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A flow cytometric micromethod for the detection of Fcϵ receptors and IgE binding factors using fluorescent microspheres
Journal oflmmunologicalMethods, 88 (1986) 25-32 Elsevier
25
JIM03845
A flow cytometric micromethod for the detection of Fce receptors and IgE binding factors using fluorescent microspheres J.Y. Bonnefoy, J. Banchereau, J.P. Aubry and J. Wijdenes UNICET, Department of lmmunology, 27 Chemin des Peupliers, 69570 Dardilly, France (Received 26 September 1985, accepted 14 November 1985)
Two assays based on the use of fluorescent microspheres have been developed in order to detect Fc~ receptors on human cells and human IgE binding factors. A direct assay using microspheres to which IgE was coupled permitted the detection of Fcc receptors on RPMI 8866 cells. However the fluorescence intensity was relatively low and made it difficult to discriminate between positive and negative cells. To increase the sensitivity of the assay, an indirect 3-step test was developed, in which the cells were subsequently incubated with soluble IgE, a polyclonal or monoclonal anti-IgE antibody and fluorescent microspheres to which anti-mouse or anti-rabbit immunoglobulin was coupled. This indirect assay resulted in an increased fluorescence intensity. Optimal discrimination between positive and negative cells was obtained. This assay permitted the detection of human IgE binding factors produced by RPMI 8866 cells. The binding of IgE was not dependent on the origin of the latter. Among the different cell lines tested, the EBV positive lymphoblastoid cells were found to express Fc~ receptors and to release IgE binding factors in their supernatants. Key words: Fcc receptor," Human lgE binding factors," Fluorescent microspheres; Flow cytometry
Introduction
Receptors for the Fc fragment of all 5 immunoglobulin (Ig) classes have been observed to be expressed on subpopulations of lymphocytes. The majority of T cells have receptors for IgM (Moretta et al., 1975). Smaller fractions of T cells have receptors for IgG (Moretta et al., 1975), IgA (Gupta et al., 1979; Lum et al., 1979), IgE (Gonzalez and Spiegelberg, 1977; Yodoi and Ishizaka, 1979) and IgD (Sj/Sberg, 1980; Coico et al., 1985). The cells bearing Fc~ receptors play a major Abbreviations: Ig, immunoglobulin; EBV, Epstein-Barr virus; BSA, bovine serum albumin; IgE BF, IgE binding factor; Mab, monoclonal antibody; Fc~R, Fc~ receptor; FACS, fluorescence-activated cell sorter.
role in the regulation of IgE production (Katz, 1984; Ishizaka, 1985). The IgE binding factors (IgE BF) are potentially useful therapeutic agents in disorders of the IgE system as evidenced by their modulating effects on the in vitro production of IgE (Deguchi et al., 1983; Katz, 1984; Leung and Geha, 1984; Ishizaka, 1985). Thus far Fcc positive cells have been detected by rosette formation with IgE coated erythrocytes. However this technique is laborious and not optimally suitable for the screening of large series. The aim of the work reported here was to set up an assay suitable for large series which would permit the rapid detection of human cells bearing Fcc receptors and human IgE binding factors. To obtain maximal sensitivity and versatility a 3-step assay is described which is based on successive incubation of cells with purified human IgE, monoclonal or
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
26 polyclonal anti-IgE antibody, and fluorescent microspheres coupled with anti-mouse or -rabbit immunoglobulin antiserum.
Materials and methods
Antibodies and proteins Three human IgE myeloma proteins (PS a gift from Dr. Ishizaka, one diagnosed in Lyon and one diagnosed in Birmingham, U.K., obtained through Dr. I. MacLennan) were purified by ion exchange chromatography (DEAE 52) using an NaC1 gradient (0.01 M-0.3 M pH 7.2). According to SDSP A G E and silver staining, the isolated IgE was pure without any other protein contamination. To detect IgE, a monoclonal anti-IgE (Hybritech, San Diego, CA) and a polyclonal anti-IgE were used. The polyclonal antiserum was prepared by immunizing rabbits with purified IgE PS, and the antiserum was rendered monospecific for IgE by passage over IgG, IgA and IgM immunoabsorbent columns (Affigel-10, Bio-Rad, Richmond, CA). A rabbit anti-mouse Ig antiserum and a goat anti-rabbit Ig antiserum have been prepared in our laboratory. Their IgG fractions were isolated by column chromatography using trisacryl G F 05 and DEAE trisacryl (lndustrie Biologique Fran~aise, Genevilliers, France) (Peterson, 1970). Human IgG, IgA and IgM have been purified from myeloma or Waldestr6m sera using ion exchange chromatography according to standard procedures (Williams and Chase, 1967). Cells WIL2WT (an EBV lymphoblastoid cell line), Daudi (a Burkitt lymphoma), Molt 4 (a T cell leukemia), HE60 (a promyelomonocytic leukemia) and U937 (a promonocytic cell line) were obtained from the ATCC (Rockville, MD). RPMI 8866 (EBV lymphoblastoid cell line) was kindly provided by Dr. K. Ishizaka. UD38 is an EBV lymphoblastoid cell line which has been established in our laboratory by Dr. F. Rousset after infection of normal peripheral blood B cells with EBV. All cells were cultured in RPMI 1640 (Flow, Irvine, Scotland) supplemented with 10% heat-in-
activated fetal calf serum (Flow), 2 mM glutamine (Flow), 100 U / m l penicillin and 100 /~g/ml streptomycin (Flow).
