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Comparison of lysis of bromelain and papain treated red cells in ADCC assays
Immunology Letters, 31 (1992) 237 - 240 Elsevier I MLET 01735
Comparison of lysis of bromelain and papain treated red cells in ADCC assays Belinda M. Kumpel I and Tibor Bak~ics 2 ~U.K. Transplant Support Services Authority, Bristol, U.K., and 2National Institute of Ontology, Budapest, Hungary (Received 3 July 1991; revision received 23 September 1991; accepted 25 September 1991)
1. Summary
Red cells were pretreated with the proteolytic enzymes bromelain or papain prior to use in antibodydependent cell-mediated cytotoxicity (ADCC) assays with lymphocytes or peripheral blood mononuclear ceils (PBMC) as effector ceils. At low concentrations of anti-D or anti-A, lysis of papaintreated cells by lymphocytes was greater than that of bromelain-treated ceils. Papain digestion resulted in both greater sensitivity to haemolysis by lymphocytes or PBMC and higher agglutination titres of anti-D-sensitised red cells than bromelain. With anti-A, however, although papain also promoted greater haemolysis, it was slightly less effective at red cell agglutination than bromelain. 2. Introduction
The sulphhydryl-activated proteolytic enzymes bromelain (EC 3.4.22.4) and papain (papainase EC 3.4.22.2) are both widely used in blood group serology for modifying the extracellular surface of red cell membranes. Treatment of red ceils removes peptides containing sialic acid, resulting in a lowering of zeta-potential [1, 2]. In antibody-dependent cell-mediated cytotoxicity (ADCC) assays utilising anti-D-sensitised red cells and peripheral blood mononuclear cells (PBMC), papain pre-treatment of red cells has been found to render them susceptiKey words: Bromelain; Papain; ADCC; Anti-D; Anti-A Correspondence to: B. M. Kumpel, U.K. Transplant Support Services Authority, Southmead Rd., Bristol BS10 5ND, U.K.
ble to lysis by lymphocytes, whereas untreated red cells are lysed by monocytes (phagocytes) [3]. Pretreatment of red cells with papain has been used in anti-D-mediated cytotoxicity assays using PBMC as effector cells at a low effector-to-red cell ratio [4]; the application of this enzyme-like kinetic model for measurement of maximal cytotoxic function has permitted reproducible and sensitive determination of the K cell activity of patients with malignant diseases. We have also found that a competition assay model has been very informative for the characterization of the lytic activity of anti-D monoclonal antibodies (manuscript in preparation), when anti-D-mediated lysis was measured in the presence of competing A Rh-negative red cells and anti-A. The "conventional" K cell ADCC assay utilises non-adherent (lymphocyte) effector cells, antibody, and either papain [5] or bromelain [6] pretreated red cells at low red cell-to-effector cell ratios. This assay (with papain) has been used to predict the severity of Rh haemolytic disease of the newborn [7] by measurement of the haemolytic potential of maternal anti-D, and (with bromelain) to compare the lytic activity of different monoclonal antibodies [6]. Lysis of red cells is mediated by K ceils in both assay systems. Individual workers have routinely used either bromelain or papain for K cell-mediated assays. It was observed by the authors that bromelain-treated red cells were not lysed in the first assay system (i.e., using a low effector-to-red cell ratio), and thus a comparison was made of the effectiveness of bromelain and papain pretreatment of red cell targets in both these assays.
