Journal oflrnmunological Methods, 30 (1979) 161--170
161
© Elsevier/North-Holland Biomedical Press
PARTICLE-LABELED ANTIBODIES. I. ANTI T-CELL ANTIBODIES ATTACHED TO PLASTIC BEADS BY POLY-L-LYSINE
J. GABRILOVAC, K. PACHMANN, H. RODT, G. JAGER and S. THIERFELDER
Institut fi~r Ha'matologie, GSF, Munich, F.R.G. (Received 15 May 1979, accepted 1 June 1979)
A simple and inexpensive method for the detection of T-ceU surface antigens is introduced using polyacrylic plastic beads (PAA-beads) as indicator ~articles. The beads are easily visible in ordinary light microscope and make the method convenient for routine laboratory use, also in laboratories possessing no special equipment. The method is an indirect test using a first, absorbed rabbit anti-T serum to sensitize the cells and a second, sandwich antiserum (goat anti-rabbit Ig) coupled to the indicator beads. Instead of glutaraldehyde used by other investigators to attach antibodies to the beads, we used poly-L-lysine which did not affect antibody titers. The F(ab)2 fragment of the sandwich goat anti-rabbit Ig antibody was employed to avoid binding of antibody-coated beads to Fc-receptor-bearing cells. Sensitivity, specificity and reproducibility of the method were tested on murine and human lymphoid cells treated with the respective anti-T serum. The data obtained show that the sensitivity of the method depends on: (a) the bead concentration, (b) the concentration of F(ab)2 fragment of the second antiserum used to coat the beads, and (c) the concentration of specific antibody used to sensitize the cells. Specificity and reproducibility of the method were found to be good. The percentage of positive cells with this method are in good agreement with those reported by means of well established immunological methods. INTRODUCTION
There are various techniques for labeling anti-T-lymphocyte antibodies, conjugation with fluorescent dyes (Raff, 1970), radioiodination(Thiel et al., 1975; Rodt et al., 1975) and labeling with peroxidase-anti-peroxidase complex (Hoffmann-Fezer et al., 1976 and 1978) have been used. These methods are relatively complicated or require expensive equipment. Polymeric plastic microspheres have been widely used in various immunological methods as immunoadsorbents or in methods based on agglutination (Malin and Edwards., 1972; Cambiaso et al., 1977). The use of polymeric plastic beads of small size to determine surface antigens on lymphoid cells has been described by Molday et al. (1974). These authors have found that the plastic beads are suitable reagents to visualize the distribution of surface antigens in scanning electron microscopy (Molday et al., 1974 and 1975). The same group of authors (Molday et al., 1977) reported later that plastic beads can serve to detect surface antigens on mouse lymphocytes in transmitted-light microscopy.
162 The indirect bead m e t h o d described by Gordon et al. (1977b) showed, however, reduced sensitivity in comparison with other established methods, especially in the detection of the theta alloantigen. The glutaraldehyde used by these authors to attach the antibodies onto the beads may diminish antib o d y activity (Avrameas et al., 1969). Since Gordon et al. (1977a, b) used whole antibody molecules, positive reaction to Fc-receptor binding cannot be excluded. In order to improve sensitivity and specificity of the bead m e t h o d we have introduced the following modifications: (a) use of poly-L-lysine for binding antibodies onto the plastic particles, (b) use of F(ab)2 fragments of sandwich antiserum to avoid non-specific binding to Fc-receptors. Specificity, sensitivity, and reproducibility of the m e t h o d were tested on murine and human lymphoid cells, using anti-T sera. The test conditions were optimized and data obtained are comparable to established immunological methods. MATERIALS AND METHODS
Polyacrylamide plastic beads (PAA-beads) PAA-beads purchased from Bofors, Nobelkrut (Sweden, Batch 750 526) were used as a carrier of the sandwich anti-antibodies. An 11% suspension of beads in distilled water served as stock solution. Large beads were separated from the relatively small ones b y centrifugation in the following way: 1.0 ml of 11% bead suspension was centrifuged at 3000 × g for 30 sec. The supernatant was removed and centrifuged for 5 min at 3000 X g. Beads from the sediment, mostly 0.2--0.8 pm in size (Jondal, 1974), were employed for coating with anti-antibodies. A n tisera Rabbit antisera against mouse or human T-lymphocytes were produced in this Institute ( R o d t et al., 1974, 1975). They were rendered T cell-specific by repeated absorptions with liver homogenate and B cells from various sources. Thereafter they were purified over DEAE-cellulose and reconcentrated at a protein concentration of 10 mg/ml. Cytotoxic, immunofluorescent and complement-fixing titers varied between 1 : 6 4 and 1 : 2 5 6 . F(ab)2 fragment of goat anti-rabbit antiserum, employed for coating PAA-beads, was prepared by peptic digestion according to the m e t h o d of Nisonoff et al. (1960). Coating o f PAA-beads PAA-beads were in the first step coated with poly-L-lysine (PLL, M.W. 200,000, Sigma, Chemical Co., St. Louis, MO., U.S.A.) which enables attachm e n t of the antibodies. PAA-beads, obtained from 1.0 ml of stock solution after two centrifugations (see above) were incubated with 400 pl of PLL solution in distilled water (100 pg/ml) for 1 h at room temperature, followed by three washings in NaC1 solution (5 min at 3000 X g). The PLL-
163
coated beads were further incubated with 100 pl of goat anti-rabbit IgF(ab)2 (= F(ab)2GaR). The u n b o u n d antibody was removed by three washings as above. Eventually free PLL-binding sites were saturated with an unrelated protein--bovine serum albumin (BSA) as follows: the antibody-coated beads were incubated with 400 gl of 25% solution of BSA in distilled water for 1 h at room temperature. After three washings the coated beads were resuspended in NaC1 solution in a final volume of 700 pl. This was regarded as the standard concentration.
