moles TNP per mole F G G ) to F G G (10 m g / m l in 0.1 M Na2CO3). The reaction mixture was stirred at room temperature for 90 min. Reagents were removed by dialysis and extent of coupling was calculated as described (Kiefer, 1979).

Cell culture The immature mouse mast cell line, C1.MC/9, was grown as described (Nabel et al., 1981a) in Dulbecco's modified Eagle's medium supplemented with 5% conditioned medium from a mouse T cell clone, D9.1 (Nabel et al., 1981b). A rat-mouse T cell hybridoma (23B6) that produces IgE-binding molecules in response to induction with rat IgE was grown as described (Huff et al., 1982).

Spleen cell preparation B A L B / c spleens were excised aseptically and a single cell suspension was made in RPMI-1640 supplemented with 10% fetal bovine serum (FCS). The cells were sequentially applied to 2 columns of Sephadex G-10 (Sigma) in phosphate-buffered saline (PBS, 0.132 M NaC1, 0.01 M N a 2 H P O 4, 0.003 M KH2PO 4, pH 7.2) supplemented with 5% FCS (Ly and Mishell, 1974). After elution from the second Sephadex G-10 column cells were suspended at a concentration of 8 × 106 cells/ml (see below).

Preparation of IgE-binding factors Induced culture supernatant from the T cell hybridoma, 23B6, was prepared and filtered exactly as described (Huff et al., 1982). Cloned IgE-binding factor cDNA from 23B6 cells was expressed transiently in COS7 cells (denoted 8.3-COS7) or stably in CHO dhfr- cells (denoted 8.3-CHO.13) and was kindly provided by Drs. C.L. Martens and K.W. Moore (Martens et al., 1985). Preparations of the expressed 8.3 clone from culture supernatants were sterile filtered and used without further purification. Mock culture supernatants (23B6 cells not incubated with IgE and COS7 cells transfected with an irrelevant cDNA clone) were used as controls. All solutions were prepared and aliquoted in polypropylene tubes, stored at - 80°C and thawed just before use.

Flow cytometry Flow cytometry was performed on a FACS IV (Becton Dickinson and Co., Sunnyvale, CA). Analysis was performed at a gain of 0.5 or 1.0. Activated fluorescent microspheres (MX green particles, 0.7/~m) were purchased from Covalent Technology, Ann Arbor, MI, and coupled to purified rat IgE or FCS according to the manufacturer's recommendations. IgE-coated beads were bound to cells according to the procedure of Sarfati et al. (1984) and 10,000 cells were analyzed. Inhibitors (IgE-binding molecules) were assayed using beads coupled to IgE at concentrations of 15 /zg/ml or 20 /~g/ml. Under these conditions - 1 0 % of

14 Sephadex G-10 depleted B A L B / c spleen cells were fluorescent (greater than 2 beads per cell) in the absence of inhibitors. A number representing the total number of cell-bound beads was obtained as the integrated fluorescence. A maximum of 30-35% of spleen cells was fluorescent using beads coupled to IgE at concentrations greater than 50/~g/ml.

