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ELISA-plaque assay for the detection of platelet antibody-secreting cells
Journal of Immunological Methods, 117 (1989) 199-203
199
Elsevier JIM05069
ELISA-plaque assay for the detection of platelet antibody-secreting cells I. Studies on optimization of reagent concentrations and culture conditions for enumeration of spot-forming cells H i r o s h i U c h i y a m a a n d Junichi A k a t s u k a Department of Pediatrics, Jikei Unioersity School of Medicine, Minato-ku, Tokyo, Japan
(Received3 August 1988, revisedreceived22 September1988, accepted 4 October 1988)
This report describes a modified ELISA-plaque assay that we have succeeded in developing and utilizing for the detection of platelet antibody-secreting cells (PASC). We investigated the effects of various culture conditions on the formation and specificity of plaques obtained from spleen cells and peripheral blood leukocytes (PBL) of mice immunized with human platelets. The values of PASC using this plaque assay were compared with the corresponding serum platelet-binding IgG (PBIgG) levels. The results revealed that plaque formation from spleen cells was well correlated with the number of inoculations, whereas that from PBL was not. The rise of PBIgG levels in sera was shown to be delayed, as compared with the plaque formation from spleen cells. Key words: ELISA-plaqueassay; Platelet antibody-secretingcell
Introduction
An ELISA-plaque assay has recently been described for the detection of specific antibodysecreting cells by Sedgwick and Holt (1983). Thereafter, we attempted to develop a method for the detection of platelet antibody-secreting cells
Correspondenceto: H. Uchiyama,Department of Pediatrics, Jikei University School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105, Japan. Abbreviations: ELISA, enzyme-linked immunosorbent assay; PASC, platelet antibody-secretingcells; PBL, peripheral blood leukocytes;PBIgG, platelet-bindingIgG; PRP, plateletrich plasma; PBS, phosphate-bufferedsaline; RBC, red blood cells; WBC, white blood cells; FCS, foetal calf serum; BSA, bovine serum albumin; 5-BCIP, 5-bromo-4-chloro-3-indolyl phosphate; ASC, antibody-secreting cells; APase, alkaline phosphatase; PFC, plaque-formingcells; SC, spleen cells.
(PASC), employing this ELISA-plaque assay. In the present study, we investigated the effects of various culture conditions on the formation and specificity of plaques. Then, the values obtained from these plaque assays, which reflected the numbers of lymphocytes producing platelet antibody, were compared with the corresponding platelet-binding IgG (PBIgG) levels in the sera. Materials and methods Preparation of antigens and immunization
Venous blood, collected 6 : 1 with a c i d / c i t r a t e / dextrose as an anticoagulant, was obtained f r o m healthy adult blood group O, Rh-positive donors. Platelet-rich plasma (PRP) was prepared by differential centrifugation (200 × g for 10 min at 22°C). The PRP was then further centrifuged
0022-1759/89/$03.50 © 1989 ElsevierScience Publishers B.V. (BiomedicalDivision)
200
(2000 × g for 5 min at 22 o C) to obtain a platelet pellet and this was resuspended in 1% aqueous ammonium oxalate for 10 min in order to lyse contaminating red cells. Platelets were then washed three times with 0.15 M phosphate-buffered saline (PBS) containing 0.01 M EDTA (pH 6.0), and resuspended in the buffer. The B A L B / c female mice used were usually inoculated three times intraperitoneally with 2 × 108 washed human platelets at 1-week intervals and killed 3 days after the last inoculation. For studies on the optimization of culture volume and incubation time, however, the mice were inoculated twice and killed 7 days later. The specificities of the ELISA plaques were also examined using mice immunized with human red blood cells (RBC) and white blood cells (WBC), of which 2 × 10 s cell suspensions were prepared as follows. A human RBC preparation was made by removing WBC from defibrinated blood using the dextran method (Maluish et al., 1986). Human WBC were also obtained by the dextran method from the defibrinated blood and contaminating RBC were lysed with distilled water. The donors of the platelets, RBC and WBC used were of blood group O, Rh-positive.