Cells Male mice, 2--3 months old, from a C3H inbred strain were used as a source of thymus, lymph node and spleen cells. When spleen cells were tested, erythrocytes were lysed with 0.83% a m m o n i u m chloride. Human peripheral blood lymphocytes (PBL) were obtained from peripheral blood of normal persons by means of separation on a Ficoll gradient (BCyum, 1968). Thymus cells were obtained from the t h y m u s of a child undergoing cardiac surgery. Erythrocytes were removed by centrifugation over Ficoll (BCyum, 1968), and small quantities of cells were frozen at --80°C in DMSO. The cells were thawed immediately before use. Cells of the MOLT-4 T-cell line, originally established by Minowada et al. (1972), were cultured in this Institute. The 1301-TK B-cell line cells were donated by Dr. R. Reisfeld, Scripps Clinic, La Jolla, CA, and further cultured in this Institute. PAA-beads assay To 10 gl of cell suspension containing 1.0 X l06 cells, 10 pl of anti-T serum in the appropriate dilution were added. The mixture was incubated for 30 min at 4°C and washed twice (10 min at 1400 X g). The supernatant was removed and 50 pl of coated beads were added to the cell sediment. To allow close contact between cells and beads the mixture was shortly centrifuged (30 sec at 3000 X g) and subsequently incubated for 30 min at 4°C. One drop of the suspension was mixed with one drop of 0.05% trypan-blue, layered onto a slide pretreated with a cover slide and immediately estimated under the light microscope. Cells binding a t least five beads were considered positive. Only living cells were counted. RESULTS
Titration o f PAA-beads The optimal bead concentration was determined at which specific attachment of antibody-labeled beads to cells was still high, w i t h o u t non-specific binding of beads to cells coated with normal rabbit serum (NRS). For this purpose PBL were treated with anti-T serum (diluted 1 : 6, a dilution found to give optimal results in the immunofluorescence test), or with normal rabbit serum of the same protein concentration, and subsequently mixed
164
100
50
O_
o~ g
~,
5
relative
1
o.5
0.2
o.t
t 0.05
bead c o n c e n t r a t i o n
Fig. 1. Peripheral blood lymphocytes (PBL) from a healthy donor, sensitized with rabbit anti-T serum (#) or with normal rabbit serum (0) and subsequently mixed with various concentrations of goat anti-rabbit-Ig coated beads. Cells with over 5 beads were recorded as positive. Bead concentration expressed as the factor of initial concentration obtained from 1.0 ml of 11% stock solution (see Materials and Methods). with coated beads of various concentrations. Since the optimal concentration o f the F(ab)2GaR on the beads had n o t y e t been determined, the beads used in this e x p e r i m e n t were coated with a c o m m o n l y used c o n c e n t r a t i o n of a n t i b o d y , i.e. 10.0 mg/ml. A plateau was obtained with concent rat i ons up to 8-fold the standard c onc e nt r at i on, mixed with specifically sensitized cells (Fig. 1). Nonspecific binding was low (less than 10% o f cells) regardless o f t h e bead co n cen t r at i on. F o r f u r t h e r work we chose the lowest c o n c e n t r a t i o n o f beads still yielding plateau values. At this c o n c e n t r a t i o n the background o f free floating beads was also minimal and thus provided easier and m o r e precise estimations of positive cells.
Titration o f F(ab )2GaR In the h o p e of improving the binding of beads to a n t i b o d y - c o a t e d cells, the beads were labeled with increasing am ou nt s of F(ab)2GaR. Various concentrations o f antibodies (F(ab)2GaR) f r om 0.3 mg/ml upwards were used. T h e dilution o f anti-T used in these experiments was again the same as t h a t e m p l o y e d in the i m m u n o f l u o r e s c e n c e test. The results obtained showed t h a t t h e highest percentage o f murine T cells (Fig. 2a) was obtained using F(ab)2GaR in a c o n c e n t r a t i o n of 4.5--18.0 mg/ml. Higher concent rat i ons o f F(ab)2GaR resulted in lower percentages o f positive cells. The range of F(ab)2GaR c o n c e n t r a t i o n , detecting the highest and const ant percentage o f h u m a n T-cells (Fig. 2b) was practically the same. In all f u r t h e r experiments beads were co ated with 10.0 mg/ml F(ab)2GaR. Titration o f rabbit anti-mouse and human T sera In order to check w h e t h e r the sensitivity a n d / o r specificity of the test
165 100
®
50
o.
04 72
36
18
9
4.5
2.3
1.2
100
0.6
@
50
o~ 36
18
concentration
9
4.5
2.3
1.2
0.6
Q3
of F(ab)2 G a R ( m g / m l )
Fig. 2. Beads coated with various concentrations of F(ab)2GaR used to determine percentage of (a) murine and (b) human T cells, a: Murine cells from thymus (e) and from lymph node (A) presensitized with anti-T serum diluted 1 : 16 (closed symbols), or with normal rabbit serum of the same protein concentration (open s y m b o l s ) - thymus cells (o), lymph node cells (A). b: Human cells: peripheral blood lymphocytes (@), MOLT-4 (~), CLL (o) and 1301-TK (~) previously sensitized with anti-T serum, diluted 1:6.