Coupling of proteins to microspheres 0.1 mg of the protein to be coupled was dissolved in 1 ml of a solution of EDAC (1-ethyl-3(3-dimethylaminopropyi)carbodiimide: 0.1 m g / m l distilled water). 100 /tl sonicated (1 min in a Bransonic 321) microspheres (Fluoresbrite carboxylate diameter 0.57 /~m Polyscience, Warrington, PA, ref. 15700) were added and the suspension incubated overnight at 4°C on a rocking table. After this incubation, the microspheres were spun down (10000 x g, 5 min) and washed 3 times with PBS-BSA (1%). The microspheres were stored at 4°C in PBS-BSA supplemented with 15 mM sodium azide. The suspension was sonicated (1 min) before each experiment. Staining protocol Two detection systems (direct and indirect) have been developed. In both assays the cells were adjusted to 107/ml in PBS-BSA and 5 x l0 s cells (50 #1) were added to each well of a 96-conical well microtiter plate (Flow, ref. no.: 7632105). (a) Direct assay. In the direct assay, sonicated IgE beads were added to the cells (50/~1 of different dilutions: 1/5 1/1000 per well) and incubated for 45 min. PBS-BSA (100 /~l/well) was added and the plate centrifuged for 7 rain at 150 x g. The supernatant was discarded and the cells resuspended in PBS-BSA (100/~l/well). This cell suspension was layered on top of 3 ml heat-inactivated filtered FCS in a conical tube (Falcon ref. 2099). After centrifugation at 100 x g for 15 min, the supernatant was slowly aspirated and PBS-BSA (150 /~l/well) added to the cell samples which, after resuspension, were analyzed by flow cytometry. All steps were carried out at 4°C. (b) Indirect assay. In the indirect assay, the cells were first incubated 45 min with monomeric IgE (0.5-2 /~g per 5 x l0 s cells) and then washed with PBS-BSA. A monoclonal anti-IgE (Hybritech, San Diego, CA) at a dilution of 1 / 5 0 - 1 / 1 6 0 or our polyclonal anti-IgE antibody at a dilution of 1 / 1 0 - 1 / 1 0 0 0 0 was then added. After 45 min, the cells were washed with PBS-BSA and the anti-mouse Ig microspheres (dilution
27 1 / 1 0 - 1 / 4 0 ) or the anti-rabbit Ig microspheres (dilution 1 / 1 0 - 1 / 8 0 ) added. The suspensions were finally processed as described above. All steps were carried out at 4°C. The assay using the monoclonal anti-IgE antibody will subsequently be referred to as the 'mouse indirect assay' while the assay using the polyclonal anti-IgE antibody will be referred to as the 'rabbit indirect assay'.
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Production, purification and assay of human IgE binding factors
Fluorescence analysis Fluorescence analysis was performed with a FACS 440 (Becton Dickinson, Sunnyvale, CA) equipped with a 5 W argon laser running at 488 nm, 0.5 W. Fluorescence parameters were collected using a built in logarithmic amplifier after gating on the combination of forward light scatter (FLS) and perpendicular light scatter (PLS) which was used to discriminate viable cells from non-viable ones. Data were stored in list mode and analyzed with a PDP 11/23 using the LYSYS program developed by R. Lefebvre.
Results
(A ) 'Direct assay For the detection of Fc~ receptors, increasing dilutions of IgE coupled microspheres were added to R P M I 8866 cells. Fig. 1A shows that maximum
................
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Culture supernatants of RPMI 8866 cells were used as a source of IgE BF as described by Sarfati et al. (1984). In the microtiter wells, 100/~1 of the supernatant were incubated with 0.5-1 /~g IgE for 1 h at room temperature and subsequently for 1 h at 4°C. 5 × 105 cells in 50/~1 were then added and all steps were performed as described above in the indirect assay. An IgE BF purified by affinity chromatography was also assayed. For this, human IgE was coupled to Affigel 10 according to the manufacturer's instructions and RPMI 8866 culture supernatant was passed overnight over the column. After washing with PBS the IgE bound fraction (which will be referred to as purified IgE BF) was eluted with 0.2 M glycine-HC1 buffer pH 2.8. Immediately after elution the pH was adjusted to 7.2 with phosphate buffer.
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Fig. 1. Direct assay. A: Effect of diluting microspheres with IgE coats on their binding to RPMI 8866 cells. The assay was performed as described in the materials and methods section using IgE beads incubated with RPMI 8866 ceils. The negative control using microspheres coated with an unrelated Ig was subtracted (8%). B: FACS histogram. The control (dark shading) using microspheres coated with a non-related Ig overlaps with the histogram obtained after incubation of RPMI 8866 cells with IgE beads diluted 1/50. The bar represents the area defining positive cells (63%).
binding was obtained at dilutions lower than 1/20. Fig. 1B shows the FACS histogram of RPMI 8866 cells reacted with IgE beads. The separation between positive and negative cells was difficult to establish and the fluorescence of positive cells was relatively low due to binding of small amounts of microspheres. This system was found to be isotype specific: preincubation of the cells with IgG, IgA and IgM did not decrease the percentage of microsphere binding cells while preincubation with 1/~g IgE dropped the number of positive cells from 58% to 25% (data not shown). Since weak fluorescence of the positive cells could be a source of
28
difficulty an indirect assay was developed in order to increase the fluorescence intensity.
(B) Indirect assay In the indirect assay RPMI 8866 cells were successively incubated with the following reagents: human monomeric IgE; monoclonal or polyclonal anti-IgE antibodies; anti-mouse or anti-rabbit Ig coupled to fluorescent beads (Fig. 2). In order to optimize the assay, the following parameters were investigated: (i) Concentration of the monoclonal anti-IgE antibody. To determine the optimal concentration of monoclonal anti-IgE antibody, 5 × 105 RPMI 8866 cells were incubated with 1 #g IgE, different concentrations of the monoclonal anti-IgE antibody, and the microspheres at a dilution of 1/10. Optimal fluorescence intensity was obtained using 1 / 5 0 - 1 / 8 0 dilutions of the monoclonal anti-IgE (10 ~ g / m l - 6 ~ g / m l ) (Fig. 3A); in all other assays the Mab was used at a dilution of 1/50. A typical FACS profile is shown in Fig. 3B in which a very clear difference between positive and negative cells can be seen. In this experiment, 95.6% of the cells bound beads whereas controls without IgE displayed only 5% positive cells. (ii) Concentration of the microspheres. The op-
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timal concentration of microspheres was determined as follows: 5 × 105 RPMI 8866 cells were incubated with 1 ~g IgE. Then the anti-IgE Mab was added at a dilution of 1/50 followed by different dilutions of the microsphere suspension. Fig. 3C demonstrates that the percentage of positive cells dropped sharply as the microspheres were diluted beyond 1/10. In all other assays the microsphere suspension was used at a dilution of
1/10. (iii) Concentration of the monomeric lgE. To determine the optimal concentration of IgE, the RPMI 8866 cells were incubated with concentra~ tions of IgE which ranged from 50 ng to 2000 ng. Other reagents were added as determined above. Fig. 3D shows that the maximum number of positive cells was obtained at concentrations of 1-2 ~g lgE. A concentration of 2 ~g of lgE was therefore used in routine assays. 17 experiments performed using optimal conditions gave a mean of 62.2% + 2.9 (SEM) positive cells (after subtraction of controis omitting IgE). (iv) Isotype specificity. In order to demonstrate that the binding of lgE to the RPMI 8866 cells was isotype specific, the effect of IgG, lgA and IgM on IgE binding was studied in 2 different ways: (a) before addition of IgE, RPMI 8866 cells were preincubated with 2 ~g of either IgG, IgA, or IgM. IgE binding was not affected (Fig. 4A); (b) RPMI 8866 cells were incubated with 2 /~g IgE and 10 t~g of either IgG, IgA, or IgM. The binding of IgE was not affected (Fig. 4B). These experiments demonstrate the specificity of the binding of IgE to the RPMI 8866 cells. (v) Assay using a polyclonal anti-IgE antibody.