Plate assay The assay was designed to produce, on the microtiter wells, 'microaggregates' of IgE that resembled IgE fixed on red blood cells. Mouse IgE specific for T N P was bound to wells coated with TNP-derivatized protein. Initially, chicken gamma globulin (FGG), chicken egg ovalbumin and keyhole limpet hemocyanin (KLH) were TNP-derivatized and tested in the assay for the ability to support IgE-specific binding of B A L B / c spleen cells to the microtiter wells. Ovalbumin was too small; few cells were bound to TNP-ovalbumin and a2-coated wells. K L H was too heterogeneous; the well-to-well variability in percent binding was high and inhibition of binding could not be obtained reproducibly. T N P - F G G supported an optimum IgE-specific binding of cells to the microtiter wells, was of intermediate size and did not bind to Fc receptors on rodent or human cells. It was derivatized to varying extents and used in subsequent experiments. Sodium azide was removed from all protein solutions used in the assay by dialysis in PBS. Solutions were dispensed to plates with an 8-channel Titertek Pipette (Flow Laboratories, McLean, VA). Pipette tips from Costar (Costar 4860, Cambridge, MA) were used throughout because they were minimally distorted by autoclaving. Coating plates with IgE and inhibitors. Falcon 3072 96-well tissue culture plates (Micro Test III, Becton Dickinson and Co.) were coated with TNPz~-FGG at a concentration of either 2 /xg/ml or 5 /~g/ml in PBS (50 /~l/well) for 2 h at 37°C. The stock solution of TNP26-FGG was diluted immediately before use. Each assay plate was routinely configured with one end column remaining uncoated as a control and the top row remaining completely blank for measuring the total number of input cells (see below). All sites on the plastic were blocked with undiluted FCS (50 t~l/well, except to the top row) for 30 min at 37°C. Wells were washed 3 times with PBS containing 5% FCS using a Nunc-Immuno Wash 12 (Vangard International, Neptune, N J). Wells were filled to the top for a wash and aspirated as completely as possible. In initial experiments wells were washed 3 times with an 8-channel pipette (100 /xl/well). Solutions were always removed from and added to the control column first. Monoclonal mouse IgE (IGEL a2) was placed in the wells (50 t~l/well) at a concentration of either 0.1 t~g/ml or 0,3/~g/ml (see Results) in PBS containing 5% FCS for 2 h at room temperature. The blank column received PBS, 5% FCS only. Plates were washed one plate at a time, as above, with PBS containing 5% FCS. When no inhibitors were employed, Hanks' balanced salt solution (HBSS, Gibco, Grand Island, NY) containing 20 mM Hepes (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid), pH 7.2 and 10% FCS was added (50 ~l/well) and the plates were placed at 4°C. When inhibitors of cell binding to IgE were to be assayed they were diluted on ice in HBSS containing 20 mM Hepes, pH 7.2 and 10% FCS in

15 microtiter wells that had been previously coated with FCS. Rat IgE was used as a standard inhibitor at concentrations between 100 /~g/ml and 6.4 ng/ml. Mock supernatants or no inhibitors (HBSS, 20 mM Hepes, pH 7.2, 10% FCS) were placed in 3 columns on each plate as a control for maximum cell binding. Inhibitors were added to washed IgE-coated plates on ice in triplicate (50 ~l/well) and incubated 2 h at 4°C. Addition of cells and quantitation of bound cells. Cells to be bound to the IgE on the plates were suspended at 8 x 10 6 cells/ml ( B A E B / c spleen, prepared as above) or 4-5 x l0 s cells/ml ( M C / 9 cells or other cultured cells, washed once) in cold HBSS containing 20 mM Hepes, pH 7.2 and 10% FCS. Cells were added to each well (100 /~l/well except for top row) while the plate was kept on ice. These cell numbers were optimum for creating an even 'monolayer' of bound cells that produced a good signal in the colorimetric assay (see below) and provided maximal sensitivity of the assay to various IgE-binding molecules. To measure the input number of cells, the same concentration of cells was prepared in RPMI-1640 supplemented with 20 mM Hepes, pH 7.2 and 10% FCS and added to 5 wells in the top row (100/~l/well). After addition of cells, plates were shaken gently (setting 3, out of 10) on a plate shaker (Micro-Shaker II, Dynatech Laboratories, Alexandria, VA) for 10 s, incubated at 37°C for 10 min, and gently shaken (setting 3) for 10 s. Wells were inspected under the microscope to ensure that cells were evenly suspended and no clumps were present in wells. Plates were incubated overnight at 4°C on a level surface in a humid chamber. One to three hours were sufficient for this step; however, overnight incubation was used for convenience. Cells not bound to the plates were removed with an 8-channel pipette as follows. A plate was vortexed on the plate shaker for 10 s, starting at setting 3 and rapidly progressing to setting 6 within the 10 s (reasonably vigorous vortexing). Immediately, the plate was tilted 1 cm on one end and 100 #1 was removed from each well except the top row (input cells). The pipette was angled to remove liquid from the lower two-thirds of the well without disturbing cells attached to the bottom of the well. A wash consisting of ice-cold HBSS containing 20 mM Hepes, pH 7.2 and 10% FCS was gently added to each well (100 /~l/well). The plate vortexing was repeated, and the full volume of liquid (150 t~l) was immediately removed from each well with the 8-channel pipette. A colorimetric assay was used to measure numbers of bound cells (Mosmann, 1983). A stock solution of 5 m g / m l MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) in PBS was added directly to the maximum wells in the top row of the plate (10 ~l/well). For the remaining wells the stock solution of MTT was diluted to 0.5 m g / m l in RPMI-1640 supplemented with 10% FCS, and 110 t~l was added to each well, including several blank wells. At this point or immediately after vortexing for a wash, plates could be briefly examined under the microscope. (Plates were then revortexed for removal of the wash medium.) Bound cells were observed as single cells; clumps were not seen, even when cultured cells that grow clumped together (RPMI-8866) were employed. Plates were incubated at 37°C for 4 h and developed by addition of 115 /~l/well acid-isopropanol as described (Mosmann, 1983). Plates were read on a Dynatech MR580 microELISA reader.