could be improved by using higher or lower concentrations of the first antisera (anti-T), the cells were sensitized with various concentrations of the appropriate antiserum and subsequently mixed with beads as described in previous sections. As shown in Fig. 3a, a critical minimal concentration of antiserum is necessary on the cells in order to obtain optimal binding of beads. Dilutions of 1 : 8 of the rabbit anti-mouse-T serum for spleen cells and 1 : 16 for t h y m u s and lymph node cells (Fig. 3a) always gave plateau values. Using lower concentrations of anti-0 serum resulted in the detection of lower percentages of positive cells. Similar results were obtained with h u m a n cells (Fig. 3b). Binding of beads to cells of the B-cell line 1301-TK and to the CLL-cells was relatively low (8% and 15%, respectively) and was
166
100
03
50
+ o
Q.
o~
0
1
2
3
4
5
6
100
7
8
(~)
;,
0
1
2
3
4
5
6
7
8
serum dilut ion (.+l/log 2.~
Fig. 3. Various concentrations of anti-human and rabbit anti-mouse anti-T sera employed to sensitize (a) murine and (b) h u m a n T-cells. Sensitized cells were subsequently mixed with beads coated with F(ab)2GaR, concentration, 10.0 mg/ml, a: Murine cells, from thymus (e), lymph nodes (4) and spleen (m) sensitized with anti-T serum; spleen cells sensitized with NRS (~). b: Human cells, from thymus (o), PBL (0), MOLT (*), 1301 (~')and CLL (o).
n o t related to the c o n c e n t r a t i o n of anti-T serum in the same range o f dilutions (i.e. up to 1 : 16). The same was true for the mouse spleen cells coat ed with NRS (Fig. 3a).
Reproducibility of the method (a) Reproducibility of coating. In order to estimate the reproducibility of t h e bead preparation, h u m a n PBL and mouse t h y m o c y t e s were tested with 7 d if f er en t bead preparations. The bead preparations were never older than one week and were stored at 4°C until use; data showed rather good reproducibility (Table 1).
(b) Stability of the coated bead preparations stored at 4°C and --80°C. F u r t h e r m o r e , we wanted to det er m i ne how long the coated beads were stable in detecting a cons t a nt percentage of positive cells, and w h e t h e r the
167 TABLE 1 R E P R O D U C I B I L I T Y O F T H E R E S U L T S O B T A I N E D WITH D I F F E R E N T B E A D PREPARATIONS Bead preparations a
1 2 3 4 5 6 7 Mean S.D.
P e r c e n t a g e o f positive cells Human PBL
Murine thymocytes
71 64 76 48 76 64 62 65.6 -+ 9.8
82 74 71 76 86 80 84 79.9 -+ 5.6
a D i f f e r e n t b e a d p r e p a r a t i o n s were e m p l o y e d f o r each test. T h e s e were n e v e r o l d e r t h a n o n e week, a n d were s t o r e d a t 4°C u n t i l use.
temperature at which the bead preparations were stored influenced their stability. For this purpose, two different bead preparations were made. Onehalf of each bead preparation was stored at 4°C and the other half at --80°C, until use. Mouse thymus cells were tested at different time intervals with both bead preparations. The percentage of T-cells detected with beads stored at 4°C, declined with the 'age' of the beads. The data obtained using beads stored at --80°C showed that the coated beads remained stable, detecting a more or less constant percentage of positive cells. The percentages of positive cells detected using t w o different bead preparations stored at the same temperature and of approximately the same 'age', did not differ significantly (Table 2). TABLE 2 S T A B I L I T Y O F B E A D P R E P A R A T I O N S W H E N S T O R E D A T 4°C A N D A T - - 8 0 ° C Bead preparations
Temp. of storage
P e r c e n t a g e s o f positive cells a o b t a i n e d w i t h b e a d p r e p a r a t i o n s o f d i f f e r e n t age (days) 1
1 2
4°C 4°C
80
1 2
--80°C --80°C
82
2
5
6
NT b 86
9
NT NT
82 86
8
17
69 NT
78 76
16
21
58 70
78 78
20
68 78
85
78
a T h y m u s - c e l l s f r o m C3H m i c e were t e s t e d at d i f f e r e n t intervals w i t h t w o b e a d preparat i o n s s t o r e d at 4°C a n d - - 8 0 ° C . b N T = n o t tested.
168 DISCUSSION In the present report we describe an inexpensive particle-labeling m e t h o d for the demonstration and evaluation of surface antigens. This m e t h o d does n o t require any special equipment since the beads used as markers are visible b y ordinary light microscopy. Under correct storage conditions, the bead preparation has shown high stability over a long time period. This makes the PAA-bead method a convenient assay for routine laboratory use. The use of plastic beads as carriers of antibody or anti-antibody to determine surface antigens on lymphoid cells has been described by two groups of authors (Gordon et al., 1977a, b; Ammann et al., 1977), using beads covalently attached to antibodies by means of glutaraldehyde or carbodiimide, respectively. We have employed PAA-beads and poly-L-lysine, found by Mazia et al. (1975) to cause attachment of proteins to plastic surfaces, for binding the antibodies onto the beads. The polycationic poly-L-lysine adheres strongly to plastic beads, leaving free cationic sites which can then be combined with anionic sites on the proteins. It is also convenient for attaching antigens to polymeric plastic beads (Pachmann and Leibold, 1976). In comparison to the use of glutaraldehyde to attach antibodies onto plastic beads, the application of poly-L-lysine has some advantages: the procedure is very easy and rapid to perform and the possible partial inactivation of antibodies caused by glutaraldehyde (Avrameas et al., 1969) is avoided. Non-specific binding of beads to the cells through unsaturated PLLsites was effectively prevented by incubating the beads with BSA at high concentrations. Control experiments, in which the beads were coated with PLL only and subsequently with BSA (without