IgE
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Fig. 2. Indirect assay: schematic representation of the different experimental steps.
In order to screen for anti-FccR Mabs and anti-IgE Mabs inhibiting the binding of IgE we investigated whether the anti-IgE Mab and the anti-mouse Ig microspheres could be replaced by a specific rabbit anti-lgE antibody and anti-rabbit Ig microspheres. Fig. 5 shows that the antibody must be sufficiently diluted to provide an optimal signal since a prozone effect is observed at low dilutions. Routinely we used the polyclonal anti-IgE antibody at a dilution of 1/2000 and the FACS histogram was similar to that obtained with the anti-IgE Mab (data not shown).
(vi) Presence of FccR on different human cell lines. Seven human cell lines were screened for
29
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Fig. 3. Mouse indirect assay. A : Effect of the monoclonal antibody concentration on the binding of microspheres to the RPMI 8866 cells. RPMI 8866 cells were successively incubated with 1 p,g IgE, different concentrations of the monoclonal anti-IgE and anti-mouse Ig coated microspheres (1/10). The negative control obtained by omitting IgE (6%) was subtracted. B: FACS histogram. RPM1 8866 cells were incubated with 2 /.tg IgE, monoclonal anti-IgE (1/50), and anti-mouse Ig coated microspheres (1/10). The dark shaded peak represents the negative control in which IgE was omitted (5%). The bar represents the area defining the positive cells (95.6%). C: Effect of microsphere concentration on binding to RPMI 8866 cells. P,PMI 8866 cells were incubated with 2 ~g IgE, monoclonal anti-IgE (1/50) and different concentrations of anti-mouse Ig coated microspheres. The negative control obtained by omitting lgE (7%) was subtracted. D: Effect of IgE concentration on the binding of microspheres to RPMI 8866 cells. RPMI 8866 cells were incubated with different concentrations of IgE, monoclonal anti-lgE (1/50) and anti-mouse Ig coated microspheres (1/10). The negative control obtained by omitting IgE (6%) was subtracted.
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the presence of Fcc receptors using the mouse indirect assay and 3 different sources of purified IgE myeloma protein. The 3 EBV lymphoblastoid cell lines RPMI 8866, WIL2WT and UD38 were
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Fig. 4. Mouse indirect assay showing lack of interference of IgG, lgA and IgM on the binding of lgE to RPMI 8866 cells. A: RPMI 8866 cells were incubated first with 2 btg IgE, lgG, IgA, IgM, then with 2 /tg lgE. Monoclonal anti-lgE (1/50) and anti-mouse Ig coated microspheres (1/10) were finally added. B: 10 ~g IgG, lgA or IgM were added together with 2 ,ttg lgE to RPMI 8866 cells. Monoclonal anti-lgE (1/50) and antimouse lg coated microspheres (1/10) were finally added. The negative control obtained by omitting lgE (6%) was subtracted.
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Fig. 5. Rabbit indirect assay showing effect of the polyclonal anti-lgE antibody concentration on the binding of microspheres to RPMI 8866 cells. RPMI 8866 cells were incubated with 1 /tg lgE, different concentrations of the polyclonal antilgE antibody, and anti-rabbit Ig coated microspheres (1/20). The negative control obtained by omitting IgE (4%) was sublracted.
found to bear F c( receptors while M O L T 4 (a T cell l y m p h o m a ) . H L 6 0 (a p r o m y e l o m o n o c y t i c cell line), Daudi (a Burkitt l y m p h o m a ) and U937 (a p r o m o n o c y t i c cell line) were negative, A l t h o u g h slight differences were found, the 3 different lg E p r e p a r a t i o n s b o u n d to a similar extent (Table 1). H e a t i n g the IgE to 5 6 ° C for 2 h ( c o n d it io n s k n o w n
TABLE I MOUSE INDIRECT ASSAY: DETECTION OF Fce RECEPTORS ON DIFFERENT HUMAN CELL LINES WITH 3 DIFFERENT lgE MYELOMA PREPARATIONS Each cell line was incubated with 2 ,ttg of each IgE, monoclonal anti-IgE (1/50) and anti-mouse lg coated microspheres (1/10). The negative control obtained by omitting IgE (6%) was subtracted. NT: not tested. Cell lines Molt 4 HL 60 Daudi U 937 RPMI 8866 Wil 2 WT UD 38
% Positive cells lgE PS
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IgE Lyon
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0 0 0 0 72 42 NT
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Fig. 6. Rabbit indirect assay showing detection of human IgE binding factors. C : negative control obtained by incubation of RPMI 8866 cells with polyclonal anti-lgE antibody (1/1000) and anti-rabbit Ig coated microspheres (1/20). C+: positive control obtained by incubation of RPMI 8866 cells with 1 /~g lgE, polyclonal anti-IgE antibody and anti-rabbit lg coated microspheres. 8866 SN IX: lgE was preincubated with 100 yI RPMI 8866 3 day culture supernatant before addition to RPM1 8866 cells. Purified IgE BF: IgE was preincubated with 50 yl of the eluate from an IgE Affigel column through which an RPMI 8866 3-day culture supernatant was passed.
to alter the Fc fragment) resulted in a decrease in the n u m b e r of cells b i n d i n g IgE from 75% to 25%.
(vii) Detection of human IgE binding factors. T o investigate whether the indirect assay could also be used for the detection of IgE b i n d i n g factors p r o d u c e d by R P M I 8866 cells, IgE was p r e i n c u b a t e d with a 3-day R P M I 8866 culture supernatant. As can be seen in Fig. 6, this prei n c u b a t i o n resulted in a small but significant decrease in the n u m b e r of cells b i n d i n g IgE. The eluate from a R P M I 8866 culture s u p e r n a t a n t passed through IgE-Affigel was found to strongly inhibit IgE b i n d i n g in both the m o u se and the rabbit indirect assay. This indicated that this assay was sensitive enough to detect h u m a n IgE b i n d i n g factors o b t a i n e d f r o m R P M I 8866 cells.