Preparation of spleen cells and peripheral blood leukocytes (PBL) Spleens were removed from immunized or control (non-immune) mice and finely minced with scissors RPMI 1640 medium containing 10% fetal calf serum (FCS) was added to each lot of spleen cells. Erythrocyte-free, single-spleen cell suspensions were prepared by the method of Sedgwick and Holt (1983). In order to obtain optimal numbers of plaques for counting, suspensions of 5 × 106, 106, 5 × 105, 105, 5 × 104, and 104 splenocytes were made up in 1 ml of RPMI-10% FCS. Heparinized blood from mice was collected directly from the abdominal aorta and PBL were isolated by centrifugation on Lymphoprep (Nycomed, Oslo) (BiSyum, 1968).
Platelet membrane protein preparation A platelet suspension was prepared as described above and platelets were homogenized by ultrasonication for 20 s, three times at an intensity of four set at 4 ° C, in an Ultrasonic Disrupter,
Model UR-200P (Tomy Co., Tokyo, Japan). Homogenized platelet aliquots were layered on top of a solution of 30% ( w / v ) sucrose and centrifuged at 4 ° C at 6 3 5 0 0 × g for 1 h. The membrane bands were isolated and pelleted by centrifugation for 45 min at 4 o C and 105 000 × g.
ELISA-plaque assay The assay was a modification of that described by Sedgwick and Holt (1983, 1986) and was performed in Nunclon multiwell tissue culture plates (24 round wells, flat bottom, 1.5 cm diameter from Nunc, Denmark).
Coating of the plates with platelet antigens. Platelet-membrane protein pellets were fully resuspended in 0.05 M carbonate-bicarbonate buffer, p H 9.6, and adjusted to a concentration of 50, 100, 500 or 1000/~g/ml by the method of Lowry et al. (1951). A 0.5 ml aliquot of the solution of platelet membrane-protein was added to each well and left overnight at 4 ° C. The coating buffer was made according to the method of Voller et al. (1976). Blocking protein. 1 h before use, the antigencoated wells were washed twice with PBS-0.05% ( v / v ) Tween 20 (Sigma Chemical Co., U.S.A.). A 0.4 ml aliquot of a 1.0 m g / m l solution of bovine serum albumin (BSA, Sigma Chemical Co., U.S.A.) in coating buffer was then added to each well and the plates were incubated for 1 h at 37 ° C. Addition of cells. The antigen-coated and BSA-blocked wells were washed twice with PBSTween followed by a third wash with normal PBS. A 1.0 ml ahquot of cell suspension was then added slowly to each well using a graduated pipette. The plates were then placed in a humid atmosphere at 37 ° C for an appropriate period. After the incubation had been completed, the plates were removed and immediately emptied by flicking over a sink. Three washes with PBS-Tween were then done. Addition of antisera. All antisera were diluted in PBS-Tween containing 1.0% ( w / v ) BSA. A 0.4 ml aliquot of an appropriate dilution (100 × , 500 × , 1000 × ) of affinity-purified rabbit anti-mouse IgG (heavy- and light-chain-specific) (Cappel Laboratories) was added to each well, and the plates were then incubated overnight at 4 ° C. After washing, 0.4 ml of an appropriate dilution (100 × , 500 × , 1000 × ) of goat anti-rabbit IgG-alkaline
201 phosphatase (APase) conjugate (Tago) was added, and the plates were incubated at room temperature for 5-6 h. Addition of substrate. After incubation with the enzyme conjugate, the wells were again washed three times with PBS-Tween and emptied. The plates were placed on a level surface, and 1 ml of warm (40 ° C) agarose-substrate mixture was added to each well. Blue dots appeared within minutes and could be readily counted by eye after 30-60 min of incubation at 37 ° C. The alkaline phosphatase substrate used in this system was 5-bromo-4-chloro-3-indolyl phosphate (5-BCIP). The warm agarose-substrate mixture was made according to the method of Sedgwick and Holt (1983). Enumeration of ASC. The plates were inverted over an X-ray viewing box, and macroscopic blue dots scored by marking the undersurface with a fine-pointed felt pen. An inverted microscope was used to distinguish occasional artefacts from zones of putative antibody.