0 1 6 5 - 2 4 7 8 / 92 / ~; ~ O0 (~3 lOq~ ]~l~evler ~elc.nt-p lanhllch,re 1~ Ttl All r;,ht . . . . . . . . . A
,'~,~,'7
3. Materials and Methods Bromelain and papain solutions, both containing cysteine, were routine blood grouping reagents supplied by the South Western Regional Transfusion Service ( S W R T C ) and International Blood G r o u p Reference L a b o r a t o r y (IBGRL), respectively. Proteolytic activity was determined by the a z o a l b u m i n m e t h o d [8]. Packed red cells were treated by incubation at 37 °C with an equal volume o f enzyme reagent for 15 min followed by washing three times in phosphate-buffered saline (PBS). Agglutination o f enzyme-treated O Rh-positive or A Rh-negative
red cells by monoclonal or polyclonal anti-D or anti-A, respectively, was assessed in microtitre plates [9]. H u m a n monoclonal anti-D antibodies were all o f the IgG1 subclass except 1A3-3 which was IgG3 [9]; murine monoclonal anti-A (IgG3) was kindly supplied by Dr. P. J u d s o n (South Western Regional Transfusion Centre, Bristol). Polyclonal anti-D was used as a 1/100 dilution o f a therapeutic IgG anti-D immunoglobulin preparation, and polyclonal h u m a n anti-A (predominantly IgG1) was obtained from Biotest. A D C C assays were p e r f o r m e d using (i) non-adherent lymphocyte effector cells from the F B M C fraction, at a 15:1
100B0 O1
_>~
60 1A3-3
40
2O' .__.__--------e O~_L 100-
~
: : : ,::::
100.
BO In
80
--~ 60,
4.0
401
BRIC 54
20
01
•
20,
100-
100,
8O
80
=2
m
"~ _>,
60
40
4O
2OI
20
0 10
100
0=
1000
Reciproc d i l u t i o n
10 of
I00
ontibody
Fig. 1. Lysis of anti-D-sensitised O R i R 1 red cells by non-adherent lymphocytes at a high (15:1) effector-to-target red cell ratio. Equal volumes of bromelain-treated (open circles), papain-treated (filled circles), and untreated (filled squares) labelled red cells (8 × 105/ml), effector ceils (12 × 106/ml) and antibody were incubated together for 18 h at 37°C. Both enzymes were used at equal proteolytic concentrations (5 U/ml). The IgG concentrations of the antibody preparations were 7.0/~g human IgG3 anti-D per ml (IA3-3), 5.0 ~g human IgG1 anti-D per ml (2B6), 32/~g murine IgG3 anti-A per ml (BRIC 54) and 4.0 ~tg IgG anti-D per ml (polyclonal anti-D, which was a 1/100 dilution of a therapeutic anti-D immunoglobulin preparation containing IgG1 and lgG3 anti-D). 238
effector (E)-to-labelled target red cell (T) ratio [6]; or (it) unseparated PBMC at a 1:3:3 effector cell (E)-to-labelled target red cell (T)-to-unlabelled competitor red cell (C) ratio (manuscript in preparation). Target cells were O Rh-positive with anti-D and A Rh-negative with anti-A, competitor cells were A Rh-negative with anti-D or O Rh-positive with anti-A. Target red cells were labelled with sodium 51chromate (Amersham plc, Bucks, UK) before use in ADCC assays, and the antibodies were in the fluid phase. 4. Results and Discussion
When used at equal proteolytic concentration, pretreatment of red cells with bromelain or papain resulted in different agglutination titres (Table 1). Anti-D gave greater titres with papain-treated red cells, whereas the anti-A titres were slightly greater with bromelain. Anti-D did not agglutinate native red cells. In an ADCC assay using non-adherent lymphocyte effector cells at a high E:T ratio (15:1), papaintreated red cells were more sensitive to lysis than bromelain-treated red cells at low antibody concentrations (Fig. 1). This effect was more marked for anti-D than for anti-A, and was not observed in antibody excess. Lysis of untreated red ceils was less than that of enzyme-treated red cells. However, in ADCC assays utilizing a lower TABLE 1 Agglutination titres of polyclonal and monoclonal anti-D and anti-A. The proteolytic activity of both enzymes was 5 U / m l . Polyclonal anti-D contained 4 ~tg IgG anti-D per ml. Monoclonal anti-D, 1A3-3, contained 7/~g h u m a n IgG 3 per ml. Polyclonal anti-A was predominantly IgG 1. Monoclonal anti-A, BRIC 54, contained 32/~g murine IgG 3 per ml. Antibody
Red cells Red cell treatment Bromelain Papain Untreated
Polyclonal anti-D Monoconal anti-D
ORIR I ORIR l
Polyclonal antioA Monoclonal anti-A
Arr Arr
4000 a 4000 16000 128
aAgglutination titre.