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Averages of triplicate wells, percentage inhibition and percentage binding of cells to the plates were calculated. We defined the units of IgE-binding molecules present in a sample as the reciprocal of the dilution of inhibitor added to the assay wells in 50 /~1 that produces half maximal inhibition of cell binding. This definition was adopted because various IgE-binding molecules (e.g. monoclonal antibodies versus IgE-binding factors from T cells) inhibited the IgE-cell interaction to different extents (see Discussion). Multiplying this number by 20 gives the units/ml of IgE-binding activity. Results

Establishment of assay conditions A wide range of IgE and TNP-FGG concentrations were found to give IgE-specific binding of cells with low-affinity Fc~ receptors to an IgE-coated microtiter plate. The design of the assay was intended to imitate as closely as possible the formation of IgE-specific rosettes by B A L B / c spleen cells. In rosetting assays, IgE fixed on red blood cells is thought to exist in an aggregated form that is believed to bind more efficiently to low-affinity lymphocyte Fc~ receptors (Spiegelberg, 1981). Therefore, the IgE we used was a TNP-specific mouse monoclonal IgE (a2) that was added to microtiter wells coated with TNP-derivatized protein to presumably produce IgE ' microaggregates'. Normal BALB/c spleen cells were used as a source of cells bearing low-affinity receptors for IgE. Adherent cells were depleted (Ly and Mishell, 1974) before adding the cell population to the IgE-coated wells. Three levels of TNP derivatization of FGG, TNPI4-FGG, TNP24 26-FGG, and TNP44-FGG were used to coat wells. The FGGs were titrated beginning at a concentration of 80/~g/ml while maintaining the IgE concentration at 0.16/~g/ml. Binding of cells to the plate was best for the TNP24_ 26-FGG at concentrations below 5 g g / m l (Table I). Additional titrations of the TNPz4_z6-FGG were made to 0.25 /~g/ml (data not shown) to determine the optimum concentrations for detecting Fc~ receptor-bearing cells (maximum bindable number of cells) and IgE-binding molecules (greatest inhibition, see below). TABLE I EFFECT OF D E G R E E OF TNP DERIVATIZATION ON SPLEEN CELL B I N D I N G TO IgECOATED WELLS Results are % binding relative to total number of cells added per well. The IgE concentration was constant at 0.16 g g / m l F G G derivatization

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Fig. 1. Binding of BALB/c spleen cells to IgE-coated wells and inhibition by IgE. Spleen cells binding to microtiter wells coated with mouse lgE were measured (indicated by the color reaction). Total input cells read 0.482 OD57o_630units (37% binding). Inhibitors were added at the indicated concentrations; O, no inhibitors; e, rat IR162 IgE; A, mouse IgG1; II, background binding to uncoated wells (average and range of 7 wells). Each point is the average of 2 replicates (rat lgE) or 3 replicates (lgG1 and no inhibitors). Left panel: data are graphed as number of input cells bound. Right panel: same data as in the left-hand panel, graphed as % inhibition of binding relative to the control wells on the same plate (no inhibitors -- defined as 0% inhibition). Virtually no cells b o u n d to uncoated wells (see Fig. 1). In addition, cells exhibited only b a c k g r o u n d binding to TNP26-FGG alone or to wells in which normal mouse serum or a digitalis-specific monoclonal mouse IgG1 was substituted for IgE (data not shown). The o p t i m u m IgE concentration was determined by titrating the a2 with constant TNP26-FGG levels. IgE-binding molecules were tested with a2 concentrations beginning at 6 / ~ g / m l and several TNP24_26-FGG concentrations (1, 2, 4, 5, 8 and 1 0 / ~ g / m l ) for their ability to inhibit binding of spleen cells to the IgE-coated wells. A concentration of a2 that gave inhibition with the highest dilutions of IgE-binding molecules (0.1 /~g/ml a2) was used in subsequent experiments. Thus, standard coating conditions for an efficient assay for IgE-binding inhibitors were 2 /~g/ml TNP26-FGG and 0.1 /~g/ml a2. W h e n the assay was utilized for detecting cells bearing high- or low-affinity Fc~ receptors, plates were coated with 5 /~g/ml T N P z 6 - F G G followed by a2 at a concentration of 0.3/~g/ml.

