Immunology Letters, 30 (1991) 87- 92 Elsevier
IMLET 01660
A mouse monoclonal antibody against glycophorin A produced by in vitro stimulation with human red cell membranes J a n Kolberg I a n d D o m i n i q u e B l a n c h a r d 2 tDepartment of Immunology, National Institute of Public Health, Oslo, Norway, and 2Centre R~gional de Transfusion Sanguine, Nantes, France (Received 10 April 1991;revision received 3 June 1991;accepted 7 June 1991)
1. Summary H u m a n erythrocyte membranes were used as antigen for production o f mouse monoclonal antibodies against blood group related structures by in vitro immunization. Culture medium supernatant of P H A and P M A stimulated mouse thymus cells was used as source of cytokines. The selected antibody designated 124,D-7 (isotype IgM) was found to directly agglutinate all human red cells, except the rare erythrocytes E n ( a - ) which lack glycophorin A. Immunoblotting showed faint bands in the positions of glycophorin A, whereas no binding occurred to glycophorin B. Inhibition o f agglutination with purified glycophorin A and peptides suggests that the epitope is located within the amino acid residues 3 5 - 4 0 . Rat and chicken erythrocytes also reacted with the antibody, whereas mouse erythrocytes were only agglutinated at very low dilutions of ascitic fluid. 2. Introduction In vitro stimulation of splenocytes for production of murine or human monoclonal antibodies is a time-saving alternative to in vivo immunization. The normal regulation of the immune response does not function in vitro, and it is thus possible to obtain a Key words: In vitro immunization; Erythrocyte membrane; GlycophorinA; Monoclonal antibody Correspondence to: Jan Kolberg, Department of Immunology, National Instituteof Public Health, Geitmyrsveien75, 0462Oslo 4, Norway.
response against structures against which antibodies cannot normally be produced. Difficulties have been reported in producing rodent mAbs to certain human antigens of diagnostic importance, such as Rh antigens [1]. We therefore decided to use in vitro stimulation to see if it was possible to get murine antibodies against blood group related determinants on the human red cell membrane. In this study we describe a mAb with specificity for glycophorin A, a major sialoglycoprotein which carries the MN antigens (for reviews see [2, 3]). 3. Materials and Methods 3.1. Antigen Haemoglobin-free ghosts from fresh human erythrocytes of blood type OR2REMNK ÷ were isolated [4]. The protein concentration was determined with reference to bovine serum albumin [5]. The membrane solution containing 7 mg/ml was sterilized by gamma radiation at 15 kGy (Institute for Energy Technology, Norway). 3.2. Basal medium and preparation o f thymocyteconditioned medium
The basal medium consisted o f a 1:1 mixture (by volume) of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12. This m~dium was supplemented with 3% heat-inactivated rabbit serum, 100 IU/ml of penicillin and 100/~g/ml streptomycin. The media and supplements were obtained from
0165-2478 / 91 / $3.50 © 1991 ElsevierSciencePublishers B.V.All rights reserved.
87
Gibco. Thymus cells were obtained from thymus glands of 12 BALB/c mice (aged up to 4 weeks). The cells (5 x 106/ml) were cultured for 48 h in 80 cm 2 plastic culture flasks (Nunc) in 50 ml medium. The basal medium was supplemented with 25 tzM 2mercaptoethanol (Biorad), 1/~g/ml of PHA (purified, HA 16, Wellcome) and 10 ng/ml of phorbol myristate acetate (PMA) (Sigma). The culture supernatants were then harvested, centrifuged to remove debris and filter-sterilized using a 0.2/~M filter. The TCM medium was stored at - 7 0 ° C until use. 3.3. In vitro stimulation Spleen cells from non-immunized male BALB/c mice aged 6 - 8 weeks were cultured for 5 days in 80 cm 2 plastic culture flasks (Nunc) in 50 ml medium at a density of 4x106/ml. The basal medium was supplemented with 40% TCM (by volume). To this medium was added rabbit serum (3%), 2mercaptoethanol (25 I~M) and pokeweed mitogen (1/~g/ml) (Sigma). The antigen was added at concentrations from 10-100 #g/ml.