16000 16000
0 0
8000 64
64 1
TABLE 2 Lysis of enzyme-treated red cells in A D C C assays at a low P B M C effectorcell-to-targetandcompetitorcellratio(E:T:C = 1:3:3). Equal volumes of effector cells (6× 106 P B M C / m l ) , labelled target red cells (18 x 106/ml), unlabelled competitor red cells (18 × 106/ml) and antibody were incubated together for 18 h at 37°C. % Lysis Bromelain, PBMC
Papain, P B M C
Monoclonal anti-D 1/8 FEF5 0 H27 - 1 AB5 - 1 R1D7 0 FC3 - 1 FEF3 0 1A3-1 - I H26 nd 2B6 - 1 1C6 0 1A3-3 1
57 53 54 61 46 54 49 41 58 60 37
Polyclonal anti-A 1/16 14407 2 Biotest 1 Merz-Dade lgG fraction Sla Kur Pic Sze
67 57 66 67 66 63
Monoelonal anti-A 1/16 BRIC37 BRIC54 BRIC56
41 46 51
Control
0
3
PBMC effector-to-target cell ratio, and in the presence of competing red cells (E:T:C = 1:3:3), bromelain-treated red cells were not lysed, whereas all samples of papain-treated red cells were lysed to some extent (Table 2). It is possible that at this relatively high target red cell concentration (22.5 times greater than in the previous ADCC assay), less antibody was bound to the target cells, and due to the presence also of the competitor red cells the relative insensitivity of bromelain-treated cells may then have been so great as to render them incapable of lysis in this system. In a separate experiment, it 239
was found that bromelain-treated red cells were not sensitive to lysis by polyclonal anti-D, nor were untreated red cells by any of the antibodies. In summary, although both bromelain and papain treatment of red cells were found to give satisfactory red cell agglutination and K cell-mediated lysis by anti-D and anti-A in the presence of excess antibody, papain treatment resulted in greater sensitivity of agglutination and haemolysis at low concentrations of anti-D. Acknowledgements The authors are grateful to Dr. M. Scott (IBGRL) and Mr. G. Poole (SWRTC) for the papain and bromelain reagents, to Mr. A. Guest for performing the azoalbumin assay and to the Central Blood Laboratories Authority and the British Council for funding.
240
References [1] van Oss, C. J. and Absolom, D. R. (1984) Vox Sang. 47, 250. [2] Kumpel, B. M. and Hadley, A. G. (1990) MoI. lmmunol. 27,247. [3] Yush 1., Frisch, B. and Goldsher, N. (1980) Eur. J. lmmunol. 10, 127. [4] Bakfics, T., Czanik, P., Kimber, 1., Ringwald, G., Moore, M. and Abraham, E. (1984) Cancer lmmunol. Immunother. 16, 170. [51 Urbaniak, S. J. (1976) Br. J. Haematol. 33,409. [61 Kumpel, B.M., Leader, K . A . , Merry, A. H., Hadley, A. G., Poole, G. D., Blancher, A., Goossens, D., HughesJones, N. C. and Bradley, B. A. (1989a) Eur. J. lmmunol. 19, 2283. [71 Urbaniak, S. J., Ayoub Griess, M., Crawtbrd, R. J. and Fergusson, M. ]. C. (1984) Vox Sang. 46, 323. [8] Lambert, R., Edwards, J. and Anstee, D. J. (1978) Med. Lab. Sci. 35,233. [91 Kumpel, B. M., Poole, G. D. and Bradley, B. A. (1989b) Br. J. Haematol. 71, 125.