Determination of serum platelet-binding IgG (PBIgG) Healthy O, Rh-positive-type platelets were fixed with 1% paraformaldehyde and-incubated with mouse serum followed by preparation according to the method of Borne et al. (platelet suspension immunofluorescence test) (1978, 1980). The antibody-treated platelets were analyzed on an automated flow cytometer (Coulter, Epics-C) (Lazarchick, 1986).
standard antigen concentration, 500 times as the standard rabbit anti-mouse Ig antisera dilution and 100 times as the standard APase-labelled antisera dilution. These standard conditions facilitated detection of the highest numbers of plaques (55 ASC/105 SC). These data represent means of triplicates, over two observations.
Optimization of culture oolume Preliminary experiments were performed by culturing spleen cells in different volumes (300/tl, 500 /zl, 750 /~1, 1 ml) of culture medium. The culture with 300/~1 of medium showed the smallest numbers of ASC (11 ASC/106 SC). The cultures with 500 /~1, 750 ~tl and 1 ml showed the same numbers of ASC (17 ASC/106 SC). Thus, 1 ml of medium was used as the working volume. Optimization of incubation time The results are shown in Fig. 1. Within only 30 min after initiation of the cultures, a few spots could be counted. Then, their number increased almost linearly with increasing incubation time and reached a maximum by 2 h. Longer incubation times yielded rather darker backgrounds. Therefore, a culture period of 2 h was selected for subsequent experiments. Determination of cell density In order to determine how the cell density affected the detection of PASC, various numbers of sensitized spleen cells were cultured in antigencoated plates. The results are shown in Fig. 2. At high cell densities (10 6 cells or more/well), partial confluence of the reactions was observed. Thus, as
Results
Optimization of reagent concentrations The spleen cells from the mice immunized with platelets and those from normal mice were used at a concentration of 1 × 105 cells/well. The optimal platelet-membrane protein concentrations for coating plates were examined over a range from 50/~g/ml to 1000/~g/ml. The optimal concentrations of rabbit anti-mouse Ig antisera and APaselabelled antisera were also examined using each antisera at dilutions ranging from 1/100 to 1 / 1000. According to the data obtained from titration of reagents, we chose 100 /zg/ml as the
2O
~ 10 n .,¢. ,~
x--~ - - I - - - ' I 2
cells
x ~~ , ~
=
Control --xspleen cells | 0 ,
4
Fig. 1. Effectof incubationtimeon
8
the
time
(hours)
ELISA-plaqueassay.
202 TABLE 1 SPECIFICITY OF SPOTS IN THE ELISA-PLAQUE ASSAY RBC "
WBC b
Plate c
ASC
3-6(5.5)/105
0-2(1)/105
6-37(32.5)/105 SC
100
" Spleen cells (SC) of mice immunized with human RBC. ~ SC of mice immunized with human WBC. c SC of mice immunized with human platelets (median).
nized mouse
cally to the coated polystyrene surface, we compared the affinities to bind antibodies secreted from platelet-sensitized splenocytes and WBC- or RBC-sensitized splenocytes. The results of this experiment are shown in Table I. The spleen cells immunized with human WBC or RBC did not form significant numbers of spots when assayed in platelet-coated plates. The formation of blue spots appeared to be due to the specificity of antibodies secreted locally by individual spleen cells.
control ~.~,,x
x ~ ; ~ -=.-----i -~ ~ X l 0 4 l0 s 5×10 ~ l0 ~
104
~ 5X10s
Number of cells plated
Fig. 2. Sensitivity of the ELISA-plaque assay for enumeration of ASC.
T i m e c o u r s e and relationship between PBIgG and A S C of spleen cells and peripheral blood leukocytes
few as 10 5 to 5 × 10 5 sensitized splenocytes were able to produce a substantial number of spots.