Discussion Th e goal of this study was to develop an assay which would permit the detection of (i) Fc~ receptors on h u m a n ceils, (ii) h u m a n I g E b i n d i n g [ac-
31 tors (IgE BF). Previously the evaluation of human Fc~ receptors and IgE BF has been achieved by rosette assays using IgE. However, such techniques require selected red blood cells that have been coated freshly with IgE. Furthermore the rosettes are relatively unstable and have to be scored microscopically, which is time consuming and laborious and therefore not suitable for large numbers of samples. We have replaced the red blood cells by fluorescent microspheres and examined binding by flow cytometry since it has been claimed that such microsphere permit the detection of receptors for the complement components on human cells (Lambris and Ross, 1982; Ross and Lambris, 1982). We first utilized IgE-coupled microspheres in a direct assay. Initially this test permitted us to detect cells that bound IgE microspheres specifically since microsphere binding was inhibitable by soluble IgE, but not by soluble IgG, IgM or IgA (data not shown). Two main reasons led us to design an indirect assay, (i) the development of a system characterized by higher binding of microspheres to cells thereby permitting better discrimination between positive and negative cells, (ii) availability of a more versatile system which permitted the detection of other Fc receptors (e.g., IgG, IgA) using the same microspheres. Fig. 3B shows a typical FACS histogram using the 3-step assay consisting of incubation of cells with monomeric IgE, monoclonal anti-IgE antibody and anti-mouse Ig microspheres. Positive cells were easily differentiated from negative cells. With the RPMI 8866 cell line we found the number of positive cells to vary between 45 and 85%. Such a variation has been described by others using the standard rosette assay. Although several hypotheses have been put forward to explain this variability (e.g., cell density or differential FccR expression during cell cycle) the reasons remain unclear. Controls omitting either IgE alone or IgE plus anti-IgE antibody resulted is 3-8% positive cells with low fluorescence intensity. Replacing the anti-IgE Mab anti-mouse Ig microspheres with anti-IgE polyclonal antibody anti-rabbit Ig microspheres yielded the same FACS histogram provided the polyclonal antibody was sufficiently diluted to avoid the prozone effect. This rabbit indirect assay was used to screen anti-RPMI 8866 Mabs inhibiting IgE binding or anti-IgE Mabs
inhibiting binding of IgE to FccR. The assay was found to be isotype specific since IgG, IgA, and IgM in excess did not alter the binding of IgE. Using this assay we found that out of 7 cell lines tested only the 3 EBV lymphoblastoid cell lines (RPMI 8866, WIL2WT and UD 38) had receptors for IgE. This correlates with a recent observation of Sarfati et al. (1984). The significance of this finding is currently under investigation in our laboratory. Our negative result with U937 is at variance with other reports (Narey-Fejes-Toth and Guyre, 1984) and could be attributed to the use of a particular subclone of the U937 cell line. The detection of Fcc receptors using fluorescent microspheres offers the/ following advantages: (i) it is not dependent on the quality of the microspheres and a 10 ml bottle of such microspheres can last for years; (ii) the micromethod permits the use of small amounts of reagent; (iii) large numbers of samples (100-150 per day) can easily be handled due to use of microplates and FACS detection; (iv) all the reagents are extremely stable, coupled beads need only be prepared every second month and there is minimum loss of the precious IgE during coupling procedures; (v) the indirect assay can very easily be modified to detect other Fc receptors. For example, the Fc~,R on Daudi cells could be easily monitored using IgG and anti-IgG antibody. Preincubation of monomeric soluble IgE with IgE BF purified from RPMI 8866 culture supernatants resulted in a drastic decrease in its binding to cells (Fig. 6). Preliminary data also showed that this assay permits the detection of IgE BF in the supernatant of an IgE pulsed human TT hybridoma. The availability of this relatively simple and rapid assay for screening human IgE BF should prove useful in the cloning of the gene for this molecule. It should also prove useful in the study of human T cells secreting these factors. Since this study has been conducted, 2 new techniques have been described for detecting Fcc receptors: (i) Kanowith-Klein and Saxon (1985) have shown that IgE immune complexes permit the detection of FccR on human cells using a cytochemical procedure, and (ii) Narey-Fejes-Toth and Guyre (1984) have demonstrated the binding of FITC-labeled IgE to U937 cells using flow cytometry.
32
Acknowledgements We thank Dr. F. Rousset for providing the UD 38 cell line, Sophie Lorkens and Muriel Vatan for typing the manuscript and Dr. J.E. De Vries for carefully reviewing this manuscript.
References Coico, R.F., B. Xue, D. Wallace, B. Pernis, G.W. Siskind and G.J. Thorbecke, 1985, Nature (London) 316, 744. Deguchi, H., M. Suemura, A. Ishizaka, Y. Osaki, S. Kishimoto, Y. Yamamura and T. Kishimoto, 1983, J. Immunol. 131, 2751. Gonzalez-Molina, A. and H.L. Spiegelberg, 1977, J. Clin. Invest. 59, 616. Gupta, S., C.D. Platsoucas, R.S. Schulof and R.A. Good, 1979, Cell. lmmunol. 45,469. Ishizaka, K., 1985, J. Immuno|. 135, 1. Kanowith-Klein, S. and A. Saxon, 1985, J. Clin. lmmunol. 5, 38.
Katz, D.H., 1984, Allergy 39, 81. Lambris, J.D. and G.D. Ross, 1982, J. Immunol. 128, 186. Leung, D.Y.M. and R.S. Geha, 1984, Clin. Immunol. Rev. 3, 1. Lum, L.G., A:V. Muchmore, D. Keren, J. Decker, I. Koski, W. Strober and R.M. Blaese, 1979, J. Immunol. 122, 105. Moretta, L., M. Ferrarini, M.L. Durante and M.C. Mingari, 1975, Eur. J. Immunol. 5, 565. Narey-Fejes-Toth, A. and P.M. Guyre, 1984, J. lmmunol. 133, 1914. Peterson, E.A., 1970, in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 2, eds. T,S. Work and E. Work (North Holland/Elsevier, Amsterdam) p. 228. Ross, G.D. and J.D. Lambris, 1982, J. Exp. Med. 155, 96. Sarfati, M., E. Rector, M. Rubio-Trujillo, K. Wong, A.H. Sehon and G. Delespesse, 1984, Immunology 53, 207. SjOberg, O., 1980, Scand. J. Immunol. 11,377. Spiegelberg, H.L., 1981, lmmunol. Rev. 56, 199. Williams, C.A. and M.W. Chase, 1967, Methods in Immunology and Immunochemistry, Vol. 1 (Academic Press. New York) p. 321. Yodoi, J. and K. Ishizaka, 1979, J. lmmunol. 122, 2577.
