Murine monoclonal antibody MB-2D10 recognizes Rh-related glycoproteins in the human red cell membrane G. MALLINSON, D.J. ANSTEE,N.D. AVENT,K. RIDGWELL,M.J.A. TANNER, G.L. DANIELS,P. TIPPET, AND A.E.G. VON DEM BORNE The human red cell membrane components reacting with monoclonal antibody MB2010 were examined by immunoblotting. The antibody bound to a diffusely staining band extending from M, 30,000 up to the high-molecular-wei ht region of the gel in normal membranes and in Rh,,,, U + membranes, but not in h,,,, U - membranes. Treatment of normal red cells with an endoglycosidase F-containing preparation destro ed the epitope recognized b MB-2D10. The reactivity of the antibody with puribd preparations of Rh-relate glycoproteins D, polypeptide, D, polypep!ide, R6A,, polypeptide, and R6&, polypeptide was also examined. Only the purified R6A4, and D,, components reacted with MB-2D10. These results show that MB2D10 recognizes a carbohydrate-dependent epitope on the R6A.45 and D,, group of Rh-related polypeptides. The results also su gest the possibility that the U antigen arises from interaction between glycophorin3! and the Rh-related components D, and R6A,,. TRANSFUSION 1990;30:222-225.
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STUDIES ON RARE PERSONS whose red cell membranes lack all the antigens of the Rh blood group system and the LW blood group system (Rh,,,, red cells) have shown that these cells are deficient in several polypeptides found in normal red cells. The antigens of the LW blood group system are associated with a glycoprotein of M , 40,000 to 47,000.1.2 The murine monoclonal antibody (MoAb) BRIC 125 identifies a glycoprotein of M , 47,000 to 52,000.3 Immunoprecipitation experiments with D antibodies have shown that a glycoprotein of M , 45,000 to 100,000 (D5o polypeptide) coprecipitates with a polypeptide of M , 30,000 to 32,000 (D30 p ~ l y p e p t i d e ) , ~ - ~ while MoAbs of the R6A type immunoprecipitate both a glycoprotein of M,35,000 to 52,000 (R6A4, polypeptide) and a polypeptide of M , 31,000 to 34,000 (R6A-32 p ~ l y p e p t i d e ) . ~Sequence .~.~ studies have shown that the D30 and R6A32 polypeptides have the same N-terminal amino acid sequence and that the R6A45 and D,, polypeptides share another N-terminal sequence.' Rh,,,, red cells either do not react at all or react only weakly with the antibodies described above. Two glycoproteins of M , similar to that of the R6A3, and R6Ad5polypeptides are immunoprecipitated by anti-c and -E ~era.~f'Other workers using human MoAbs have concluded that c, D, and E From the Blood Group Referencc Laboratory, South Westcrn Regional Blood Transfusion Centre, Southmead, Bristol, UK; the Department of Biochemistry, School of Medical Sciences, University of Bristol; thc MRC Blood Group Unit, University College London, London, UK; and the Central Laboratory of the Netherlands Rcd Cross, Amsterdam, The Netherlands. Supported in part by a grant from the Medical Rcscarch Council. Received for publication May 25, 1989; rcvision rcccivcd Scplernber 6, 1989, and accepted Septcrnber 7, 1989.
antigens are on three related but distinct polypeptides with M , similar to that of the D30 and R6A32 polypeptide^.^ Although these results show that Rh,,,, red cells are deficient in a number of different polypeptides, the exact number of polypeptides involved is still unclear. Furthermore, there is evidence that glycophorin B (syn: sialoglycoprotein 6), which carries Ss blood group antigens, is also reduced in amount in Rh,,,, red cells and that some of the determinants recognized by Duclos serum are associated with this glycoprotein.'O.ll The glycoprotein carrying Duffy blood group antigens may also be involved because Rh,,,, red cells lack the Fy5 antigen.12 Recently, three murine MoAbs directed against a novel antigen in red cells have been described (von dem Borne, et al., submitted for publication). The antibodies failed to react with Rh,,,, cells lacking the U blood group antigen, but they did react with Rh,,,, red cells having the U blood group antigen. (Rh,,,, samples with dramatically depressed expression of U antigen that fail to react with the majority of anti-U sera in direct tests are designated Rh,,,, U- .) These antibodies, therefore, show some similarity in specificity to Duclos serum13 and provide further tools for probing the nature of the abnormalities in Rh,,,, red cells. In this article, we show that one of these antibodies (MB-2D10) reacts with a carbohydratedependent epitope found on both the R6A4, and the D5o polypeptides. Materials and Methods LW(a - b - ) red cells (donor Big.) were from B.P.L. Moore, Canadian Blood Transfusion Service, Toronto; Fy(a - b - ) red cells (donors J.B. and S.F.) were from M. Leak, South London Transfusion Centre, Tooting, and G. Poole, South Western