antibodies), have shown that beads coated in this way have practically no affinity for the cells (less than 5% binding). In contrast to Gordon et al. (1977a, b) and Amman et al. (1977) who coated their beads with whole antibody molecules, we used the F(ab)2 fragment of goat anti-rabbit globulin which considerably reduced the nonspecific binding to B-cells, probably via the Fc-receptor. The sensitivity of the method depends on the concentration of coated beads mixed with the cells. A critical minimal concentration of beads is necessary in order to obtain optimal sensitivity. Further increase of the concentration up to 8-fold does not influence the results (see the long plateau in Fig. 1). As the method described is an indirect test it depends also on the concentration of both the specific antiserum used to sensitize the cells, and of the anti-antiserum (goat anti-rabbit) used to coat the beads. The dilution curve of F(ab)2GaR had a maximum with supraoptimal concentrations resulting in lower sensitivity which could be explained as a prozone effect. When the cells were sensitized with various concentrations of anti-mouse or human T serum, a rather long plateau was obtained with human thymus cells and PBL,
169
as well as with murine thymus and lymph node cells. A significantly shorter plateau (two dilution steps) was obtained with mouse spleen cells. This probably reflects the lower concentration of T-antigen on spleen cells (Raft, 1971). Further experiments have shown that the method can distinguish between cells with high and low antigen concentration, as fewer beads bind to murine T-cells from the spleen than from the thymus (manuscript in preparation). The percentage of positive cells that we detected among MOLT-4 cells was relatively low but in accordance with immunofluorescence testing (result n o t shown). The low percentage of positive MOLT-4 cells, and the shorter plateaus obtained both with various concentrations of F(ab)2GaR and anti-T, could be due to the fact that the cells were in different stages of maturation and therefore did n o t carry equal amounts of surface T-antigen. In general, the m e t h o d presented here determines in murine lymphoid tissues, as well as in human PBL, percentages of T-cells which are in good agreement with the data obtained by other authors by means of other immunological methods (Raff, 1971; Jondal et al., 1973; R o d t et al., 1975). Non-specific binding of beads to the cells coated with NRS was low (up to 10%) and was independent, over a wide range, of the amount of antibodies used to coat the beads. The low percentage of positive cells in the B-cell line 1301-TK as well as in CLL (8% and 15%) did n o t depend on the concentration of goat anti-rabbit antibodies. Theoretically, binding of anti-T antibodies used as whole molecules toFc-receptors may occur. However, control experiments showed that the percentage of positive cells determined could n o t be decreased when the Fc-receptors were blocked by aggregated IgG (data n o t shown). Furthermore, the fact that the percentage of positive CLLand 1301-TK cells was practically independent of the concentration of anti-T serum argues against the binding through Fc-receptors. The reproducibility of the method was found to be good over a period of at least three weeks, when the bead preparations were stored at --80°C. This stability of the coated beads is a further advantage of the method, since it enables coating of larger amounts of beads ready for use. We have described here a simple and sensitive method using PAA-beads. Compared to others using plastic beads, we detected higher percentages of T-antigen bearing cells and low non-specific binding through Fc-receptors. This may be due to either (a) the use of the F(ab)2 fragment as sandwich antibody instead of the whole antibody, or (b) the higher amount of functionally active antibodies coupled to the beads by means of poly-L-lysine, or both. REFERENCES Ammann, A.J., D. Borg, L. K o n d o and D.W. Wara, 1977, J. Immunol. Methods 17, 365. Avrameas, S., B. Taudou and S. Chuilon, 1969, Immunochemistry 6, 67. B~yum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 31. Carnbiaso, C.L., A.E. Leek, F . de Steenwinkel, J. Billin and P.L. Masson, 1977, J. Immunol. Methods 18, 33.
170 Gordon, I.L., W.J. Dreyer, S.P.S. Yen and A. Rembaum, 1977a, Clin. Immunol. Immunopath. 8, 51. Gordon, I.L., W.J. Dreyer, S.P.S. Yen and A. Rembaum, 1977b, Cell. Immunol. 28, 307. Hoffmann-Fezer, G., H. Rodt, M. Eulitz and S. Thierfelder, 1976, J. Immunol. Methods 13, 261. Hoffmann-Fezer, G., K. Pielsticker, H. Rodt and S. Thierfelder, 1978, Verh. Dtsch. Ges. Path. 62, 371. Jondal, M., H. Wigzell and F. Aiuti, 1973, Transplant. Rev. 16, 163. Jondal, M., 1974, Scand. J. Immunol. 3, 269. Malin, S. and J. Edwards, 1972, Nature 235, 182. Mazia, D., G. Schatten and W. Sale, 1975, J. Cell. Biol. 66, 198. Minowada, J., T. Ahnuma and G.E. Moor, 1972, J. Nat. Cancer Inst. 49, 891. Molday, R.S., W.J. Dreyer, A. Rembaum and S.P.S. Yen, 1974, Nature 249, 81. Molday, R.S., W.J. Dreyer, A. Rembaum and S.P.S. Yen, 1975, J. Cell. Biol. 64, 75. Molday, R.S., S.P.S. Yen and A. Rembaum, 1977, Nature 268, 43. Nisonoff, A., F.C. Wissler, L.N. Lipman and D.L. Woernley, 1960, Arch. Biophys. 89, 230. Pachmann, K. and W. Leibold, 1976, J. Immunol. Methods 12, 89. Raft, M.C., 1970, Immunology 19, 637. Raft, M.C., 1971, in: Cell Interactions and Receptor Antibodies in Immune Responses, eds. O. M~kel~, A. Cross and T.V. Kosunen (Academic Press, New York) p. 83. Rodt, H., S. Thierfelder and M. Eulitz, 1974, Europ. J. Immunol. 4, 25. Rodt H., S. Thierfelder, E. Thiel, D. GStze, B. Netzel and M. Eulitz, 1975, Immunogenetics 2,411. Thiel, E., P. DSrmer and S. Thierfelder, 1975, J. Immunol. Methods 6, 317.