3.5. Agglutination assays and enzyme treatments E n ( a - ) cells (G.W.) were kindly provided by Dr. Juhani Leikola, Helsingfors, Finland. Packed erythrocytes (0.2 ml) were suspended in 0.4 ml phosphate-buffered saline (PBS) pH 7.4 containing 0.8 mg trypsin (Type XI from bovine pancreas, Sigma) and incubated for 1 h at 37 °C. Sialidase treatment was performed as previously described [6]. Two-fold serial dilutions of the antibodies were performed in microplates with U-bottomed wells in 50 #1PBS (pH 7.4). To each well was added 50/zl 1% red cell suspension in PBS containing 2% bovine serum albumin. Agglutination was read without centrifugation.
3.6. Glycophorins and peptides Glycophorins A and B were extracted fro,n red cell membranes and purified by high performance liquid chromatography [7]. Primary tryptic and chymotryptic fragments from glycophorin A and B were purified as previously described [7, 8]. 3.7. SDS-gel electrophoresis and immunoblotting
3.4. Cell fusion and cloning The stimulated spleen cells were harvested and mixed with NSO myeloma cells at a ratio of 2:1. The fusion was performed by standard methods with polyethylene glycol 1450 (Eastman Kodak Company, USA) as the fusing agent. HECS (human endothelial culture supernatant) from Costar Europe Ltd was used instead of feeder cells. Screening was performed by transferring 50 #1 of each supernatant to wells in microtitre plates followed by addition of 50/zl of a 1% solution of erythrocytes from the donor whose red cells had been used to produce antigen. Hybrids producing antibodies were cloned by limiting dilution. Cloned hybridoma cells were injected into pristane primed BALB/c mice for production of ascitic fluid. MAb in cell culture medium was bound to wells coated with erythrocyte membranes and isotyped using a kit (93-6550) from Zymed Lab. Inc. USA with biotinylated anti-mouse antibodies and a streptavidin conjugate.
88
Red cell membrane proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis in a 10%0 separating gel and blotted to nitrocellulose papers essentially as described previously [9]. Blocked nitrocellulose papers were incubated overnight with the antibodies diluted in 10 mM Tris-HCl with 0.15 M NaC1 (pH 7.4) and 1% BSA. The papers were washed and incubated with alkaline phosphatase labelled goat anti-mouse Ig (Bioatlantic, France) diluted 1:I000, and stained with nitro blue tetrazolium. 4. Results
4.1. Stimulation conditions and fusion efficiencies Seven in vitro immunizations were performed with human red cell membranes as antigen and agglutination was used as the screening assay. The average fusion efficiency (No. wells with hybridomas/No, wells seeded) was 75% whereas the specific efficiency (No. wells with anti-red blood cell antibodies/No, wells with hybridomas) was usually less
than 1%. The antigen concentration was varied from 10 to 100/zg/ml and within this range there was no clear dose-response relationship. In two experiments the pooled splenocytes from 12 mice were divided into two groups, one with addition of antigen and the other without antigen. The two group of cultures yielded after fusion a similar number of growing hybrids, but agglutinating antibodies were only produced by hybrids from antigen stimulated splenocytes. 4.2. Agglutination assays Strongly reacting antibodies in the initial screening were further examined by agglutination with red cells from standard blood bank panels. All antibodies except one reacted with human erythrocytes without showing any specificity against blood group related antigens. The mAb designated 124,D-7 (isotype IgM) was obtained from a fusion with splenocytes stimulated with 100 #g/ml of the antigen. The mAb caused direct agglutination with all human erythrocytes, but treatment of these erythrocytes with sialidase or trypsin destroyed their reactivities with the antibody (Table 1). No difference was observed between MM and NN erythrocytes. E n ( a - ) cells, which lack glycophorin A, failed to react with the antibody. The mAb agglutination of human erythrocytes were inhibited by a low concentration of purified glycophorin A, whereas a high concentration was needed of the only peptide (35 - 64) that was inhibitory (Table 2). Glycophorin B and its Nterminal peptide had no effect. Erythrocytes from rat and chicken were also agTABLE 1 Agglutination of untreated and enzyme-treated erythrocytes by mAb 124,D-7. Titre a Untreated Human normal En(a - ) Rat Hen Mouse
32 000 0 16000 8000 32
Trypsin
Sialidase
2
0
0 0 64
32000 64000 0
a Starting with ascitic fluid diluted 1:10.