The mice of group A were sensitized once, those of group B twice and those of group C three times at 1-week intervals and all mice were killed 3 days after the last inoculation. As shown in Fig. 3, the plaque formation from SC was well corre-
Specificity of the spots To rule out the possibility that immunoglobulins secreted in this system might bind non-specifi-
PBIgG CONTROL SAMPLE NO = 22PBIGT.24 PBIgG IMMUNIZED 62.7.31 (GroupC) SAMPLE NO = 22PBIG7.24 ~~ .................
,; ~i
i=~ PBIgG IMMUNIZED 6 2 . 7 . 2 4 (Group6) SAMPLE NO = 22P61G7.24 PBIgG iMMUNIZED 62.7.17 (GroupA) SAMPLE ND =22PBIGT.24 _~. . . . . . . . . . . . . . . . . . . . . .
A S C / 5 xl0Ssc
i
,'=
!
Group C
50"
St !i
Group 6
Group A
I
2
3
~
4
5
6
7
8
9
10
I1
12
13
14
Fig. 3. T i m e c o u r s e and relationship b e t w e e n P B I g G and A S C o f SC and PBL.
15
16
17
day
203
lated with the number of inoculations, but that from PBL was not. In group A, PASC in sensitized splenocytes were 8.4 per 5 × 105 SC, whereas PASC in control splenocytes and PBIgG levels in the sera were not detectable. Thus, the times until the rise in PBIgG levels in the sera were shown to be delayed, as compared with the plaque formation from SC.
advice and Dr. K. Fujisawa for performing measurements of PBIgG. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, and in part by a grant from the Research Committee on Idiopathic Disorders of Haematopoietic Organs, Ministry of Welfare, Japan.
References Discussion Enumeration of specific antibody-secreting cells (ASC) is commonly done by hemolytic plaque-forming cell (PFC) assay (Jerne et al., 1974). Difficulty in coupling antigens with erythrocytes, which provide suitable targets with an appropriate density of antigenic determinants and sensitivity to antibody-mediated complement lysis, is probably the main limitation of haemolytic plaque assays (Golub et al., 1968; Pasanen and MLkel~i, 1969; Jerne et al., 1974). We have made several attempts to develop specific hemolytic PFC assays for detecting PASC, using different coupling agents with which to coat the red cells with platelet-membrane proteins. The major problems we encountered were: insufficient coating of live red cells with platelet-membrane proteins and the instability of the coupled red cells. In contrast, adsorption of macromolecules to a hydrophobic plastic surface is simple, does not require a cumbersome coupling step, and is usually stable for a long period of time. The immunospot assay thus seems to be a simpler and more versatile method than conventional PFC assays. This spot assay was shown to be useful method as an immunological parameter for assessing the production of antiplatelet antibodies at the cellular level in mice, and its clinical application is suggested.
Acknowledgements The authors thank Professor K. Maekawa, who kindly reviewed the manuscript. We would also like to thank Dr. P.G. Holt for his kind technical
B~yum, A. (1968) Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Stand. J. Clin. Lab. Invest. 21 (suppl. 97), 77. Golub, E.S., Mishell, R.I., Wiegle, W.O. and Dutton, R.W. (1968) A modification of the hemolytic plaque assay for use with protein antigens. J. Immunol. 100, 133. Jerne, N.K., Henry, C., Nordin, A.A., Fuji, H., Koros, A.M.C. and Lefkovits, I. (1974) Plaque forming cells: Methodology and Theory. Transplant. Rev. 18, 130. Lazarchick, J. and Hall, S.A. (1986) Platelet-associated IgG assay using flow cytometric analysis. J. Immunol. Methods 87, 257. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265. Maluish, A.E. and Strong, D.M. (1986) Lymphocyte Proliferation. In: N.R. Rose, H. Friedman and J.L. Fahey (Eds.), Manual of Clinical Laboratory Immunology 3rd ed. American Society for Microbiology, Washington, DC, p. 277. Pasanen, V.J. and M~ikel~, O. (1969) Effect of the number of haptens coupled to each erythrocyte on haemolytic plaque formation. Immunology 16, 399. Sedgwick, J.D. and Holt, P.G. (1983) A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J. Immunol. Methods 57, 301. Sedgwick, .I.D. and Holt, P.G. (1986) The ELISA-plaque assay for the detection and enumeration of antibody-secreting cells. J. Immunol. Methods 87, 37. Voller, A., Bidwell, D. and Badett A. (1976) In: H.R. Rose and H. Friedman (Eds.), Manual of Clinical Immunology. American Society for Microbiology, Washington, DC, ch. 69. Von dem Borne, A.E.G.Kr., Verheugt, F.W.A., Oosterhof, F., Von Riesz, E., Brntel de la Rivi6re, A. and Engelfriet, C.P. (1978) A simple immunofluorescence test for the detection of platelet antibodies. Br..i. Haematol. 39, 195. Von dem Borne, A.E.G.Kr., Hermerhorst, F.M., Van Leeuwen, E.F., Pegels, H.G., Von Riesz, E. and Engelfriet, C.P. (1980) Autoimmune thrombocytopenia: detection of platelet autoantibodies with the suspension immunofluorescence test. Br. J. Haematol. 45, 319.