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Regional Blood Transfusion Centre, Bristol, UK, respectively. Rh,,,, red cells (donors H.H., A.L., and Y.T.) were from G. Garratty, American Red Cross Blood Services, Los Angeles, CAYJ. Moulds, Gamma Biologicals, Houston, TX, and B. Macdonald, Queensland Blood Transfusion Service, Brisbane, Australia, respectively. Rh,,,, U + red cells14 (donor S.M.) were from D.G. Gabra and M. Bruce, Glasgow and West of Scotland Blood Transfusion Service, Carluke, Scotland. Red cells of Bombay phenotype (0,) were from V. Ray, Blood Bank, K.E.M. Hospital, Bombay, India. MkMkcells21 were obtained from Y. Okubo, Osaka Red Cross Blood Center, Osaka, Japan. MoAb MB-2DlO was as described by von dern Borne et al. (submitted for publication), monoclonal anti-LWb BS56 was as described by Sonneborn et al.,ls and immunoblotting with human anti-Fy’ was as described by Tanner et al.16 MoAb BRIC 198 was shown to have anti-H activity by its failure to agglutinate 0, red cells, and it was absorbed by H type 11, but not H type I, a synthetic A/B substance (Synsorb, Chembiomed, Edmonton, Alberta, Canada). BRIC 198 was cloned from a fusion that used spleen cells from a Balb/c mouse given two intraperitoneal injections of group 0 human red cells (100 pL of 50% suspension in PBS, pH 7.3) 11 days apart. Fusion was performed 3 days after the second injection. The preparation of D30,D,,, R6A,,, and R6A4, polypeptides was as described,’ as were the production and use of endo-pgalactosidase (F. kerafolyficus)and endoglycosidase F (Endo F) preparations (F. rneningosepficum).lsSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of red cell membranes was carried out by the method of Laemmli” on gels containing 10 percent acrylamide with a 3 percent overlay. Immunoblotting was as described by Mallinson et al.,’ except that 5 percent (wt/vol) bovine milk powder was used as the blocking agent. Red cells were treated with typsin and sialidase as in Mallinson et al.‘ and with chymotrypsin as in Judson and Anstee.18
Results The reactivity of MB-2DlO with whole red cell membranes We examined the component(s) reacting with MB-2D10 by immunoblotting the electrophoretically separated components of normal red cell membranes. A diffusely staining band extending from M, 30,000 to the high-molecular-weight region of the gel was obtained whether the membranes were electrophoresed under reducing or non-reducing conditions. Rh,,,, U + membranes (donor S.M., Fig. I-b) also gave the diffusely staining band, but Rh,,,, U - membranes (donors Y.T., A.L., and H.H.; Fig. I-c) did not. Trypsin treatment of normal red cells did not alter the band, but it was almost completely abolished by pretreatment of normal red cells with chymotrypsin (Fig. 1-d,-e). MB-2D10 still reacted on immunoblotting of both sialidase-treated red cells and endo-p-galactosidase-treated red cells (data not shown). When normal red cells were treated with low concentrations of an endoglycosidase F-containing preparation (Endo F), no reactive band was detected upon subsequent immunoblotting with MB-2D10 (Fig. 1-f). Both the diffuse nature of the band and its extreme sensitivity to Endo F are similar to properties shown by the Fyn-active glycoprotein.16 Therefore, Fy(a - b - ) red cell membranes were also examined by immunoblotting with MB-2Dl0, and a normal staining pattern was observed (Fig. 1-g). LW(a - b - ) red cell membranes also gave a normal staining pattern with MB-2D10 (Fig. 1-h), as did membranes from MkMkred cells (which lack
FIG. 1. Identification of red cell membrane components reacting with MB-2D10 by immunoblotring: red ccll membrane samples were solubilized under nonrcducing conditions and immunoblottcd with MB2D10. a) normal membranes, b) Rh,,,, U + mcrnbranes (donor S.M.), c) Rh,,,, U - membranes (donor H.H.), d) trypsin-treated normal membranes, e) chymotrypsin-treated normal mcrnbranes, f ) Endo Ftreated normal membranes, g) Fy(a - b -) membranes (donor S.F.), h) L W ( a - b - ) rnernbrancs (donor Big.), and i) MkMk membranes (donor K.M.). All membrane samples werc run on the same gel and immunoblotted at the samc time.
glycophorins A and B as well as the U blood group antige~~)’~.’~ (Fig. 14).