TABLE 2 Agglutination inhibition of mAb 124,D-7 by glycophorins and peptides purified from glycophorins. Inhibitor
Inhibitory activity (mg/ml)
Butanol extract Glycophorin A Dansylated glycophorin A Glycophorin A-C1 (1 - 64) Glycophorin A-C3 ( 3 5 - 64) Glycophorin A-T3 (40 - 61) Glycophorin B Glycophorin B-T1 (1 - 35)
0.013 0.0625 0.02 > 10 5 > 10 > 10 >5
C and T stand for chymotrypsin and trypsin, respectively.
glutinated by the antibody, but the reaction patterns were different from that with human red cells since the agglutination of these animal cells was not sensitive to sialidase-treatment. Erythrocytes from mouse were agglutinated at very low dilutions of ascitic fluid (Table 1), whereas red cells from pig and rabbit were non-reactive (results not shown). The native mouse red cells were not agglutinated by 6 other ascitic fluids containing antibodies against irrelevant antigens, but this does not necessarily exclude the possibility of a contaminating antibody in the 124,D-7 ascitic fluid. 4.4. Immunoblotting Faint bands were visible in the positions of glycophorin A and its homo- (glycophorin A x 2) and heterodimers (glycophorin A+B) whereas no staining of glycophorin B and asialoglycophorin A could be demonstrated (results not shown). 5. Discussion
The medium used in our in vitro stimulation system with erythrocyte membranes as antigen was supplemented with supernatants from PHA and PMA activated murine thymocytes. Others have used both thymoma cells (EL 4) conditioned medium and supernatants from mixed lymphocyte cultures [10]. The high numbers of wells with growing hybridoma ceils indicate that our medium contained sufficient factors for B cell growth and differentiation. However, the percentage of specific antibody89
producing hybrid cells was very low, but this might reflect that agglutination was used for screening. This technique most likely favours the selection of antibodies of isotype IgM with high affinity for red cell receptors whereas antibodies with low affinities probably do not cause agglutination. Our finding that in vitro stimulations with and without antigen yielded a similar number of growing hybrids after fusion is in accordance with that reported before [11]. MAb 124,D-7 agglutinated all normal human erythrocytes irrespective of blood type. Lack of reactivity with the rare erythrocytes E n ( a - ) which do not contain glycophorin A [12, 13] indicated that the epitope could be on this membrane protein. The major human erythrocyte sialoglycoproteins, glycophorins A and B have been extensively characterized (for reviews, see [2, 3]). Glycophorin A expresses M or N antigen, and this expression is governed by the nature of the amino acid residues at positions I and 5. Glycophorin B also has N activity. The complete amino acid sequences of both these proteins have been determined and the N-terminal residues were found to be identical [7, 14]. Agglutination with mAb 124,D-7 was inhibited by purified glycophorin A, whereas glycophorin B had no effect. These findings were supported by immunoblotring of red cell membrane proteins. The epitope therefore cannot be located in the N-terminal homologous sequence domain (residues 1-26) of the glycophorins A and B. This and the sensitivity to trypsin treatment of the erythrocytes suggest that the epitope is located within amino acid residue 27 and the trypsin cleavage site at position 39 [14]. The glycophorin A peptide (residues 35-64) inhibited agglutination, but a high concentration was needed. We therefore