Comparison of lysis of bromelain and papain treated red cells in ADCC assays Belinda M. Kumpel I and Tibor Bak~ics 2 ~U.K. Transplant Support Services Authority, Bristol, U.K., and 2National Institute of Ontology, Budapest, Hungary (Received 3 July 1991; revision received 23 September 1991; accepted 25 September 1991)
1. Summary
Red cells were pretreated with the proteolytic enzymes bromelain or papain prior to use in antibodydependent cell-mediated cytotoxicity (ADCC) assays with lymphocytes or peripheral blood mononuclear ceils (PBMC) as effector ceils. At low concentrations of anti-D or anti-A, lysis of papaintreated cells by lymphocytes was greater than that of bromelain-treated ceils. Papain digestion resulted in both greater sensitivity to haemolysis by lymphocytes or PBMC and higher agglutination titres of anti-D-sensitised red cells than bromelain. With anti-A, however, although papain also promoted greater haemolysis, it was slightly less effective at red cell agglutination than bromelain. 2. Introduction
The sulphhydryl-activated proteolytic enzymes bromelain (EC 3.4.22.4) and papain (papainase EC 3.4.22.2) are both widely used in blood group serology for modifying the extracellular surface of red cell membranes. Treatment of red ceils removes peptides containing sialic acid, resulting in a lowering of zeta-potential [1, 2]. In antibody-dependent cell-mediated cytotoxicity (ADCC) assays utilising anti-D-sensitised red cells and peripheral blood mononuclear cells (PBMC), papain pre-treatment of red cells has been found to render them susceptiKey words: Bromelain; Papain; ADCC; Anti-D; Anti-A Correspondence to: B. M. Kumpel, U.K. Transplant Support Services Authority, Southmead Rd., Bristol BS10 5ND, U.K.
ble to lysis by lymphocytes, whereas untreated red cells are lysed by monocytes (phagocytes) [3]. Pretreatment of red cells with papain has been used in anti-D-mediated cytotoxicity assays using PBMC as effector cells at a low effector-to-red cell ratio [4]; the application of this enzyme-like kinetic model for measurement of maximal cytotoxic function has permitted reproducible and sensitive determination of the K cell activity of patients with malignant diseases. We have also found that a competition assay model has been very informative for the characterization of the lytic activity of anti-D monoclonal antibodies (manuscript in preparation), when anti-D-mediated lysis was measured in the presence of competing A Rh-negative red cells and anti-A. The "conventional" K cell ADCC assay utilises non-adherent (lymphocyte) effector cells, antibody, and either papain [5] or bromelain [6] pretreated red cells at low red cell-to-effector cell ratios. This assay (with papain) has been used to predict the severity of Rh haemolytic disease of the newborn [7] by measurement of the haemolytic potential of maternal anti-D, and (with bromelain) to compare the lytic activity of different monoclonal antibodies [6]. Lysis of red cells is mediated by K ceils in both assay systems. Individual workers have routinely used either bromelain or papain for K cell-mediated assays. It was observed by the authors that bromelain-treated red cells were not lysed in the first assay system (i.e., using a low effector-to-red cell ratio), and thus a comparison was made of the effectiveness of bromelain and papain pretreatment of red cell targets in both these assays.