25
JIM03845
A flow cytometric micromethod for the detection of Fce receptors and IgE binding factors using fluorescent microspheres J.Y. Bonnefoy, J. Banchereau, J.P. Aubry and J. Wijdenes UNICET, Department of lmmunology, 27 Chemin des Peupliers, 69570 Dardilly, France (Received 26 September 1985, accepted 14 November 1985)
Two assays based on the use of fluorescent microspheres have been developed in order to detect Fc~ receptors on human cells and human IgE binding factors. A direct assay using microspheres to which IgE was coupled permitted the detection of Fcc receptors on RPMI 8866 cells. However the fluorescence intensity was relatively low and made it difficult to discriminate between positive and negative cells. To increase the sensitivity of the assay, an indirect 3-step test was developed, in which the cells were subsequently incubated with soluble IgE, a polyclonal or monoclonal anti-IgE antibody and fluorescent microspheres to which anti-mouse or anti-rabbit immunoglobulin was coupled. This indirect assay resulted in an increased fluorescence intensity. Optimal discrimination between positive and negative cells was obtained. This assay permitted the detection of human IgE binding factors produced by RPMI 8866 cells. The binding of IgE was not dependent on the origin of the latter. Among the different cell lines tested, the EBV positive lymphoblastoid cells were found to express Fc~ receptors and to release IgE binding factors in their supernatants. Key words: Fcc receptor," Human lgE binding factors," Fluorescent microspheres; Flow cytometry
Introduction
Receptors for the Fc fragment of all 5 immunoglobulin (Ig) classes have been observed to be expressed on subpopulations of lymphocytes. The majority of T cells have receptors for IgM (Moretta et al., 1975). Smaller fractions of T cells have receptors for IgG (Moretta et al., 1975), IgA (Gupta et al., 1979; Lum et al., 1979), IgE (Gonzalez and Spiegelberg, 1977; Yodoi and Ishizaka, 1979) and IgD (Sj/Sberg, 1980; Coico et al., 1985). The cells bearing Fc~ receptors play a major Abbreviations: Ig, immunoglobulin; EBV, Epstein-Barr virus; BSA, bovine serum albumin; IgE BF, IgE binding factor; Mab, monoclonal antibody; Fc~R, Fc~ receptor; FACS, fluorescence-activated cell sorter.
role in the regulation of IgE production (Katz, 1984; Ishizaka, 1985). The IgE binding factors (IgE BF) are potentially useful therapeutic agents in disorders of the IgE system as evidenced by their modulating effects on the in vitro production of IgE (Deguchi et al., 1983; Katz, 1984; Leung and Geha, 1984; Ishizaka, 1985). Thus far Fcc positive cells have been detected by rosette formation with IgE coated erythrocytes. However this technique is laborious and not optimally suitable for the screening of large series. The aim of the work reported here was to set up an assay suitable for large series which would permit the rapid detection of human cells bearing Fcc receptors and human IgE binding factors. To obtain maximal sensitivity and versatility a 3-step assay is described which is based on successive incubation of cells with purified human IgE, monoclonal or
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
26 polyclonal anti-IgE antibody, and fluorescent microspheres coupled with anti-mouse or -rabbit immunoglobulin antiserum.
Materials and methods
Antibodies and proteins Three human IgE myeloma proteins (PS a gift from Dr. Ishizaka, one diagnosed in Lyon and one diagnosed in Birmingham, U.K., obtained through Dr. I. MacLennan) were purified by ion exchange chromatography (DEAE 52) using an NaC1 gradient (0.01 M-0.3 M pH 7.2). According to SDSP A G E and silver staining, the isolated IgE was pure without any other protein contamination. To detect IgE, a monoclonal anti-IgE (Hybritech, San Diego, CA) and a polyclonal anti-IgE were used. The polyclonal antiserum was prepared by immunizing rabbits with purified IgE PS, and the antiserum was rendered monospecific for IgE by passage over IgG, IgA and IgM immunoabsorbent columns (Affigel-10, Bio-Rad, Richmond, CA). A rabbit anti-mouse Ig antiserum and a goat anti-rabbit Ig antiserum have been prepared in our laboratory. Their IgG fractions were isolated by column chromatography using trisacryl G F 05 and DEAE trisacryl (lndustrie Biologique Fran~aise, Genevilliers, France) (Peterson, 1970). Human IgG, IgA and IgM have been purified from myeloma or Waldestr6m sera using ion exchange chromatography according to standard procedures (Williams and Chase, 1967). Cells WIL2WT (an EBV lymphoblastoid cell line), Daudi (a Burkitt lymphoma), Molt 4 (a T cell leukemia), HE60 (a promyelomonocytic leukemia) and U937 (a promonocytic cell line) were obtained from the ATCC (Rockville, MD). RPMI 8866 (EBV lymphoblastoid cell line) was kindly provided by Dr. K. Ishizaka. UD38 is an EBV lymphoblastoid cell line which has been established in our laboratory by Dr. F. Rousset after infection of normal peripheral blood B cells with EBV. All cells were cultured in RPMI 1640 (Flow, Irvine, Scotland) supplemented with 10% heat-in-
activated fetal calf serum (Flow), 2 mM glutamine (Flow), 100 U / m l penicillin and 100 /~g/ml streptomycin (Flow).
Coupling of proteins to microspheres 0.1 mg of the protein to be coupled was dissolved in 1 ml of a solution of EDAC (1-ethyl-3(3-dimethylaminopropyi)carbodiimide: 0.1 m g / m l distilled water). 100 /tl sonicated (1 min in a Bransonic 321) microspheres (Fluoresbrite carboxylate diameter 0.57 /~m Polyscience, Warrington, PA, ref. 15700) were added and the suspension incubated overnight at 4°C on a rocking table. After this incubation, the microspheres were spun down (10000 x g, 5 min) and washed 3 times with PBS-BSA (1%). The microspheres were stored at 4°C in PBS-BSA supplemented with 15 mM sodium azide. The suspension was sonicated (1 min) before each experiment. Staining protocol Two detection systems (direct and indirect) have been developed. In both assays the cells were adjusted to 107/ml in PBS-BSA and 5 x l0 s cells (50 #1) were added to each well of a 96-conical well microtiter plate (Flow, ref. no.: 7632105). (a) Direct assay. In the direct assay, sonicated IgE beads were added to the cells (50/~1 of different dilutions: 1/5 1/1000 per well) and incubated for 45 min. PBS-BSA (100 /~l/well) was added and the plate centrifuged for 7 rain at 150 x g. The supernatant was discarded and the cells resuspended in PBS-BSA (100/~l/well). This cell suspension was layered on top of 3 ml heat-inactivated filtered FCS in a conical tube (Falcon ref. 2099). After centrifugation at 100 x g for 15 min, the supernatant was slowly aspirated and PBS-BSA (150 /~l/well) added to the cell samples which, after resuspension, were analyzed by flow cytometry. All steps were carried out at 4°C. (b) Indirect assay. In the indirect assay, the cells were first incubated 45 min with monomeric IgE (0.5-2 /~g per 5 x l0 s cells) and then washed with PBS-BSA. A monoclonal anti-IgE (Hybritech, San Diego, CA) at a dilution of 1 / 5 0 - 1 / 1 6 0 or our polyclonal anti-IgE antibody at a dilution of 1 / 1 0 - 1 / 1 0 0 0 0 was then added. After 45 min, the cells were washed with PBS-BSA and the anti-mouse Ig microspheres (dilution
27 1 / 1 0 - 1 / 4 0 ) or the anti-rabbit Ig microspheres (dilution 1 / 1 0 - 1 / 8 0 ) added. The suspensions were finally processed as described above. All steps were carried out at 4°C. The assay using the monoclonal anti-IgE antibody will subsequently be referred to as the 'mouse indirect assay' while the assay using the polyclonal anti-IgE antibody will be referred to as the 'rabbit indirect assay'.