199
Elsevier JIM05069
ELISA-plaque assay for the detection of platelet antibody-secreting cells I. Studies on optimization of reagent concentrations and culture conditions for enumeration of spot-forming cells H i r o s h i U c h i y a m a a n d Junichi A k a t s u k a Department of Pediatrics, Jikei Unioersity School of Medicine, Minato-ku, Tokyo, Japan
(Received3 August 1988, revisedreceived22 September1988, accepted 4 October 1988)
This report describes a modified ELISA-plaque assay that we have succeeded in developing and utilizing for the detection of platelet antibody-secreting cells (PASC). We investigated the effects of various culture conditions on the formation and specificity of plaques obtained from spleen cells and peripheral blood leukocytes (PBL) of mice immunized with human platelets. The values of PASC using this plaque assay were compared with the corresponding serum platelet-binding IgG (PBIgG) levels. The results revealed that plaque formation from spleen cells was well correlated with the number of inoculations, whereas that from PBL was not. The rise of PBIgG levels in sera was shown to be delayed, as compared with the plaque formation from spleen cells. Key words: ELISA-plaqueassay; Platelet antibody-secretingcell
Introduction
An ELISA-plaque assay has recently been described for the detection of specific antibodysecreting cells by Sedgwick and Holt (1983). Thereafter, we attempted to develop a method for the detection of platelet antibody-secreting cells
Correspondenceto: H. Uchiyama,Department of Pediatrics, Jikei University School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105, Japan. Abbreviations: ELISA, enzyme-linked immunosorbent assay; PASC, platelet antibody-secretingcells; PBL, peripheral blood leukocytes;PBIgG, platelet-bindingIgG; PRP, plateletrich plasma; PBS, phosphate-bufferedsaline; RBC, red blood cells; WBC, white blood cells; FCS, foetal calf serum; BSA, bovine serum albumin; 5-BCIP, 5-bromo-4-chloro-3-indolyl phosphate; ASC, antibody-secreting cells; APase, alkaline phosphatase; PFC, plaque-formingcells; SC, spleen cells.