propose that the epitope for mAb 124,D-7 is localized to residues 35-40. The antibody did not agglutinate sialidase-treated human erythrocytes and failed to bind to asialoglycophorin A. The native protein is heavily glycosylated with numerous O-glycans, the predominant form of which is a disialotetrasaccharide. The region (35-40) contains only one oligosaccharide unit linked to thr in position 37 [3] and the sialic acid residues in this unit might be part of the epitope. It is also possible that the determinant lacks sialic acid at the combining site but is sialic acid dependent because its conformation depends on the presence of 90
sialic acids at some distant residues. There are some other reports of blood group unrelated mAbs against glycophorin A (15 -20). Eighteen of these mAbs were tested against sialidase as well as trypsin-treated erythrocytes, but only BRIC 119 (isotype IgG) [19] showed a reaction pattern similar to mAb 124,D-7. The epitope for mAb BRIC 119 was proposed to be in the region 37- 39. The antibody described here (124,D-7) is of isotype IgM and therefore more useful in agglutination assays. This is important because the glycophorin A expression is restricted to the erythroid lineage [21]. This antibody, reacting with a sialidase-sensitive epitope, can therefore be used together with mAbs against sialidase-resistant epitopes on glycophorin A to detect erythroid differentiation by leukemia ceils. MAb 124,D-7 can also be used to remove contaminating erythroid lineage cells from Ficoll-Paque leukocytes [22]. The primary structure of a major mouse glycophorin deduced from cDNA clones showed a strong homology with human glycophorin A only in the transmembrane domain [23]. We found that the mAb 124,D-7 reacted only with mouse erythrocytes at very low dilutions of the antibody. It is therefore unlikely that the selected hybridoma cell line 124,D-7 produces natural mouse antibodies against a self antigen. Most likely the mAb is a result of a primary, antigen specific activation of B lymphocytes in culture.
Acknowledgements We thank Gunnhild Voile, Torunn Steen and Val6rie Bruneau for skillful technical assistance.
References [1] [2] [3] [4]
Thompson, K. M. (1988) Immunol. Today 9, 113. Anstee, D. J. (1990) Vox Sang. 58, 1. Blanchard, D. (1990) Transfusion Med. Rev. 3, 170. Dodge, J. T., Mitchell, C. and Hanahan, D. T. (1963) Arch. Biochem. Biophys. 100, 119. [5] Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265. [6] Kolberg, J., Michaelsen, T. E. and Sletten, K. (1983) HoppeSeyler's Z. Physiol. Chem. 364, 655. [7] Blanchard, D., Dahr, W., Hummel, M., Latron, E, Beyreuther, K. and Cartoon, J.-P. (1987) J. Biol. Chem. 262, 5808.
[8] Dahr, W., Muller, T., Moulds, J., Baumeister, G., Issitt, P. D., Wilkinson, S. and Garratty, G. (1985) Biol. Chem. HoppeSeyler 366, 41. [9] E1-Maliki, B., Blanchard, D., Dahr, W., Beyreuther, K. and Cartron, J.-P. (1989) Eur. J. Biochem. 183, 639. [10] Borrebaeck, C. A. K. (1986) Trends Biotechnol. 4, 147. [11] M611er, S. A. and Borrebaeck, C. A. K. (1988) in: In Vitro Immunization in Hybridoma Technology (C. A. K. Borrebaeck, Ed.) pp. 3-22. Elsevier, Amsterdam. [12] Dahr, W., Uhlenbruck, G., Leikola, J., Wagstaff, W. and Landfried, K. (1976) J. Immunogenet. 3, 329. [13] Gahmberg, C. G., Myllyla, G., Leikola, J., Pirkola, A. and Nordling, S. (1976) J. Biol. Chem. 251, 6108. [14] Tomita, M., Furthmayr, H. and Marchesi, V. T. (1978) Biochemistry 17, 4756. [15] Anstee, D. J. and Edwards, P. A. W. (1982) Eur. J. Immunol. 12, 228.