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Production, purification and assay of human IgE binding factors
Fluorescence analysis Fluorescence analysis was performed with a FACS 440 (Becton Dickinson, Sunnyvale, CA) equipped with a 5 W argon laser running at 488 nm, 0.5 W. Fluorescence parameters were collected using a built in logarithmic amplifier after gating on the combination of forward light scatter (FLS) and perpendicular light scatter (PLS) which was used to discriminate viable cells from non-viable ones. Data were stored in list mode and analyzed with a PDP 11/23 using the LYSYS program developed by R. Lefebvre.
Results
(A ) 'Direct assay For the detection of Fc~ receptors, increasing dilutions of IgE coupled microspheres were added to R P M I 8866 cells. Fig. 1A shows that maximum
................
30
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Culture supernatants of RPMI 8866 cells were used as a source of IgE BF as described by Sarfati et al. (1984). In the microtiter wells, 100/~1 of the supernatant were incubated with 0.5-1 /~g IgE for 1 h at room temperature and subsequently for 1 h at 4°C. 5 × 105 cells in 50/~1 were then added and all steps were performed as described above in the indirect assay. An IgE BF purified by affinity chromatography was also assayed. For this, human IgE was coupled to Affigel 10 according to the manufacturer's instructions and RPMI 8866 culture supernatant was passed overnight over the column. After washing with PBS the IgE bound fraction (which will be referred to as purified IgE BF) was eluted with 0.2 M glycine-HC1 buffer pH 2.8. Immediately after elution the pH was adjusted to 7.2 with phosphate buffer.
A
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150
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Fig. 1. Direct assay. A: Effect of diluting microspheres with IgE coats on their binding to RPMI 8866 cells. The assay was performed as described in the materials and methods section using IgE beads incubated with RPMI 8866 ceils. The negative control using microspheres coated with an unrelated Ig was subtracted (8%). B: FACS histogram. The control (dark shading) using microspheres coated with a non-related Ig overlaps with the histogram obtained after incubation of RPMI 8866 cells with IgE beads diluted 1/50. The bar represents the area defining positive cells (63%).
binding was obtained at dilutions lower than 1/20. Fig. 1B shows the FACS histogram of RPMI 8866 cells reacted with IgE beads. The separation between positive and negative cells was difficult to establish and the fluorescence of positive cells was relatively low due to binding of small amounts of microspheres. This system was found to be isotype specific: preincubation of the cells with IgG, IgA and IgM did not decrease the percentage of microsphere binding cells while preincubation with 1/~g IgE dropped the number of positive cells from 58% to 25% (data not shown). Since weak fluorescence of the positive cells could be a source of
28
difficulty an indirect assay was developed in order to increase the fluorescence intensity.
(B) Indirect assay In the indirect assay RPMI 8866 cells were successively incubated with the following reagents: human monomeric IgE; monoclonal or polyclonal anti-IgE antibodies; anti-mouse or anti-rabbit Ig coupled to fluorescent beads (Fig. 2). In order to optimize the assay, the following parameters were investigated: (i) Concentration of the monoclonal anti-IgE antibody. To determine the optimal concentration of monoclonal anti-IgE antibody, 5 × 105 RPMI 8866 cells were incubated with 1 #g IgE, different concentrations of the monoclonal anti-IgE antibody, and the microspheres at a dilution of 1/10. Optimal fluorescence intensity was obtained using 1 / 5 0 - 1 / 8 0 dilutions of the monoclonal anti-IgE (10 ~ g / m l - 6 ~ g / m l ) (Fig. 3A); in all other assays the Mab was used at a dilution of 1/50. A typical FACS profile is shown in Fig. 3B in which a very clear difference between positive and negative cells can be seen. In this experiment, 95.6% of the cells bound beads whereas controls without IgE displayed only 5% positive cells. (ii) Concentration of the microspheres. The op-
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timal concentration of microspheres was determined as follows: 5 × 105 RPMI 8866 cells were incubated with 1 ~g IgE. Then the anti-IgE Mab was added at a dilution of 1/50 followed by different dilutions of the microsphere suspension. Fig. 3C demonstrates that the percentage of positive cells dropped sharply as the microspheres were diluted beyond 1/10. In all other assays the microsphere suspension was used at a dilution of
1/10. (iii) Concentration of the monomeric lgE. To determine the optimal concentration of IgE, the RPMI 8866 cells were incubated with concentra~ tions of IgE which ranged from 50 ng to 2000 ng. Other reagents were added as determined above. Fig. 3D shows that the maximum number of positive cells was obtained at concentrations of 1-2 ~g lgE. A concentration of 2 ~g of lgE was therefore used in routine assays. 17 experiments performed using optimal conditions gave a mean of 62.2% + 2.9 (SEM) positive cells (after subtraction of controis omitting IgE). (iv) Isotype specificity. In order to demonstrate that the binding of lgE to the RPMI 8866 cells was isotype specific, the effect of IgG, lgA and IgM on IgE binding was studied in 2 different ways: (a) before addition of IgE, RPMI 8866 cells were preincubated with 2 ~g of either IgG, IgA, or IgM. IgE binding was not affected (Fig. 4A); (b) RPMI 8866 cells were incubated with 2 /~g IgE and 10 t~g of either IgG, IgA, or IgM. The binding of IgE was not affected (Fig. 4B). These experiments demonstrate the specificity of the binding of IgE to the RPMI 8866 cells. (v) Assay using a polyclonal anti-IgE antibody.