(PASC), employing this ELISA-plaque assay. In the present study, we investigated the effects of various culture conditions on the formation and specificity of plaques. Then, the values obtained from these plaque assays, which reflected the numbers of lymphocytes producing platelet antibody, were compared with the corresponding platelet-binding IgG (PBIgG) levels in the sera. Materials and methods Preparation of antigens and immunization
Venous blood, collected 6 : 1 with a c i d / c i t r a t e / dextrose as an anticoagulant, was obtained f r o m healthy adult blood group O, Rh-positive donors. Platelet-rich plasma (PRP) was prepared by differential centrifugation (200 × g for 10 min at 22°C). The PRP was then further centrifuged
0022-1759/89/$03.50 © 1989 ElsevierScience Publishers B.V. (BiomedicalDivision)
200
(2000 × g for 5 min at 22 o C) to obtain a platelet pellet and this was resuspended in 1% aqueous ammonium oxalate for 10 min in order to lyse contaminating red cells. Platelets were then washed three times with 0.15 M phosphate-buffered saline (PBS) containing 0.01 M EDTA (pH 6.0), and resuspended in the buffer. The B A L B / c female mice used were usually inoculated three times intraperitoneally with 2 × 108 washed human platelets at 1-week intervals and killed 3 days after the last inoculation. For studies on the optimization of culture volume and incubation time, however, the mice were inoculated twice and killed 7 days later. The specificities of the ELISA plaques were also examined using mice immunized with human red blood cells (RBC) and white blood cells (WBC), of which 2 × 10 s cell suspensions were prepared as follows. A human RBC preparation was made by removing WBC from defibrinated blood using the dextran method (Maluish et al., 1986). Human WBC were also obtained by the dextran method from the defibrinated blood and contaminating RBC were lysed with distilled water. The donors of the platelets, RBC and WBC used were of blood group O, Rh-positive.
Preparation of spleen cells and peripheral blood leukocytes (PBL) Spleens were removed from immunized or control (non-immune) mice and finely minced with scissors RPMI 1640 medium containing 10% fetal calf serum (FCS) was added to each lot of spleen cells. Erythrocyte-free, single-spleen cell suspensions were prepared by the method of Sedgwick and Holt (1983). In order to obtain optimal numbers of plaques for counting, suspensions of 5 × 106, 106, 5 × 105, 105, 5 × 104, and 104 splenocytes were made up in 1 ml of RPMI-10% FCS. Heparinized blood from mice was collected directly from the abdominal aorta and PBL were isolated by centrifugation on Lymphoprep (Nycomed, Oslo) (BiSyum, 1968).
Platelet membrane protein preparation A platelet suspension was prepared as described above and platelets were homogenized by ultrasonication for 20 s, three times at an intensity of four set at 4 ° C, in an Ultrasonic Disrupter,
Model UR-200P (Tomy Co., Tokyo, Japan). Homogenized platelet aliquots were layered on top of a solution of 30% ( w / v ) sucrose and centrifuged at 4 ° C at 6 3 5 0 0 × g for 1 h. The membrane bands were isolated and pelleted by centrifugation for 45 min at 4 o C and 105 000 × g.
ELISA-plaque assay The assay was a modification of that described by Sedgwick and Holt (1983, 1986) and was performed in Nunclon multiwell tissue culture plates (24 round wells, flat bottom, 1.5 cm diameter from Nunc, Denmark).
Coating of the plates with platelet antigens. Platelet-membrane protein pellets were fully resuspended in 0.05 M carbonate-bicarbonate buffer, p H 9.6, and adjusted to a concentration of 50, 100, 500 or 1000/~g/ml by the method of Lowry et al. (1951). A 0.5 ml aliquot of the solution of platelet membrane-protein was added to each well and left overnight at 4 ° C. The coating buffer was made according to the method of Voller et al. (1976). Blocking protein. 