[16] Ochiai, Y., Furthmayr, H. and Marcus, D. M. (1983) J. Immunol. 131, 864. [17] Wasniowska, K., Schroer, K. R., McGinniss, M., Reichert, C. and Zopf, D. (1988) Hybridoma 7, 49. [18] Bigbee, W. L., Langlois, R. G., Vanderlaan, M. and Jensen, R. H. (1984) J. Immunol. t33, 3149. [19] Gardner, B., Parsons, S. E, Merry, A. H. and Anstee, D. J. (1989) Immunology 68, 283. [20] Anstee, D. J. and Lisowska, E. (1990) J. Immunogenet. 17, 301. [21] Gahmberg, C. G., Jokinen, M. and Andersson, L. C. (1978) Blood 52, 379. [22] Heldrup, J. (1990) Scand. J. Immunol. 31, 289. [23] Matsui, Y., Natori, S. and Obinata, M. (1989) Gene 77, 325.
91
IMLET 01660
A mouse monoclonal antibody against glycophorin A produced by in vitro stimulation with human red cell membranes J a n Kolberg I a n d D o m i n i q u e B l a n c h a r d 2 tDepartment of Immunology, National Institute of Public Health, Oslo, Norway, and 2Centre R~gional de Transfusion Sanguine, Nantes, France (Received 10 April 1991;revision received 3 June 1991;accepted 7 June 1991)
1. Summary H u m a n erythrocyte membranes were used as antigen for production o f mouse monoclonal antibodies against blood group related structures by in vitro immunization. Culture medium supernatant of P H A and P M A stimulated mouse thymus cells was used as source of cytokines. The selected antibody designated 124,D-7 (isotype IgM) was found to directly agglutinate all human red cells, except the rare erythrocytes E n ( a - ) which lack glycophorin A. Immunoblotting showed faint bands in the positions of glycophorin A, whereas no binding occurred to glycophorin B. Inhibition o f agglutination with purified glycophorin A and peptides suggests that the epitope is located within the amino acid residues 3 5 - 4 0 . Rat and chicken erythrocytes also reacted with the antibody, whereas mouse erythrocytes were only agglutinated at very low dilutions of ascitic fluid. 2. Introduction In vitro stimulation of splenocytes for production of murine or human monoclonal antibodies is a time-saving alternative to in vivo immunization. The normal regulation of the immune response does not function in vitro, and it is thus possible to obtain a Key words: In vitro immunization; Erythrocyte membrane; GlycophorinA; Monoclonal antibody Correspondence to: Jan Kolberg, Department of Immunology, National Instituteof Public Health, Geitmyrsveien75, 0462Oslo 4, Norway.
response against structures against which antibodies cannot normally be produced. Difficulties have been reported in producing rodent mAbs to certain human antigens of diagnostic importance, such as Rh antigens [1]. We therefore decided to use in vitro stimulation to see if it was possible to get murine antibodies against blood group related determinants on the human red cell membrane. In this study we describe a mAb with specificity for glycophorin A, a major sialoglycoprotein which carries the MN antigens (for reviews see [2, 3]). 3. Materials and Methods 3.1. Antigen Haemoglobin-free ghosts from fresh human erythrocytes of blood type OR2REMNK ÷ were isolated [4]. The protein concentration was determined with reference to bovine serum albumin [5]. The membrane solution containing 7 mg/ml was sterilized by gamma radiation at 15 kGy (Institute for Energy Technology, Norway). 3.2. Basal medium and preparation o f thymocyteconditioned medium
The basal medium consisted o f a 1:1 mixture (by volume) of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12. This m~dium was supplemented with 3% heat-inactivated rabbit serum, 100 IU/ml of penicillin and 100/~g/ml streptomycin. The media and supplements were obtained from
0165-2478 / 91 / $3.50 © 1991 ElsevierSciencePublishers B.V.All rights reserved.