IgE
Y
Anti IgE
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Mlcrospheres coupled to anti Ig ( mouse or rabbit )
Fig. 2. Indirect assay: schematic representation of the different experimental steps.
In order to screen for anti-FccR Mabs and anti-IgE Mabs inhibiting the binding of IgE we investigated whether the anti-IgE Mab and the anti-mouse Ig microspheres could be replaced by a specific rabbit anti-lgE antibody and anti-rabbit Ig microspheres. Fig. 5 shows that the antibody must be sufficiently diluted to provide an optimal signal since a prozone effect is observed at low dilutions. Routinely we used the polyclonal anti-IgE antibody at a dilution of 1/2000 and the FACS histogram was similar to that obtained with the anti-IgE Mab (data not shown).
(vi) Presence of FccR on different human cell lines. Seven human cell lines were screened for
29
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Fig. 3. Mouse indirect assay. A : Effect of the monoclonal antibody concentration on the binding of microspheres to the RPMI 8866 cells. RPMI 8866 cells were successively incubated with 1 p,g IgE, different concentrations of the monoclonal anti-IgE and anti-mouse Ig coated microspheres (1/10). The negative control obtained by omitting IgE (6%) was subtracted. B: FACS histogram. RPM1 8866 cells were incubated with 2 /.tg IgE, monoclonal anti-IgE (1/50), and anti-mouse Ig coated microspheres (1/10). The dark shaded peak represents the negative control in which IgE was omitted (5%). The bar represents the area defining the positive cells (95.6%). C: Effect of microsphere concentration on binding to RPMI 8866 cells. P,PMI 8866 cells were incubated with 2 ~g IgE, monoclonal anti-IgE (1/50) and different concentrations of anti-mouse Ig coated microspheres. The negative control obtained by omitting lgE (7%) was subtracted. D: Effect of IgE concentration on the binding of microspheres to RPMI 8866 cells. RPMI 8866 cells were incubated with different concentrations of IgE, monoclonal anti-lgE (1/50) and anti-mouse Ig coated microspheres (1/10). The negative control obtained by omitting IgE (6%) was subtracted.
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the presence of Fcc receptors using the mouse indirect assay and 3 different sources of purified IgE myeloma protein. The 3 EBV lymphoblastoid cell lines RPMI 8866, WIL2WT and UD38 were
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Fig. 4. Mouse indirect assay showing lack of interference of IgG, lgA and IgM on the binding of lgE to RPMI 8866 cells. A: RPMI 8866 cells were incubated first with 2 btg IgE, lgG, IgA, IgM, then with 2 /tg lgE. Monoclonal anti-lgE (1/50) and anti-mouse Ig coated microspheres (1/10) were finally added. B: 10 ~g IgG, lgA or IgM were added together with 2 ,ttg lgE to RPMI 8866 cells. Monoclonal anti-lgE (1/50) and antimouse lg coated microspheres (1/10) were finally added. The negative control obtained by omitting lgE (6%) was subtracted.
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Fig. 5. Rabbit indirect assay showing effect of the polyclonal anti-lgE antibody concentration on the binding of microspheres to RPMI 8866 cells. RPMI 8866 cells were incubated with 1 /tg lgE, different concentrations of the polyclonal antilgE antibody, and anti-rabbit Ig coated microspheres (1/20). The negative control obtained by omitting IgE (4%) was sublracted.
found to bear F c( receptors while M O L T 4 (a T cell l y m p h o m a ) . H L 6 0 (a p r o m y e l o m o n o c y t i c cell line), Daudi (a Burkitt l y m p h o m a ) and U937 (a p r o m o n o c y t i c cell line) were negative, A l t h o u g h slight differences were found, the 3 different lg E p r e p a r a t i o n s b o u n d to a similar extent (Table 1). H e a t i n g the IgE to 5 6 ° C for 2 h ( c o n d it io n s k n o w n
TABLE I MOUSE INDIRECT ASSAY: DETECTION OF Fce RECEPTORS ON DIFFERENT HUMAN CELL LINES WITH 3 DIFFERENT lgE MYELOMA PREPARATIONS Each cell line was incubated with 2 ,ttg of each IgE, monoclonal anti-IgE (1/50) and anti-mouse lg coated microspheres (1/10). The negative control obtained by omitting IgE (6%) was subtracted. NT: not tested. Cell lines Molt 4 HL 60 Daudi U 937 RPMI 8866 Wil 2 WT UD 38
% Positive cells lgE PS
IgE Birmingham
IgE Lyon
0 0 0 0 81 58 52
0 0 0 0 72 42 NT
0 0 0 0 55 38 NT
C+
8866 SN IX
PURIFIED IgEBF
Fig. 6. Rabbit indirect assay showing detection of human IgE binding factors. C : negative control obtained by incubation of RPMI 8866 cells with polyclonal anti-lgE antibody (1/1000) and anti-rabbit Ig coated microspheres (1/20). C+: positive control obtained by incubation of RPMI 8866 cells with 1 /~g lgE, polyclonal anti-IgE antibody and anti-rabbit lg coated microspheres. 8866 SN IX: lgE was preincubated with 100 yI RPMI 8866 3 day culture supernatant before addition to RPM1 8866 cells. Purified IgE BF: IgE was preincubated with 50 yl of the eluate from an IgE Affigel column through which an RPMI 8866 3-day culture supernatant was passed.
to alter the Fc fragment) resulted in a decrease in the n u m b e r of cells b i n d i n g IgE from 75% to 25%.
(vii) Detection of human IgE binding factors. T o investigate whether the indirect assay could also be used for the detection of IgE b i n d i n g factors p r o d u c e d by R P M I 8866 cells, IgE was p r e i n c u b a t e d with a 3-day R P M I 8866 culture supernatant. As can be seen in Fig. 6, this prei n c u b a t i o n resulted in a small but significant decrease in the n u m b e r of cells b i n d i n g IgE. The eluate from a R P M I 8866 culture s u p e r n a t a n t passed through IgE-Affigel was found to strongly inhibit IgE b i n d i n g in both the m o u se and the rabbit indirect assay. This indicated that this assay was sensitive enough to detect h u m a n IgE b i n d i n g factors o b t a i n e d f r o m R P M I 8866 cells.