Correspondence of the plate assay with other assays and systems Results from a binding experiment that employed the assay conditions for inhibitors are shown in Fig. 1. The left panel shows data obtained from the readout of the M T T metabolized by the b o u n d B A L B / c spleen cells. The absorbance is linearly proportional to cell n u m b e r (Mosmann, 1983). F r o m the signal of the total input cells (see legend to Fig. 1) it was calculated that under these assay conditions 37% of the cells were binding to the a2-coated plate. Rosetting of adherent-depleted B A L B / c spleen cells using ox red cells coated with rat IgE in 4 separate experiments

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gave 36% + 2% rosette-forming cells. Under these rosetting conditions, 18% of spleen or mesenteric lymph node cells from Lewis rats infected with Nippostrongylus brasiliensis (Nb) formed IgE-rosettes. Other investigators have observed 31-36% (Vander-Mallie et al., 1982) or 15-17% (Uede et al., 1983) IgE-rosette-forming cells from normal B A L B / c spleen, and variations of 16-22% (Spiegelberg, 1981), 22-30% (Iwata et al., 1983) and 14-27% (Yodoi and Ishizaka, 1980) in the ability of spleen and mesenteric lymph node cells from Nb-infected rats to form IgE-rosettes. Clearly variation exists in methods of cell preparation and assays and among cell populations. The percentage of B A L B / c spleen cells that were binding to the a2-coated plate (37%) was consistent with our rosetting data on these populations and with the data of Vander-Mallie et al. (1982), who used TNP-specific IgE to coat TNP-coupled ox red cells. Thus, we seemed to be detecting a population of cells that was similar in frequency to the total IgE-rosetting population. Rat IgE at initial concentrations of 20 t~g/ml or greater completely inhibited the binding of spleen cells to the plate (Fig. 1). In contrast, in the absence of inhibitors or in the presence of mouse lgG1 as an 'inhibitor', the number of cells binding to the

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Fig. 2. Binding of a murine mast cell line to IgE-coated wells and inhibition by lgE and monoclonal antibodies against IgE. M C / 9 cells binding to wells coated with mouse IgE and TNP15-FGG were measured. Inhibitors were added at the indicated concentrations (IgE and IgG1) or dilutions (supernatants of rat-mouse hybridomas specific for mouse IgE) as follows: C), no inhibitors; e, rat IR162 lgE; A, mouse IgG1; [], culture supernatant from hybridoma EM-126.6; z~, culture supernatant from hybridoma EM-95.3; II, culture supernatant from hybridoma EM-51.3. Each point is the average of 3 replicates. Left panel: data are graphed as number of input cells bound, m, background binding to uncoated wells (average and range of 14 wells); D, background binding to wells coated with TNP15-FGG and normal mouse serum (average and range of 14 wells). Binding in the absence of inhibitors increased at the bottom rows of the plate. This was often observed on certain microtiter plates. For this reason, control wells (no inhibitors) were always included in each row across the plate. Right panel: data for rat IgE (from left panel) and for supernatants of hybridoma lines are graphed as % inhibition of binding as in Fig. 1.

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wells remained relatively constant. Thus, the binding of cells to the microtiter wells appeared to be IgE-specific. Concentrations of rat IgE between 0 . 1 6 / ~ g / m l and 0.8 ~ g / m l normally resulted in a 50-60% inhibition of the binding of cells to the plate, but inhibition by as much as - 7 0 % was occasionally observed (see Fig. 1). A titration of rat IgE as an inhibitor of binding was included in each experiment as a reproducible positive control in all assays. Several experiments were performed to verify that by measuring inhibition of binding of cells to the a2-coated plate we were assaying for IgE-binding properties similar to those that would inhibit direct binding of IgE to the cell surface. Fig. 2 shows the results of an experiment in which cloned IL-3-dependent mast cells ( M C / 9 ) were bound to the a2-coated plate via their high-affinity receptors for IgE. The left panel shows that rat IgE but not mouse IgG1 inhibited binding of M C / 9 cells to the wells. M C / 9 cells did not bind significantly to uncoated wells or to wells coated only with T N P - F G G and normal mouse serum. Three rat monoclonal antibodies of the IgG class that are directed against mouse IgE (Baniyash and Eshhar, 1984) were tested in the assay as inhibitors of the binding of M C / 9 cells to the IgE-coated wells. It is known that all 3 monoclonal antibodies inhibit direct binding of radioiodinated mouse IgE to high-affinity receptors for IgE