87
Gibco. Thymus cells were obtained from thymus glands of 12 BALB/c mice (aged up to 4 weeks). The cells (5 x 106/ml) were cultured for 48 h in 80 cm 2 plastic culture flasks (Nunc) in 50 ml medium. The basal medium was supplemented with 25 tzM 2mercaptoethanol (Biorad), 1/~g/ml of PHA (purified, HA 16, Wellcome) and 10 ng/ml of phorbol myristate acetate (PMA) (Sigma). The culture supernatants were then harvested, centrifuged to remove debris and filter-sterilized using a 0.2/~M filter. The TCM medium was stored at - 7 0 ° C until use. 3.3. In vitro stimulation Spleen cells from non-immunized male BALB/c mice aged 6 - 8 weeks were cultured for 5 days in 80 cm 2 plastic culture flasks (Nunc) in 50 ml medium at a density of 4x106/ml. The basal medium was supplemented with 40% TCM (by volume). To this medium was added rabbit serum (3%), 2mercaptoethanol (25 I~M) and pokeweed mitogen (1/~g/ml) (Sigma). The antigen was added at concentrations from 10-100 #g/ml.
3.5. Agglutination assays and enzyme treatments E n ( a - ) cells (G.W.) were kindly provided by Dr. Juhani Leikola, Helsingfors, Finland. Packed erythrocytes (0.2 ml) were suspended in 0.4 ml phosphate-buffered saline (PBS) pH 7.4 containing 0.8 mg trypsin (Type XI from bovine pancreas, Sigma) and incubated for 1 h at 37 °C. Sialidase treatment was performed as previously described [6]. Two-fold serial dilutions of the antibodies were performed in microplates with U-bottomed wells in 50 #1PBS (pH 7.4). To each well was added 50/zl 1% red cell suspension in PBS containing 2% bovine serum albumin. Agglutination was read without centrifugation.
3.6. Glycophorins and peptides Glycophorins A and B were extracted fro,n red cell membranes and purified by high performance liquid chromatography [7]. Primary tryptic and chymotryptic fragments from glycophorin A and B were purified as previously described [7, 8]. 3.7. SDS-gel electrophoresis and immunoblotting
3.4. Cell fusion and cloning The stimulated spleen cells were harvested and mixed with NSO myeloma cells at a ratio of 2:1. The fusion was performed by standard methods with polyethylene glycol 1450 (Eastman Kodak Company, USA) as the fusing agent. HECS (human endothelial culture supernatant) from Costar Europe Ltd was used instead of feeder cells. Screening was performed by transferring 50 #1 of each supernatant to wells in microtitre plates followed by addition of 50/zl of a 1% solution of erythrocytes from the donor whose red cells had been used to produce antigen. Hybrids producing antibodies were cloned by limiting dilution. Cloned hybridoma cells were injected into pristane primed BALB/c mice for production of ascitic fluid. MAb in cell culture medium was bound to wells coated with erythrocyte membranes and isotyped using a kit (93-6550) from Zymed Lab. Inc. USA with biotinylated anti-mouse antibodies and a streptavidin conjugate.