0 1 6 5 - 2 4 7 8 / 92 / ~; ~ O0 (~3 lOq~ ]~l~evler ~elc.nt-p lanhllch,re 1~ Ttl All r;,ht . . . . . . . . . A
,'~,~,'7
3. Materials and Methods Bromelain and papain solutions, both containing cysteine, were routine blood grouping reagents supplied by the South Western Regional Transfusion Service ( S W R T C ) and International Blood G r o u p Reference L a b o r a t o r y (IBGRL), respectively. Proteolytic activity was determined by the a z o a l b u m i n m e t h o d [8]. Packed red cells were treated by incubation at 37 °C with an equal volume o f enzyme reagent for 15 min followed by washing three times in phosphate-buffered saline (PBS). Agglutination o f enzyme-treated O Rh-positive or A Rh-negative
red cells by monoclonal or polyclonal anti-D or anti-A, respectively, was assessed in microtitre plates [9]. H u m a n monoclonal anti-D antibodies were all o f the IgG1 subclass except 1A3-3 which was IgG3 [9]; murine monoclonal anti-A (IgG3) was kindly supplied by Dr. P. J u d s o n (South Western Regional Transfusion Centre, Bristol). Polyclonal anti-D was used as a 1/100 dilution o f a therapeutic IgG anti-D immunoglobulin preparation, and polyclonal h u m a n anti-A (predominantly IgG1) was obtained from Biotest. A D C C assays were p e r f o r m e d using (i) non-adherent lymphocyte effector cells from the F B M C fraction, at a 15:1
100B0 O1
_>~
60 1A3-3
40
2O' .__.__--------e O~_L 100-
~
: : : ,::::
100.
BO In
80
--~ 60,
4.0
401
BRIC 54
20
01
•
20,
100-
100,
8O
80
=2
m
"~ _>,
60
40
4O
2OI
20
0 10
100
0=
1000
Reciproc d i l u t i o n
10 of
I00
ontibody
Fig. 1. Lysis of anti-D-sensitised O R i R 1 red cells by non-adherent lymphocytes at a high (15:1) effector-to-target red cell ratio. Equal volumes of bromelain-treated (open circles), papain-treated (filled circles), and untreated (filled squares) labelled red cells (8 × 105/ml), effector ceils (12 × 106/ml) and antibody were incubated together for 18 h at 37°C. Both enzymes were used at equal proteolytic concentrations (5 U/ml). The IgG concentrations of the antibody preparations were 7.0/~g human IgG3 anti-D per ml (IA3-3), 5.0 ~g human IgG1 anti-D per ml (2B6), 32/~g murine IgG3 anti-A per ml (BRIC 54) and 4.0 ~tg IgG anti-D per ml (polyclonal anti-D, which was a 1/100 dilution of a therapeutic anti-D immunoglobulin preparation containing IgG1 and lgG3 anti-D). 238
effector (E)-to-labelled target red cell (T) ratio [6]; or (it) unseparated PBMC at a 1:3:3 effector cell (E)-to-labelled target red cell (T)-to-unlabelled competitor red cell (C) ratio (manuscript in preparation). Target cells were O Rh-positive with anti-D and A Rh-negative with anti-A, competitor cells were A Rh-negative with anti-D or O Rh-positive with anti-A. Target red cells were labelled with sodium 51chromate (Amersham plc, Bucks, UK) before use in ADCC assays, and the antibodies were in the fluid phase. 4. Results and Discussion
When used at equal proteolytic concentration, pretreatment of red cells with bromelain or papain resulted in different agglutination titres (Table 1). Anti-D gave greater titres with papain-treated red cells, whereas the anti-A titres were slightly greater with bromelain. Anti-D did not agglutinate native red cells. In an ADCC assay using non-adherent lymphocyte effector cells at a high E:T ratio (15:1), papaintreated red cells were more sensitive to lysis than bromelain-treated red cells at low antibody concentrations (Fig. 1). This effect was more marked for anti-D than for anti-A, and was not observed in antibody excess. Lysis of untreated red ceils was less than that of enzyme-treated red cells. However, in ADCC assays utilizing a lower TABLE 1 Agglutination titres of polyclonal and monoclonal anti-D and anti-A. The proteolytic activity of both enzymes was 5 U / m l . Polyclonal anti-D contained 4 ~tg IgG anti-D per ml. Monoclonal anti-D, 1A3-3, contained 7/~g h u m a n IgG 3 per ml. Polyclonal anti-A was predominantly IgG 1. Monoclonal anti-A, BRIC 54, contained 32/~g murine IgG 3 per ml. Antibody
Red cells Red cell treatment Bromelain Papain Untreated
Polyclonal anti-D Monoconal anti-D
ORIR I ORIR l
Polyclonal antioA Monoclonal anti-A
Arr Arr
4000 a 4000 16000 128
aAgglutination titre.