1 h before use, the antigencoated wells were washed twice with PBS-0.05% ( v / v ) Tween 20 (Sigma Chemical Co., U.S.A.). A 0.4 ml aliquot of a 1.0 m g / m l solution of bovine serum albumin (BSA, Sigma Chemical Co., U.S.A.) in coating buffer was then added to each well and the plates were incubated for 1 h at 37 ° C. Addition of cells. The antigen-coated and BSA-blocked wells were washed twice with PBSTween followed by a third wash with normal PBS. A 1.0 ml ahquot of cell suspension was then added slowly to each well using a graduated pipette. The plates were then placed in a humid atmosphere at 37 ° C for an appropriate period. After the incubation had been completed, the plates were removed and immediately emptied by flicking over a sink. Three washes with PBS-Tween were then done. Addition of antisera. All antisera were diluted in PBS-Tween containing 1.0% ( w / v ) BSA. A 0.4 ml aliquot of an appropriate dilution (100 × , 500 × , 1000 × ) of affinity-purified rabbit anti-mouse IgG (heavy- and light-chain-specific) (Cappel Laboratories) was added to each well, and the plates were then incubated overnight at 4 ° C. After washing, 0.4 ml of an appropriate dilution (100 × , 500 × , 1000 × ) of goat anti-rabbit IgG-alkaline
201 phosphatase (APase) conjugate (Tago) was added, and the plates were incubated at room temperature for 5-6 h. Addition of substrate. After incubation with the enzyme conjugate, the wells were again washed three times with PBS-Tween and emptied. The plates were placed on a level surface, and 1 ml of warm (40 ° C) agarose-substrate mixture was added to each well. Blue dots appeared within minutes and could be readily counted by eye after 30-60 min of incubation at 37 ° C. The alkaline phosphatase substrate used in this system was 5-bromo-4-chloro-3-indolyl phosphate (5-BCIP). The warm agarose-substrate mixture was made according to the method of Sedgwick and Holt (1983). Enumeration of ASC. The plates were inverted over an X-ray viewing box, and macroscopic blue dots scored by marking the undersurface with a fine-pointed felt pen. An inverted microscope was used to distinguish occasional artefacts from zones of putative antibody.
Determination of serum platelet-binding IgG (PBIgG) Healthy O, Rh-positive-type platelets were fixed with 1% paraformaldehyde and-incubated with mouse serum followed by preparation according to the method of Borne et al. (platelet suspension immunofluorescence test) (1978, 1980). The antibody-treated platelets were analyzed on an automated flow cytometer (Coulter, Epics-C) (Lazarchick, 1986).
standard antigen concentration, 500 times as the standard rabbit anti-mouse Ig antisera dilution and 100 times as the standard APase-labelled antisera dilution. These standard conditions facilitated detection of the highest numbers of plaques (55 ASC/105 SC). These data represent means of triplicates, over two observations.
Optimization of culture oolume Preliminary experiments were performed by culturing spleen cells in different volumes (300/tl, 500 /zl, 750 /~1, 1 ml) of culture medium. The culture with 300/~1 of medium showed the smallest numbers of ASC (11 ASC/106 SC). The cultures with 500 /~1, 750 ~tl and 1 ml showed the same numbers of ASC (17 ASC/106 SC). Thus, 1 ml of medium was used as the working volume. Optimization of incubation time The results are shown in Fig. 1. Within only 30 min after initiation of the cultures, a few spots could be counted. Then, their number increased almost linearly with increasing incubation time and reached a maximum by 2 h. Longer incubation times yielded rather darker backgrounds. Therefore, a culture period of 2 h was selected for subsequent experiments. Determination of cell density In order to determine how the cell density affected the detection of PASC, various numbers of sensitized spleen cells were cultured in antigencoated plates. The results are shown in Fig. 2. At high cell densities (10 6 cells or more/well), partial confluence of the reactions was observed. Thus, as
Results
Optimization of reagent concentrations The spleen cells from the mice immunized with platelets and those from normal mice were used at a concentration of 1 × 105 cells/well. The optimal platelet-membrane protein concentrations for coating plates were examined over a range from 50/~g/ml to 1000/~g/ml. The optimal concentrations of rabbit anti-mouse Ig antisera and APaselabelled antisera were also examined using each antisera at dilutions ranging from 1/100 to 1 / 1000. According to the data obtained from titration of reagents, we chose 100 /zg/ml as the
2O
~ 10 n .,¢. ,~
x--~ - - I - - - ' I 2
cells
x ~~ , ~
=
Control --xspleen cells | 0 ,
4
Fig. 1. Effectof incubationtimeon
8
the
time
(hours)
ELISA-plaqueassay.