161
© Elsevier/North-Holland Biomedical Press
PARTICLE-LABELED ANTIBODIES. I. ANTI T-CELL ANTIBODIES ATTACHED TO PLASTIC BEADS BY POLY-L-LYSINE
J. GABRILOVAC, K. PACHMANN, H. RODT, G. JAGER and S. THIERFELDER
Institut fi~r Ha'matologie, GSF, Munich, F.R.G. (Received 15 May 1979, accepted 1 June 1979)
A simple and inexpensive method for the detection of T-ceU surface antigens is introduced using polyacrylic plastic beads (PAA-beads) as indicator ~articles. The beads are easily visible in ordinary light microscope and make the method convenient for routine laboratory use, also in laboratories possessing no special equipment. The method is an indirect test using a first, absorbed rabbit anti-T serum to sensitize the cells and a second, sandwich antiserum (goat anti-rabbit Ig) coupled to the indicator beads. Instead of glutaraldehyde used by other investigators to attach antibodies to the beads, we used poly-L-lysine which did not affect antibody titers. The F(ab)2 fragment of the sandwich goat anti-rabbit Ig antibody was employed to avoid binding of antibody-coated beads to Fc-receptor-bearing cells. Sensitivity, specificity and reproducibility of the method were tested on murine and human lymphoid cells treated with the respective anti-T serum. The data obtained show that the sensitivity of the method depends on: (a) the bead concentration, (b) the concentration of F(ab)2 fragment of the second antiserum used to coat the beads, and (c) the concentration of specific antibody used to sensitize the cells. Specificity and reproducibility of the method were found to be good. The percentage of positive cells with this method are in good agreement with those reported by means of well established immunological methods. INTRODUCTION
There are various techniques for labeling anti-T-lymphocyte antibodies, conjugation with fluorescent dyes (Raff, 1970), radioiodination(Thiel et al., 1975; Rodt et al., 1975) and labeling with peroxidase-anti-peroxidase complex (Hoffmann-Fezer et al., 1976 and 1978) have been used. These methods are relatively complicated or require expensive equipment. Polymeric plastic microspheres have been widely used in various immunological methods as immunoadsorbents or in methods based on agglutination (Malin and Edwards., 1972; Cambiaso et al., 1977). The use of polymeric plastic beads of small size to determine surface antigens on lymphoid cells has been described by Molday et al. (1974). These authors have found that the plastic beads are suitable reagents to visualize the distribution of surface antigens in scanning electron microscopy (Molday et al., 1974 and 1975). The same group of authors (Molday et al., 1977) reported later that plastic beads can serve to detect surface antigens on mouse lymphocytes in transmitted-light microscopy.
162 The indirect bead m e t h o d described by Gordon et al. (1977b) showed, however, reduced sensitivity in comparison with other established methods, especially in the detection of the theta alloantigen. The glutaraldehyde used by these authors to attach the antibodies onto the beads may diminish antib o d y activity (Avrameas et al., 1969). Since Gordon et al. (1977a, b) used whole antibody molecules, positive reaction to Fc-receptor binding cannot be excluded. In order to improve sensitivity and specificity of the bead m e t h o d we have introduced the following modifications: (a) use of poly-L-lysine for binding antibodies onto the plastic particles, (b) use of F(ab)2 fragments of sandwich antiserum to avoid non-specific binding to Fc-receptors. Specificity, sensitivity, and reproducibility of the m e t h o d were tested on murine and human lymphoid cells, using anti-T sera. The test conditions were optimized and data obtained are comparable to established immunological methods. MATERIALS AND METHODS
Polyacrylamide plastic beads (PAA-beads) PAA-beads purchased from Bofors, Nobelkrut (Sweden, Batch 750 526) were used as a carrier of the sandwich anti-antibodies. An 11% suspension of beads in distilled water served as stock solution. Large beads were separated from the relatively small ones b y centrifugation in the following way: 1.0 ml of 11% bead suspension was centrifuged at 3000 × g for 30 sec. The supernatant was removed and centrifuged for 5 min at 3000 X g. Beads from the sediment, mostly 0.2--0.8 pm in size (Jondal, 1974), were employed for coating with anti-antibodies. A n tisera Rabbit antisera against mouse or human T-lymphocytes were produced in this Institute ( R o d t et al., 1974, 1975). They were rendered T cell-specific by repeated absorptions with liver homogenate and B cells from various sources. Thereafter they were purified over DEAE-cellulose and reconcentrated at a protein concentration of 10 mg/ml. Cytotoxic, immunofluorescent and complement-fixing titers varied between 1 : 6 4 and 1 : 2 5 6 . F(ab)2 fragment of goat anti-rabbit antiserum, employed for coating PAA-beads, was prepared by peptic digestion according to the m e t h o d of Nisonoff et al. (1960). Coating o f PAA-beads PAA-beads were in the first step coated with poly-L-lysine (PLL, M.W. 200,000, Sigma, Chemical Co., St. Louis, MO., U.S.A.) which enables attachm e n t of the antibodies. PAA-beads, obtained from 1.0 ml of stock solution after two centrifugations (see above) were incubated with 400 pl of PLL solution in distilled water (100 pg/ml) for 1 h at room temperature, followed by three washings in NaC1 solution (5 min at 3000 X g). The PLL-
163
coated beads were further incubated with 100 pl of goat anti-rabbit IgF(ab)2 (= F(ab)2GaR). The u n b o u n d antibody was removed by three washings as above. Eventually free PLL-binding sites were saturated with an unrelated protein--bovine serum albumin (BSA) as follows: the antibody-coated beads were incubated with 400 gl of 25% solution of BSA in distilled water for 1 h at room temperature. After three washings the coated beads were resuspended in NaC1 solution in a final volume of 700 pl. This was regarded as the standard concentration.