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Fig. 3. Binding of spleen cells to lgE-coated wells is inhibited by rodent IgE-binding molecules and a monoclonal antibody against mouse IgE. Various dilutions of culture supernatant were added as inhibitors (each inhibitor was tested in a separate experiment). Percent inhibition is relative to the binding of cells in control wells. O, supernatant from 23B6 hyhridoma cells induced with IgE. Total input cells: 0.640 OD57o_630 units; background binding to uncoated wells: 0.064 OD570_630 units (2 replicates of each point). ©, supernatant from 8.3-CHO.13 cells. Total input cells: 0.401 OD57o_630 units; background: 0.006 OD570_630 units (2 replicates of each point). A, supernatant from hybridoma EM-95.3. Total input cells: 0.482 OD570_630 units (39% binding); background: 0.011 OD570_630 units (3 replicates of each point).

20 on b a s o p h i l s / m a s t cells (Baniyash and Eshhar, 1984). In the right panel of Fig. 2, titration curves of the inhibition by the monoclonal supernatants (EM-51.3, EM-95.3 and EM-126.6) are compared to the inhibition produced by rat IgE. EM-95.3 and EM-126.6 culture supernatants efficiently inhibited (60-70%) the binding of M C / 9 cells to the IgE-coated wells. This inhibition titrated out with increasing dilution of the supernatant medium. The EM-51.3 antibody gave only partial inhibition (25%) that quickly titrated. These results were comparable with direct binding studies in which inhibition of binding of IgE to rat basophilic leukemia cells was 54%, 82% and 87% for EM-51.3, EM-95.3 and EM-126.6, respectively (Baniyash and Eshhar, 1984; Figs. 2 and 3). This experiment demonstrated that for detecting cells bearing high-affinity IgE receptors and for identifying molecules that inhibit the binding of [gE to these cells, the microtiter plate experiments were able to measure features similar to those observed in other IgE-binding assays. Note that the microtiter plate assay was not designed for quantitating ligand affinities or numbers of receptors per cell.

Assays of low-affinity IgE-binding molecules Confirmation that the plate assay could detect IgE-binding activities that would inhibit rosette formation was obtained by testing different IgE-binding molecules in 2 additional assays. Several of our preparations of low-affinity IgE-binding molecules were tested in our plate assay on B A L B / c spleen cells, for inhibition of binding of IgE-coated fluorescent beads to spleen cells (Sarfati et al., 1984) and, in the laboratory of Dr. K. Ishizaka, for rosette-inhibitory activity. We obtained good correspondence between our plate assay and the rosette assay with all samples tested. IgE-binding activity that was detectable in undiluted culture supernatants by the fluorescent bead assay (15-40% decrease in total cell-bound fluorescence) also gave inhibition in the microtiter plate assay.

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II:3000 1 / 2 0 0 0 1/1000 1/100 Supernatant dilution

Fig. 4. Titration of transiently expressed rodent IgE-binding molecule on spleen cells in the plate assay. Supernatant from COS7 cells transfected with cDNA clone 8.3 (8.3-COS7, Martens et al., 1985) was titrated as an inhibitor. Two separate titration experiments on the same culture supernatant are shown: e, total input cells: 0.276 0 0 5 7 0 _ 6 3 0 ; background: 0.018 0 0 5 7 0 _ 6 3 0 units (3 replicates of each point); C), total input cells: 0,395 O O 5 7 0 _ 6 3 0 units; background: 0.042 0 0 5 7 0 _ 6 3 0 units (2 replicates of each point).