16000 16000
0 0
8000 64
64 1
TABLE 2 Lysis of enzyme-treated red cells in A D C C assays at a low P B M C effectorcell-to-targetandcompetitorcellratio(E:T:C = 1:3:3). Equal volumes of effector cells (6× 106 P B M C / m l ) , labelled target red cells (18 x 106/ml), unlabelled competitor red cells (18 × 106/ml) and antibody were incubated together for 18 h at 37°C. % Lysis Bromelain, PBMC
Papain, P B M C
Monoclonal anti-D 1/8 FEF5 0 H27 - 1 AB5 - 1 R1D7 0 FC3 - 1 FEF3 0 1A3-1 - I H26 nd 2B6 - 1 1C6 0 1A3-3 1
57 53 54 61 46 54 49 41 58 60 37
Polyclonal anti-A 1/16 14407 2 Biotest 1 Merz-Dade lgG fraction Sla Kur Pic Sze
67 57 66 67 66 63
Monoelonal anti-A 1/16 BRIC37 BRIC54 BRIC56
41 46 51
Control
0
3
PBMC effector-to-target cell ratio, and in the presence of competing red cells (E:T:C = 1:3:3), bromelain-treated red cells were not lysed, whereas all samples of papain-treated red cells were lysed to some extent (Table 2). It is possible that at this relatively high target red cell concentration (22.5 times greater than in the previous ADCC assay), less antibody was bound to the target cells, and due to the presence also of the competitor red cells the relative insensitivity of bromelain-treated cells may then have been so great as to render them incapable of lysis in this system. In a separate experiment, it 239
was found that bromelain-treated red cells were not sensitive to lysis by polyclonal anti-D, nor were untreated red cells by any of the antibodies. In summary, although both bromelain and papain treatment of red cells were found to give satisfactory red cell agglutination and K cell-mediated lysis by anti-D and anti-A in the presence of excess antibody, papain treatment resulted in greater sensitivity of agglutination and haemolysis at low concentrations of anti-D. Acknowledgements The authors are grateful to Dr. M. Scott (IBGRL) and Mr. G. Poole (SWRTC) for the papain and bromelain reagents, to Mr. A. Guest for performing the azoalbumin assay and to the Central Blood Laboratories Authority and the British Council for funding.
240
References [1] van Oss, C. J. and Absolom, D. R. (1984) Vox Sang. 47, 250. [2] Kumpel, B. M. and Hadley, A. G. (1990) MoI. lmmunol. 27,247. [3] Yush 1., Frisch, B. and Goldsher, N. (1980) Eur. J. lmmunol. 10, 127. [4] Bakfics, T., Czanik, P., Kimber, 1., Ringwald, G., Moore, M. and Abraham, E. (1984) Cancer lmmunol. Immunother. 16, 170. [51 Urbaniak, S. J. (1976) Br. J. Haematol. 33,409. [61 Kumpel, B.M., Leader, K . A . , Merry, A. H., Hadley, A. G., Poole, G. D., Blancher, A., Goossens, D., HughesJones, N. C. and Bradley, B. A. (1989a) Eur. J. lmmunol. 19, 2283. [71 Urbaniak, S. J., Ayoub Griess, M., Crawtbrd, R. J. and Fergusson, M. ]. C. (1984) Vox Sang. 46, 323. [8] Lambert, R., Edwards, J. and Anstee, D. J. (1978) Med. Lab. Sci. 35,233. [91 Kumpel, B. M., Poole, G. D. and Bradley, B. A. (1989b) Br. J. Haematol. 71, 125.