Cells Male mice, 2--3 months old, from a C3H inbred strain were used as a source of thymus, lymph node and spleen cells. When spleen cells were tested, erythrocytes were lysed with 0.83% a m m o n i u m chloride. Human peripheral blood lymphocytes (PBL) were obtained from peripheral blood of normal persons by means of separation on a Ficoll gradient (BCyum, 1968). Thymus cells were obtained from the t h y m u s of a child undergoing cardiac surgery. Erythrocytes were removed by centrifugation over Ficoll (BCyum, 1968), and small quantities of cells were frozen at --80°C in DMSO. The cells were thawed immediately before use. Cells of the MOLT-4 T-cell line, originally established by Minowada et al. (1972), were cultured in this Institute. The 1301-TK B-cell line cells were donated by Dr. R. Reisfeld, Scripps Clinic, La Jolla, CA, and further cultured in this Institute. PAA-beads assay To 10 gl of cell suspension containing 1.0 X l06 cells, 10 pl of anti-T serum in the appropriate dilution were added. The mixture was incubated for 30 min at 4°C and washed twice (10 min at 1400 X g). The supernatant was removed and 50 pl of coated beads were added to the cell sediment. To allow close contact between cells and beads the mixture was shortly centrifuged (30 sec at 3000 X g) and subsequently incubated for 30 min at 4°C. One drop of the suspension was mixed with one drop of 0.05% trypan-blue, layered onto a slide pretreated with a cover slide and immediately estimated under the light microscope. Cells binding a t least five beads were considered positive. Only living cells were counted. RESULTS
Titration o f PAA-beads The optimal bead concentration was determined at which specific attachment of antibody-labeled beads to cells was still high, w i t h o u t non-specific binding of beads to cells coated with normal rabbit serum (NRS). For this purpose PBL were treated with anti-T serum (diluted 1 : 6, a dilution found to give optimal results in the immunofluorescence test), or with normal rabbit serum of the same protein concentration, and subsequently mixed
164
100
50
O_
o~ g
~,
5
relative
1
o.5
0.2
o.t
t 0.05
bead c o n c e n t r a t i o n
Fig. 1. Peripheral blood lymphocytes (PBL) from a healthy donor, sensitized with rabbit anti-T serum (#) or with normal rabbit serum (0) and subsequently mixed with various concentrations of goat anti-rabbit-Ig coated beads. Cells with over 5 beads were recorded as positive. Bead concentration expressed as the factor of initial concentration obtained from 1.0 ml of 11% stock solution (see Materials and Methods). with coated beads of various concentrations. Since the optimal concentration o f the F(ab)2GaR on the beads had n o t y e t been determined, the beads used in this e x p e r i m e n t were coated with a c o m m o n l y used c o n c e n t r a t i o n of a n t i b o d y , i.e. 10.0 mg/ml. A plateau was obtained with concent rat i ons up to 8-fold the standard c onc e nt r at i on, mixed with specifically sensitized cells (Fig. 1). Nonspecific binding was low (less than 10% o f cells) regardless o f t h e bead co n cen t r at i on. F o r f u r t h e r work we chose the lowest c o n c e n t r a t i o n o f beads still yielding plateau values. At this c o n c e n t r a t i o n the background o f free floating beads was also minimal and thus provided easier and m o r e precise estimations of positive cells.
Titration o f F(ab )2GaR In the h o p e of improving the binding of beads to a n t i b o d y - c o a t e d cells, the beads were labeled with increasing am ou nt s of F(ab)2GaR. Various concentrations o f antibodies (F(ab)2GaR) f r om 0.3 mg/ml upwards were used. T h e dilution o f anti-T used in these experiments was again the same as t h a t e m p l o y e d in the i m m u n o f l u o r e s c e n c e test. The results obtained showed t h a t t h e highest percentage o f murine T cells (Fig. 2a) was obtained using F(ab)2GaR in a c o n c e n t r a t i o n of 4.5--18.0 mg/ml. Higher concent rat i ons o f F(ab)2GaR resulted in lower percentages o f positive cells. The range of F(ab)2GaR c o n c e n t r a t i o n , detecting the highest and const ant percentage o f h u m a n T-cells (Fig. 2b) was practically the same. In all f u r t h e r experiments beads were co ated with 10.0 mg/ml F(ab)2GaR. Titration o f rabbit anti-mouse and human T sera In order to check w h e t h e r the sensitivity a n d / o r specificity of the test
165 100
®
50
o.
04 72
36
18
9
4.5
2.3
1.2
100
0.6
@
50
o~ 36
18
concentration
9
4.5
2.3
1.2
0.6
Q3
of F(ab)2 G a R ( m g / m l )
Fig. 2. Beads coated with various concentrations of F(ab)2GaR used to determine percentage of (a) murine and (b) human T cells, a: Murine cells from thymus (e) and from lymph node (A) presensitized with anti-T serum diluted 1 : 16 (closed symbols), or with normal rabbit serum of the same protein concentration (open s y m b o l s ) - thymus cells (o), lymph node cells (A). b: Human cells: peripheral blood lymphocytes (@), MOLT-4 (~), CLL (o) and 1301-TK (~) previously sensitized with anti-T serum, diluted 1:6.
could be improved by using higher or lower concentrations of the first antisera (anti-T), the cells were sensitized with various concentrations of the appropriate antiserum and subsequently mixed with beads as described in previous sections. As shown in Fig. 3a, a critical minimal concentration of antiserum is necessary on the cells in order to obtain optimal binding of beads. Dilutions of 1 : 8 of the rabbit anti-mouse-T serum for spleen cells and 1 : 16 for t h y m u s and lymph node cells (Fig. 3a) always gave plateau values. Using lower concentrations of anti-0 serum resulted in the detection of lower percentages of positive cells. Similar results were obtained with h u m a n cells (Fig. 3b). Binding of beads to cells of the B-cell line 1301-TK and to the CLL-cells was relatively low (8% and 15%, respectively) and was
166
100
03
50
+ o
Q.