88
Red cell membrane proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis in a 10%0 separating gel and blotted to nitrocellulose papers essentially as described previously [9]. Blocked nitrocellulose papers were incubated overnight with the antibodies diluted in 10 mM Tris-HCl with 0.15 M NaC1 (pH 7.4) and 1% BSA. The papers were washed and incubated with alkaline phosphatase labelled goat anti-mouse Ig (Bioatlantic, France) diluted 1:I000, and stained with nitro blue tetrazolium. 4. Results
4.1. Stimulation conditions and fusion efficiencies Seven in vitro immunizations were performed with human red cell membranes as antigen and agglutination was used as the screening assay. The average fusion efficiency (No. wells with hybridomas/No, wells seeded) was 75% whereas the specific efficiency (No. wells with anti-red blood cell antibodies/No, wells with hybridomas) was usually less
than 1%. The antigen concentration was varied from 10 to 100/zg/ml and within this range there was no clear dose-response relationship. In two experiments the pooled splenocytes from 12 mice were divided into two groups, one with addition of antigen and the other without antigen. The two group of cultures yielded after fusion a similar number of growing hybrids, but agglutinating antibodies were only produced by hybrids from antigen stimulated splenocytes. 4.2. Agglutination assays Strongly reacting antibodies in the initial screening were further examined by agglutination with red cells from standard blood bank panels. All antibodies except one reacted with human erythrocytes without showing any specificity against blood group related antigens. The mAb designated 124,D-7 (isotype IgM) was obtained from a fusion with splenocytes stimulated with 100 #g/ml of the antigen. The mAb caused direct agglutination with all human erythrocytes, but treatment of these erythrocytes with sialidase or trypsin destroyed their reactivities with the antibody (Table 1). No difference was observed between MM and NN erythrocytes. E n ( a - ) cells, which lack glycophorin A, failed to react with the antibody. The mAb agglutination of human erythrocytes were inhibited by a low concentration of purified glycophorin A, whereas a high concentration was needed of the only peptide (35 - 64) that was inhibitory (Table 2). Glycophorin B and its Nterminal peptide had no effect. Erythrocytes from rat and chicken were also agTABLE 1 Agglutination of untreated and enzyme-treated erythrocytes by mAb 124,D-7. Titre a Untreated Human normal En(a - ) Rat Hen Mouse
32 000 0 16000 8000 32
Trypsin
Sialidase
2
0
0 0 64
32000 64000 0
a Starting with ascitic fluid diluted 1:10.
TABLE 2 Agglutination inhibition of mAb 124,D-7 by glycophorins and peptides purified from glycophorins. Inhibitor
Inhibitory activity (mg/ml)
Butanol extract Glycophorin A Dansylated glycophorin A Glycophorin A-C1 (1 - 64) Glycophorin A-C3 ( 3 5 - 64) Glycophorin A-T3 (40 - 61) Glycophorin B Glycophorin B-T1 (1 - 35)
0.013 0.0625 0.02 > 10 5 > 10 > 10 >5
C and T stand for chymotrypsin and trypsin, respectively.
glutinated by the antibody, but the reaction patterns were different from that with human red cells since the agglutination of these animal cells was not sensitive to sialidase-treatment. Erythrocytes from mouse were agglutinated at very low dilutions of ascitic fluid (Table 1), whereas red cells from pig and rabbit were non-reactive (results not shown). The native mouse red cells were not agglutinated by 6 other ascitic fluids containing antibodies against irrelevant antigens, but this does not necessarily exclude the possibility of a contaminating antibody in the 124,D-7 ascitic fluid. 4.4. Immunoblotting Faint bands were visible in the positions of glycophorin A and its homo- (glycophorin A x 2) and heterodimers (glycophorin A+B) whereas no staining of glycophorin B and asialoglycophorin A could be demonstrated (results not shown). 5. Discussion
The medium used in our in vitro stimulation system with erythrocyte membranes as antigen was supplemented with supernatants from PHA and PMA activated murine thymocytes. Others have used both thymoma cells (EL 4) conditioned medium and supernatants from mixed lymphocyte cultures [10]. The high numbers of wells with growing hybridoma ceils indicate that our medium contained sufficient factors for B cell growth and differentiation. However, the percentage of specific antibody89