Discussion Th e goal of this study was to develop an assay which would permit the detection of (i) Fc~ receptors on h u m a n ceils, (ii) h u m a n I g E b i n d i n g [ac-
31 tors (IgE BF). Previously the evaluation of human Fc~ receptors and IgE BF has been achieved by rosette assays using IgE. However, such techniques require selected red blood cells that have been coated freshly with IgE. Furthermore the rosettes are relatively unstable and have to be scored microscopically, which is time consuming and laborious and therefore not suitable for large numbers of samples. We have replaced the red blood cells by fluorescent microspheres and examined binding by flow cytometry since it has been claimed that such microsphere permit the detection of receptors for the complement components on human cells (Lambris and Ross, 1982; Ross and Lambris, 1982). We first utilized IgE-coupled microspheres in a direct assay. Initially this test permitted us to detect cells that bound IgE microspheres specifically since microsphere binding was inhibitable by soluble IgE, but not by soluble IgG, IgM or IgA (data not shown). Two main reasons led us to design an indirect assay, (i) the development of a system characterized by higher binding of microspheres to cells thereby permitting better discrimination between positive and negative cells, (ii) availability of a more versatile system which permitted the detection of other Fc receptors (e.g., IgG, IgA) using the same microspheres. Fig. 3B shows a typical FACS histogram using the 3-step assay consisting of incubation of cells with monomeric IgE, monoclonal anti-IgE antibody and anti-mouse Ig microspheres. Positive cells were easily differentiated from negative cells. With the RPMI 8866 cell line we found the number of positive cells to vary between 45 and 85%. Such a variation has been described by others using the standard rosette assay. Although several hypotheses have been put forward to explain this variability (e.g., cell density or differential FccR expression during cell cycle) the reasons remain unclear. Controls omitting either IgE alone or IgE plus anti-IgE antibody resulted is 3-8% positive cells with low fluorescence intensity. Replacing the anti-IgE Mab anti-mouse Ig microspheres with anti-IgE polyclonal antibody anti-rabbit Ig microspheres yielded the same FACS histogram provided the polyclonal antibody was sufficiently diluted to avoid the prozone effect. This rabbit indirect assay was used to screen anti-RPMI 8866 Mabs inhibiting IgE binding or anti-IgE Mabs
inhibiting binding of IgE to FccR. The assay was found to be isotype specific since IgG, IgA, and IgM in excess did not alter the binding of IgE. Using this assay we found that out of 7 cell lines tested only the 3 EBV lymphoblastoid cell lines (RPMI 8866, WIL2WT and UD 38) had receptors for IgE. This correlates with a recent observation of Sarfati et al. (1984). The significance of this finding is currently under investigation in our laboratory. Our negative result with U937 is at variance with other reports (Narey-Fejes-Toth and Guyre, 1984) and could be attributed to the use of a particular subclone of the U937 cell line. The detection of Fcc receptors using fluorescent microspheres offers the/ following advantages: (i) it is not dependent on the quality of the microspheres and a 10 ml bottle of such microspheres can last for years; (ii) the micromethod permits the use of small amounts of reagent; (iii) large numbers of samples (100-150 per day) can easily be handled due to use of microplates and FACS detection; (iv) all the reagents are extremely stable, coupled beads need only be prepared every second month and there is minimum loss of the precious IgE during coupling procedures; (v) the indirect assay can very easily be modified to detect other Fc receptors. For example, the Fc~,R on Daudi cells could be easily monitored using IgG and anti-IgG antibody. Preincubation of monomeric soluble IgE with IgE BF purified from RPMI 8866 culture supernatants resulted in a drastic decrease in its binding to cells (Fig. 6). Preliminary data also showed that this assay permits the detection of IgE BF in the supernatant of an IgE pulsed human TT hybridoma. The availability of this relatively simple and rapid assay for screening human IgE BF should prove useful in the cloning of the gene for this molecule. It should also prove useful in the study of human T cells secreting these factors. Since this study has been conducted, 2 new techniques have been described for detecting Fcc receptors: (i) Kanowith-Klein and Saxon (1985) have shown that IgE immune complexes permit the detection of FccR on human cells using a cytochemical procedure, and (ii) Narey-Fejes-Toth and Guyre (1984) have demonstrated the binding of FITC-labeled IgE to U937 cells using flow cytometry.
32
Acknowledgements We thank Dr. F. Rousset for providing the UD 38 cell line, Sophie Lorkens and Muriel Vatan for typing the manuscript and Dr. J.E. De Vries for carefully reviewing this manuscript.
References Coico, R.F., B. Xue, D. Wallace, B. Pernis, G.W. Siskind and G.J. Thorbecke, 1985, Nature (London) 316, 744. Deguchi, H., M. Suemura, A. Ishizaka, Y. Osaki, S. Kishimoto, Y. Yamamura and T. Kishimoto, 1983, J. Immunol. 131, 2751. Gonzalez-Molina, A. and H.L. Spiegelberg, 1977, J. Clin. Invest. 59, 616. Gupta, S., C.D. Platsoucas, R.S. Schulof and R.A. Good, 1979, Cell. lmmunol. 45,469. Ishizaka, K., 1985, J. Immuno|. 135, 1. Kanowith-Klein, S. and A. Saxon, 1985, J. Clin. lmmunol. 5, 38.
Katz, D.H., 1984, Allergy 39, 81. Lambris, J.D. and G.D. Ross, 1982, J. Immunol. 128, 186. Leung, D.Y.M. and R.S. Geha, 1984, Clin. Immunol. Rev. 3, 1. Lum, L.G., A:V. Muchmore, D. Keren, J. Decker, I. Koski, W. Strober and R.M. Blaese, 1979, J. Immunol. 122, 105. Moretta, L., M. Ferrarini, M.L. Durante and M.C. Mingari, 1975, Eur. J. Immunol. 5, 565. Narey-Fejes-Toth, A. and P.M. Guyre, 1984, J. lmmunol. 133, 1914. Peterson, E.A., 1970, in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 2, eds. T,S. Work and E. Work (North Holland/Elsevier, Amsterdam) p. 228. Ross, G.D. and J.D. Lambris, 1982, J. Exp. Med. 155, 96. Sarfati, M., E. Rector, M. Rubio-Trujillo, K. Wong, A.H. Sehon and G. Delespesse, 1984, Immunology 53, 207. SjOberg, O., 1980, Scand. J. Immunol. 11,377. Spiegelberg, H.L., 1981, lmmunol. Rev. 56, 199. Williams, C.A. and M.W. Chase, 1967, Methods in Immunology and Immunochemistry, Vol. 1 (Academic Press. New York) p. 321. Yodoi, J. and K. Ishizaka, 1979, J. lmmunol. 122, 2577.