o~
0
1
2
3
4
5
6
100
7
8
(~)
;,
0
1
2
3
4
5
6
7
8
serum dilut ion (.+l/log 2.~
Fig. 3. Various concentrations of anti-human and rabbit anti-mouse anti-T sera employed to sensitize (a) murine and (b) h u m a n T-cells. Sensitized cells were subsequently mixed with beads coated with F(ab)2GaR, concentration, 10.0 mg/ml, a: Murine cells, from thymus (e), lymph nodes (4) and spleen (m) sensitized with anti-T serum; spleen cells sensitized with NRS (~). b: Human cells, from thymus (o), PBL (0), MOLT (*), 1301 (~')and CLL (o).
n o t related to the c o n c e n t r a t i o n of anti-T serum in the same range o f dilutions (i.e. up to 1 : 16). The same was true for the mouse spleen cells coat ed with NRS (Fig. 3a).
Reproducibility of the method (a) Reproducibility of coating. In order to estimate the reproducibility of t h e bead preparation, h u m a n PBL and mouse t h y m o c y t e s were tested with 7 d if f er en t bead preparations. The bead preparations were never older than one week and were stored at 4°C until use; data showed rather good reproducibility (Table 1).
(b) Stability of the coated bead preparations stored at 4°C and --80°C. F u r t h e r m o r e , we wanted to det er m i ne how long the coated beads were stable in detecting a cons t a nt percentage of positive cells, and w h e t h e r the
167 TABLE 1 R E P R O D U C I B I L I T Y O F T H E R E S U L T S O B T A I N E D WITH D I F F E R E N T B E A D PREPARATIONS Bead preparations a
1 2 3 4 5 6 7 Mean S.D.
P e r c e n t a g e o f positive cells Human PBL
Murine thymocytes
71 64 76 48 76 64 62 65.6 -+ 9.8
82 74 71 76 86 80 84 79.9 -+ 5.6
a D i f f e r e n t b e a d p r e p a r a t i o n s were e m p l o y e d f o r each test. T h e s e were n e v e r o l d e r t h a n o n e week, a n d were s t o r e d a t 4°C u n t i l use.
temperature at which the bead preparations were stored influenced their stability. For this purpose, two different bead preparations were made. Onehalf of each bead preparation was stored at 4°C and the other half at --80°C, until use. Mouse thymus cells were tested at different time intervals with both bead preparations. The percentage of T-cells detected with beads stored at 4°C, declined with the 'age' of the beads. The data obtained using beads stored at --80°C showed that the coated beads remained stable, detecting a more or less constant percentage of positive cells. The percentages of positive cells detected using t w o different bead preparations stored at the same temperature and of approximately the same 'age', did not differ significantly (Table 2). TABLE 2 S T A B I L I T Y O F B E A D P R E P A R A T I O N S W H E N S T O R E D A T 4°C A N D A T - - 8 0 ° C Bead preparations
Temp. of storage
P e r c e n t a g e s o f positive cells a o b t a i n e d w i t h b e a d p r e p a r a t i o n s o f d i f f e r e n t age (days) 1
1 2
4°C 4°C
80
1 2
--80°C --80°C
82
2
5
6
NT b 86
9
NT NT
82 86
8
17
69 NT
78 76
16
21
58 70
78 78
20
68 78
85
78
a T h y m u s - c e l l s f r o m C3H m i c e were t e s t e d at d i f f e r e n t intervals w i t h t w o b e a d preparat i o n s s t o r e d at 4°C a n d - - 8 0 ° C . b N T = n o t tested.
168 DISCUSSION In the present report we describe an inexpensive particle-labeling m e t h o d for the demonstration and evaluation of surface antigens. This m e t h o d does n o t require any special equipment since the beads used as markers are visible b y ordinary light microscopy. Under correct storage conditions, the bead preparation has shown high stability over a long time period. This makes the PAA-bead method a convenient assay for routine laboratory use. The use of plastic beads as carriers of antibody or anti-antibody to determine surface antigens on lymphoid cells has been described by two groups of authors (Gordon et al., 1977a, b; Ammann et al., 1977), using beads covalently attached to antibodies by means of glutaraldehyde or carbodiimide, respectively. We have employed PAA-beads and poly-L-lysine, found by Mazia et al. (1975) to cause attachment of proteins to plastic surfaces, for binding the antibodies onto the beads. The polycationic poly-L-lysine adheres strongly to plastic beads, leaving free cationic sites which can then be combined with anionic sites on the proteins. It is also convenient for attaching antigens to polymeric plastic beads (Pachmann and Leibold, 1976). In comparison to the use of glutaraldehyde to attach antibodies onto plastic beads, the application of poly-L-lysine has some advantages: the procedure is very easy and rapid to perform and the possible partial inactivation of antibodies caused by glutaraldehyde (Avrameas et al., 1969) is avoided. Non-specific binding of beads to the cells through unsaturated PLLsites was effectively prevented by incubating the beads with BSA at high concentrations. Control experiments, in which the beads were coated with PLL only and subsequently with BSA (without antibodies), have shown that beads coated in this way have practically no affinity for the cells (less than 5% binding). In contrast to Gordon et al. (1977a, b) and Amman et al. (1977) who coated their beads with whole antibody molecules, we used the F(ab)2 fragment of goat anti-rabbit globulin which considerably reduced the nonspecific binding to B-cells, probably via the Fc-receptor. The sensitivity of the method depends on the concentration of coated beads mixed with the cells. A critical minimal concentration of beads is necessary in order to obtain optimal sensitivity. Further increase of the concentration up to 8-fold does not influence the results (see the long plateau in Fig. 1). As the method described is an indirect test it depends also on the concentration of both the specific antiserum used to sensitize the cells, and of the anti-antiserum (goat anti-rabbit) used to coat the beads. The dilution curve of F(ab)2GaR had a maximum with supraoptimal concentrations resulting in lower sensitivity which could be explained as a prozone effect. When the cells were sensitized with various concentrations of anti-mouse or human T serum, a rather long plateau was obtained with human thymus cells and PBL,