21 Assay data on several of these preparations are shown in Figs. 3 and 4. In the plate assay, inhibition by a filtered culture supernatant derived from the IgE-induced (24 h culture) T cell hybridoma, 23B6, was observed with the undiluted preparation but could not be detected at dilutions greater than 4-fold (Fig. 3). This corresponds well with the sensitivity of the rosette-inhibition assay for detecting similar IgE-binding molecules at maximum dilutions of 1 : 4 or ] : 8 (Huff et al., 1982). In addition, the percent inhibition of binding of cells to the wells by low-affinity IgE-binding molecules was always in the same range as the percent inhibition of rosettes (15-40% inhibition). Culture supernatant from COS7 cells transiently expressing a cloned cDNA (8.3-COS7) that encodes a molecule with low-affinity IgE-binding activity (Martens et al., 1985) was also assayed. The results of 2 separate experiments titrating 8.3-COS7 supernatant are shown in Fig. 4. IgE-binding activity was titratable to a dilution of 1 : 2000. The maximum percent inhibition was, again, comparable to that observed in rosette inhibition assays (average of 29% for dilutions up to 1 : 1000). By our definition of a unit of IgE-binding activity in the plate assay (see Materials and Methods), the 8.3-COS7 material assayed in Fig. 4 had - 4 6 , 0 0 0 units/ml of IgE-binding activity. Culture supernatant from a clone of CHO cells stably expressing cDNA clone 8.3 (8.3-CHO.13) also was titrated in the microtiter plate assay (Fig. 3). This material had approximately 4000 units/ml of IgE-binding activity. In contrast, IgE-induced supernatant from 23B6 cells contained - 1 2 0 units/ml of IgE-binding activity (Fig. 3). Thus we were able to document that the transient expression level of cDNA clone 8.3 was high in COS7 cells and approximately 10-fold lower in stably transformed CHO cells. The plate assay thus seemed to be suitable as a method for monitoring different cell lines for levels of expression of IgE-binding molecules. The rat monoclonal antibodies specific for mouse IgE (EM-126.6, EM-95.3 and EM-51.3) were also tested for their ability to inhibit binding of IgE to the low-affinity Fc~ receptor on spleen cells. Only EM-95.3 was found .to inhibit binding. (Fig. 3, data not shown for EM-126.6 and EM-51.3.) This inhibition suggests that the sites on IgE that bind to low- and high-affinity F% receptors appear to be different but may overlap, Well-to-well variability in cell binding and in percentage inhibition by IgE-binding molecules was always observed (Figs. 1-4), and occasionally 'bad' wells or data points were obtained (Fig. 3). Experiments repeating the EM-95.3 inhibition shown in Fig. 3 gave titration plateaus and were reproducible to +5% of the percent inhibition shown (except for the 1 : 5 dilution). This variability was not significantly different from rosetting experiments and was greater when a heterogeneous cell population (spleen or lymph node) was employed. Experiments that utilized cultured cell lines ( M C / 9 , RPMI-8866 and others) gave results that exhibited about 10% variation within an experiment, for example, 50.3 _+ 5.8% binding for 12 wells of M C / 9 cells and 50.3 _+ 4.7% binding for 16 wells of RPMI-8866 cells. Average binding was reproducible among experiments. Several lots of microtiter plates gave less variability than others (see Fig. 2). This seemed to be the result of more uniform adsorption of protein.

22 Discussion

We have developed a microtiter plate assay for cells bearing Fc~ receptors and for various IgE-binding molecules. Numbers of Fc~ receptor-bearing lymphocytes that were bound to microtiter wells coated with IgE were measured colorimetrically. Two conditions were important for the success of the assay. First, the relative concentrations of T N P - F G G and IgE (the size of the 'microaggregate' of IgE) seemed to be critical in order to obtain reproducible cell binding and to detect inhibition of binding of lymphocytes by low-affinity IgE-binding molecules. Second, an optimum number of cells per well (that was empirically determined for each cell type because the optimum varied according to receptor affinity and number, clonality of the population and metabolic activity of the cells) increased the sensitivity of the assay for inhibition, For measurement of the number of receptor-bearing cells, higher concentrations of T N P - F G G and IgE were used on the plate to increase the density of IgE microaggregates. This ensured that the maximum number of Fc~ receptor-positive cells in a population would be detected. High-affinity IgE-binding molecules (i.e. monoclonal antibodies) could be detected at these higher concentrations of TNPF G G and IgE, but with a sacrifice in the level of sensitivity. We demonstrated that the microtiter plate assay was comparable in sensitivity to the rosette-inhibition assay for detecting immunoglobulin-binding molecules. Plate assays for inhibition of lymphocyte binding by IgE-binding molecules confirmed results obtained in other assays (e.g. binding radiolabeled IgE directly to the cell surface). As with the rosetting assay, cells bearing low-affinity receptors for IgE could be detected and enumerated. Like the rosette assay, the microtiter plate assay was not suitable for measuring ligand-receptor affinities or the total number of receptors on the cell surface. The variability in measuring percent inhibition was also comparable to the rosette-inhibition assay. Some of this variation was due to the use of a heterogeneous cell population, and some variability was the result of the method and its similarity to rosetting. The microtiter plate assay provides several advantages over other assays for detecting IgE-binding molecules and cells bearing Fc~ receptors. First, many samples can be assayed at once. Replicates and titrations are easily performed. We found that an assay comprising up to 5 microtiter plates could be performed reproducibly and provided adequate sample space. Second, the results obtained are easy to read out. Absorbance of the metabolized MTT is made on an automated plate reader and indicates the number of cells bound to the plate. Thus, the percentage of Fc~ receptor-positive cells or a titration of IgE-binding molecules in a particular sample can be determined reproducibly. Finally, the assay conditions, including ligands and receptors, can be easily altered to detect different binding molecules and cells bearing different surface receptors (see below). It is interesting that, in the plate assay, the maximum observable inhibition of binding of spleen cells by low-affinity IgE-binding molecules was 35-40%. In contrast, IgE was able to completely inhibit cell binding. Identical results have been obtained in rosette-inhibition assays. In both cases a heterogeneous cell population