producing hybrid cells was very low, but this might reflect that agglutination was used for screening. This technique most likely favours the selection of antibodies of isotype IgM with high affinity for red cell receptors whereas antibodies with low affinities probably do not cause agglutination. Our finding that in vitro stimulations with and without antigen yielded a similar number of growing hybrids after fusion is in accordance with that reported before [11]. MAb 124,D-7 agglutinated all normal human erythrocytes irrespective of blood type. Lack of reactivity with the rare erythrocytes E n ( a - ) which do not contain glycophorin A [12, 13] indicated that the epitope could be on this membrane protein. The major human erythrocyte sialoglycoproteins, glycophorins A and B have been extensively characterized (for reviews, see [2, 3]). Glycophorin A expresses M or N antigen, and this expression is governed by the nature of the amino acid residues at positions I and 5. Glycophorin B also has N activity. The complete amino acid sequences of both these proteins have been determined and the N-terminal residues were found to be identical [7, 14]. Agglutination with mAb 124,D-7 was inhibited by purified glycophorin A, whereas glycophorin B had no effect. These findings were supported by immunoblotring of red cell membrane proteins. The epitope therefore cannot be located in the N-terminal homologous sequence domain (residues 1-26) of the glycophorins A and B. This and the sensitivity to trypsin treatment of the erythrocytes suggest that the epitope is located within amino acid residue 27 and the trypsin cleavage site at position 39 [14]. The glycophorin A peptide (residues 35-64) inhibited agglutination, but a high concentration was needed. We therefore propose that the epitope for mAb 124,D-7 is localized to residues 35-40. The antibody did not agglutinate sialidase-treated human erythrocytes and failed to bind to asialoglycophorin A. The native protein is heavily glycosylated with numerous O-glycans, the predominant form of which is a disialotetrasaccharide. The region (35-40) contains only one oligosaccharide unit linked to thr in position 37 [3] and the sialic acid residues in this unit might be part of the epitope. It is also possible that the determinant lacks sialic acid at the combining site but is sialic acid dependent because its conformation depends on the presence of 90
sialic acids at some distant residues. There are some other reports of blood group unrelated mAbs against glycophorin A (15 -20). Eighteen of these mAbs were tested against sialidase as well as trypsin-treated erythrocytes, but only BRIC 119 (isotype IgG) [19] showed a reaction pattern similar to mAb 124,D-7. The epitope for mAb BRIC 119 was proposed to be in the region 37- 39. The antibody described here (124,D-7) is of isotype IgM and therefore more useful in agglutination assays. This is important because the glycophorin A expression is restricted to the erythroid lineage [21]. This antibody, reacting with a sialidase-sensitive epitope, can therefore be used together with mAbs against sialidase-resistant epitopes on glycophorin A to detect erythroid differentiation by leukemia ceils. MAb 124,D-7 can also be used to remove contaminating erythroid lineage cells from Ficoll-Paque leukocytes [22]. The primary structure of a major mouse glycophorin deduced from cDNA clones showed a strong homology with human glycophorin A only in the transmembrane domain [23]. We found that the mAb 124,D-7 reacted only with mouse erythrocytes at very low dilutions of the antibody. It is therefore unlikely that the selected hybridoma cell line 124,D-7 produces natural mouse antibodies against a self antigen. Most likely the mAb is a result of a primary, antigen specific activation of B lymphocytes in culture.
Acknowledgements We thank Gunnhild Voile, Torunn Steen and Val6rie Bruneau for skillful technical assistance.
References [1] [2] [3] [4]
Thompson, K. M. (1988) Immunol. Today 9, 113. Anstee, D. J. (1990) Vox Sang. 58, 1. Blanchard, D. (1990) Transfusion Med. Rev. 3, 170. Dodge, J. T., Mitchell, C. and Hanahan, D. T. (1963) Arch. Biochem. Biophys. 100, 119. [5] Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265. [6] Kolberg, J., Michaelsen, T. E. and Sletten, K. (1983) HoppeSeyler's Z. Physiol. Chem. 364, 655. [7] Blanchard, D., Dahr, W., Hummel, M., Latron, E, Beyreuther, K. and Cartoon, J.-P. (1987) J. Biol. Chem. 262, 5808.
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