169
as well as with murine thymus and lymph node cells. A significantly shorter plateau (two dilution steps) was obtained with mouse spleen cells. This probably reflects the lower concentration of T-antigen on spleen cells (Raft, 1971). Further experiments have shown that the method can distinguish between cells with high and low antigen concentration, as fewer beads bind to murine T-cells from the spleen than from the thymus (manuscript in preparation). The percentage of positive cells that we detected among MOLT-4 cells was relatively low but in accordance with immunofluorescence testing (result n o t shown). The low percentage of positive MOLT-4 cells, and the shorter plateaus obtained both with various concentrations of F(ab)2GaR and anti-T, could be due to the fact that the cells were in different stages of maturation and therefore did n o t carry equal amounts of surface T-antigen. In general, the m e t h o d presented here determines in murine lymphoid tissues, as well as in human PBL, percentages of T-cells which are in good agreement with the data obtained by other authors by means of other immunological methods (Raff, 1971; Jondal et al., 1973; R o d t et al., 1975). Non-specific binding of beads to the cells coated with NRS was low (up to 10%) and was independent, over a wide range, of the amount of antibodies used to coat the beads. The low percentage of positive cells in the B-cell line 1301-TK as well as in CLL (8% and 15%) did n o t depend on the concentration of goat anti-rabbit antibodies. Theoretically, binding of anti-T antibodies used as whole molecules toFc-receptors may occur. However, control experiments showed that the percentage of positive cells determined could n o t be decreased when the Fc-receptors were blocked by aggregated IgG (data n o t shown). Furthermore, the fact that the percentage of positive CLLand 1301-TK cells was practically independent of the concentration of anti-T serum argues against the binding through Fc-receptors. The reproducibility of the method was found to be good over a period of at least three weeks, when the bead preparations were stored at --80°C. This stability of the coated beads is a further advantage of the method, since it enables coating of larger amounts of beads ready for use. We have described here a simple and sensitive method using PAA-beads. Compared to others using plastic beads, we detected higher percentages of T-antigen bearing cells and low non-specific binding through Fc-receptors. This may be due to either (a) the use of the F(ab)2 fragment as sandwich antibody instead of the whole antibody, or (b) the higher amount of functionally active antibodies coupled to the beads by means of poly-L-lysine, or both. REFERENCES Ammann, A.J., D. Borg, L. K o n d o and D.W. Wara, 1977, J. Immunol. Methods 17, 365. Avrameas, S., B. Taudou and S. Chuilon, 1969, Immunochemistry 6, 67. B~yum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 31. Carnbiaso, C.L., A.E. Leek, F . de Steenwinkel, J. Billin and P.L. Masson, 1977, J. Immunol. Methods 18, 33.
170 Gordon, I.L., W.J. Dreyer, S.P.S. Yen and A. Rembaum, 1977a, Clin. Immunol. Immunopath. 8, 51. Gordon, I.L., W.J. Dreyer, S.P.S. Yen and A. Rembaum, 1977b, Cell. Immunol. 28, 307. Hoffmann-Fezer, G., H. Rodt, M. Eulitz and S. Thierfelder, 1976, J. Immunol. Methods 13, 261. Hoffmann-Fezer, G., K. Pielsticker, H. Rodt and S. Thierfelder, 1978, Verh. Dtsch. Ges. Path. 62, 371. Jondal, M., H. Wigzell and F. Aiuti, 1973, Transplant. Rev. 16, 163. Jondal, M., 1974, Scand. J. Immunol. 3, 269. Malin, S. and J. Edwards, 1972, Nature 235, 182. Mazia, D., G. Schatten and W. Sale, 1975, J. Cell. Biol. 66, 198. Minowada, J., T. Ahnuma and G.E. Moor, 1972, J. Nat. Cancer Inst. 49, 891. Molday, R.S., W.J. Dreyer, A. Rembaum and S.P.S. Yen, 1974, Nature 249, 81. Molday, R.S., W.J. Dreyer, A. Rembaum and S.P.S. Yen, 1975, J. Cell. Biol. 64, 75. Molday, R.S., S.P.S. Yen and A. Rembaum, 1977, Nature 268, 43. Nisonoff, A., F.C. Wissler, L.N. Lipman and D.L. Woernley, 1960, Arch. Biophys. 89, 230. Pachmann, K. and W. Leibold, 1976, J. Immunol. Methods 12, 89. Raft, M.C., 1970, Immunology 19, 637. Raft, M.C., 1971, in: Cell Interactions and Receptor Antibodies in Immune Responses, eds. O. M~kel~, A. Cross and T.V. Kosunen (Academic Press, New York) p. 83. Rodt, H., S. Thierfelder and M. Eulitz, 1974, Europ. J. Immunol. 4, 25. Rodt H., S. Thierfelder, E. Thiel, D. GStze, B. Netzel and M. Eulitz, 1975, Immunogenetics 2,411. Thiel, E., P. DSrmer and S. Thierfelder, 1975, J. Immunol. Methods 6, 317.