23 (lymph node cells or splenic lymphocytes) was used as the source of Fc~ receptorbearing cells. It is possible that individual cells or different cell types in the spleen have surface Fc~ receptors that differ in their affinity for lgE (Vander-Mallie et al., 1982; Spiegelberg, 1984). Low-affinity IgE-binding molecules would effectively compete for IgE with only a subset of lymphocytes bearing the appropriate type of Fc~ receptor. Alternatively, the effect is merely due to the low concentrations of IgE-binding molecules present in culture supernatants. Since the assays are only suitable for titrating inhibition due to low-affinity IgE-binding molecules relative to IgE and not for measuring the molar amounts in the preparations, either or both possibilities are likely. The assay was used for examining the properties of several monoclonal antibodies directed against mouse IgE. The 3 antibodies we tested were previously found to bind to distinct determinants on mouse IgE and to inhibit the interaction of IgE with the high-affinity IgE receptor on basophils/mast cells (Baniyash and Eshhar, 1984). We confirmed these observations using a mouse mast cell line and in addition observed that only one of the antibodies inhibited the interaction of IgE with low affinity lymphoCyte Fc~ receptors. This implies that the sites on IgE molecules that are recognized by the 2 classes of IgE receptor are different. The results presented above also suggest that the binding sites on IgE for the 2 receptor classes may overlap to some degree. This is consistent with the observations of Conrad and Peterson (1984) that ascribe different biochemical properties to the polypeptides comprising the lymphocyte and basophil/mast cell receptor for IgE. Thus, additional general applications for the plate assay may include screening for monoclonal antibodies directed against the binding regions of either ligands or receptors. Recent modifications of the assay suggest several ways in which the plate assay may be used to detect other cells bearing Fc receptors. The assay has been readily adapted to detecting IgE-binding molecules and FcF receptor-bearing cells of human origin. A human lymphoblastoid B cell line which bears - 200,000 Fc~ receptors per cell, RPMI-8866 (Spiegelberg and Melewicz, 1980), was bound to wells coated with human IgE. The binding was inhibited by 2 preparations known to contain IgEbinding molecules of human origin: culture supernatant from confluent RPMI-8866 cells (Sarfati et al., 1984) and culture supernatant from a human T cell hybridoma that was induced with human IgE (Huff and Ishizaka, 1984) (unpublished results). Thus, it seems feasible to utilize cells of different species and to assay both Fc receptors for various classes of immunoglobulin molecules and immunoglobulinbinding molecules specific for other classes.

Acknowledgements We thank Dr. T. Mosmann for his initial observation and demonstration that B A L B / c spleen cells bind to IgE-coated mierotiter plates, Drs. C. Martens and K. Moore for generously and continuously providing culture supernatants from cells transfected with a cloned IgE-binding molecule, Drs. P. Jardieu and K. Ishizaka for assaying our samples by rosette inhibition and for encouragement at the initial

24 stages of assay development, and

G.

Burget

for e x c e l l e n t p r e p a r a t i o n

of the

m a n u s c r i p t . S p e c i a l t h a n k s to D r . Z. E s h h a r f o r his g e n e r o u s gifts o f rat m o n o c l o n a l antibodies and helpful discussions.

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