202 TABLE 1 SPECIFICITY OF SPOTS IN THE ELISA-PLAQUE ASSAY RBC "
WBC b
Plate c
ASC
3-6(5.5)/105
0-2(1)/105
6-37(32.5)/105 SC
100
" Spleen cells (SC) of mice immunized with human RBC. ~ SC of mice immunized with human WBC. c SC of mice immunized with human platelets (median).
nized mouse
cally to the coated polystyrene surface, we compared the affinities to bind antibodies secreted from platelet-sensitized splenocytes and WBC- or RBC-sensitized splenocytes. The results of this experiment are shown in Table I. The spleen cells immunized with human WBC or RBC did not form significant numbers of spots when assayed in platelet-coated plates. The formation of blue spots appeared to be due to the specificity of antibodies secreted locally by individual spleen cells.
control ~.~,,x
x ~ ; ~ -=.-----i -~ ~ X l 0 4 l0 s 5×10 ~ l0 ~
104
~ 5X10s
Number of cells plated
Fig. 2. Sensitivity of the ELISA-plaque assay for enumeration of ASC.
T i m e c o u r s e and relationship between PBIgG and A S C of spleen cells and peripheral blood leukocytes
few as 10 5 to 5 × 10 5 sensitized splenocytes were able to produce a substantial number of spots.
The mice of group A were sensitized once, those of group B twice and those of group C three times at 1-week intervals and all mice were killed 3 days after the last inoculation. As shown in Fig. 3, the plaque formation from SC was well corre-
Specificity of the spots To rule out the possibility that immunoglobulins secreted in this system might bind non-specifi-
PBIgG CONTROL SAMPLE NO = 22PBIGT.24 PBIgG IMMUNIZED 62.7.31 (GroupC) SAMPLE NO = 22PBIG7.24 ~~ .................
,; ~i
i=~ PBIgG IMMUNIZED 6 2 . 7 . 2 4 (Group6) SAMPLE NO = 22P61G7.24 PBIgG iMMUNIZED 62.7.17 (GroupA) SAMPLE ND =22PBIGT.24 _~. . . . . . . . . . . . . . . . . . . . . .
A S C / 5 xl0Ssc
i
,'=
!
Group C
50"
St !i
Group 6
Group A
I
2
3
~
4
5
6
7
8
9
10
I1
12
13
14
Fig. 3. T i m e c o u r s e and relationship b e t w e e n P B I g G and A S C o f SC and PBL.
15
16
17
day
203
lated with the number of inoculations, but that from PBL was not. In group A, PASC in sensitized splenocytes were 8.4 per 5 × 105 SC, whereas PASC in control splenocytes and PBIgG levels in the sera were not detectable. Thus, the times until the rise in PBIgG levels in the sera were shown to be delayed, as compared with the plaque formation from SC.
advice and Dr. K. Fujisawa for performing measurements of PBIgG. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, and in part by a grant from the Research Committee on Idiopathic Disorders of Haematopoietic Organs, Ministry of Welfare, Japan.
References Discussion Enumeration of specific antibody-secreting cells (ASC) is commonly done by hemolytic plaque-forming cell (PFC) assay (Jerne et al., 1974). Difficulty in coupling antigens with erythrocytes, which provide suitable targets with an appropriate density of antigenic determinants and sensitivity to antibody-mediated complement lysis, is probably the main limitation of haemolytic plaque assays (Golub et al., 1968; Pasanen and MLkel~i, 1969; Jerne et al., 1974). We have made several attempts to develop specific hemolytic PFC assays for detecting PASC, using different coupling agents with which to coat the red cells with platelet-membrane proteins. The major problems we encountered were: insufficient coating of live red cells with platelet-membrane proteins and the instability of the coupled red cells. In contrast, adsorption of macromolecules to a hydrophobic plastic surface is simple, does not require a cumbersome coupling step, and is usually stable for a long period of time. The immunospot assay thus seems to be a simpler and more versatile method than conventional PFC assays. This spot assay was shown to be useful method as an immunological parameter for assessing the production of antiplatelet antibodies at the cellular level in mice, and its clinical application is suggested.
Acknowledgements The authors thank Professor K. Maekawa, who kindly reviewed the manuscript. We would also like to thank Dr. P.G. Holt for his kind technical
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