The reactivity of MB-2D10 with purified Rh-related components The diffuse nature of the component(s) reacting with MB2D10 on normal red cell membranes suggested that the antibody might be reacting with the diffusely migrating, glycosylated components immunoprecipitated by anti-D (Mr45,000100,000) and MoAbs of the R6A type (Mr 35,000-52,000), designated D,, polypeptide and R6A4, polypeptide, respectively.’ Immunoblotting of the purified D,, and D,, .components showed that only the D,, component reacted with MB2D10 (Fig. 2-b,-c). Similar experiments with purified R6A,, and R6A4, polypeptides showed that only the R6Ad5 component reacted with the antibody (Fig. 2-d,-e). MoAb BS56 (antiLWb)and human anti-Fy” did not react with any of these components by immunoblotting (data not shown), which indicates that the MB-2D10-reactive material does not correspond to either the Fyn glycoprotein or the LW glycoproteins, which have M,of 38,500 to 90,000 and 37,000 to 47,000, respective1y.’J6
Discussion Serologic studies (von dem Borne et al., submitted for publication) have established that MB-2D10 reacts with a chymotrypsin-sensitive, sialidase-resistant epitope on
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FIG. 2. Reactivity of MB-2D10 with purified D and R6A polypeptides by immunoblotting. a) normal red cell membranes, b) D, polypeptide, c) D,, polypeptide, d) R6A3, polypeptide. e) R6A,, polypeptide. Tracks a, b, and c are from one gel, and tracks d and e are from another.
normal red cells. The present work using immunoblotting confirms these data and also shows that the antibody recognizes an N-glycan-dependent epitope, because the epitope is destroyed by treatment with Endo F preparations. These studies (von dem Borne et al., submitted for publication) also showed that MB-2D10 reacts with Fy(a - b -), LW(a - b -), and MkMkred cells in agglutination tests. These results were confirmed by immunoblotting in the present study. Previously reported immunoprecipitation studies with MB-2D10 gave a very complex pattern of bands.21 The immunoprecipitation contained the D,, polypeptide, a diffusely migrating, higher-molecular-weight component, and, under certain circumstances, a polypeptide of M,25,000, which, it was suggested, corresponds to glycophorin B. Our results with immunoblotting clearly show that the MB-2D10 epitope is located on the D5o and the R6A4, components, and it is therefore likely that the diffuse higher-molecular-weight components observed by Bloy et correspond to the D50 and R6A45 components. The D,, and R6A4, polypeptides are known to coprecipitate with the D30 polypeptide and the R6A-32 polypeptides, respectively, in immunoprecipitation exp e r i m e n t ~ ,which ~ . ~ accounts for the presence of the D,, polypeptides in the immunoprecipitates obtained by Bloy et It seems likely that these immunoprecipitates also contain the R6A32 polypeptide. The D50 and R6A4, preparations used in this study were derived from red cells of blood group 0 and were found to carry blood group H antigen activity on immunoblotting with murine MoAb BRIC 198 (data not shown). This observation confirms that the D50 and R6A45
polypeptides correspond to the ABH-active glycosylated components that are found in immunoprecipitates obtained using anti-D and anti-c/-E, respectively.6 The presence of the MB-2D10 epitope on both the R6A45 and D50 polypeptides is not unexpected, as they share a common N-terminal ~ e q u e n c e . ~ Our results clearly show that the R6A4, and D5o polypeptides are present in Rh,,,, U + red cells. Although the MB-2D10 epitope is absent from the Rh,,,, U- red cells, it is not known whether this represents the complete absence of the R6A4, and D5o polypeptides or whether these polypeptides are altered so as not to express the MB-2D10 epitope. In this study, we have shown that the MB-2D10 epitope is located on the D,, and R6A4, polypeptides and not on the D30 or R6A32 polypeptides or, apparently, glycophorin B. Our data also suggest that the polypeptides expressing the MB-2D10 epitope are the same as the ABH-active Rh glycoproteins described by Moore and Green.6 The results point to the possibility that the U antigen arises from the interaction between MB-2D10reactive material (D5, and R6A4,) and glycophorin By because there is a known association between the absence of U antigen and the absence of glycophorin B.22
References 1. Moorc S. Identification of rcd ccll membrane componcnts associatcd with rhcsus blood group antigcn cxprcssion. In: Cartron
2.
3.
4. 5.
6. 7.
8. 9.
JP, Rougcr P, Salmon C, cds. Red ccll membranc glycoconjugates and rclatcd gcnctic markers. Paris: Librairic Arncttc, 1983:97106. Mallinson G, Martin PG, Anstee DJ, et al. ldcntification and partial charactcrization of thc human crythrocyte mcmbranc componcnt(s) that cxpress the antigcns of thc LW blood-group systcm. Biochcm J 1986;234:649-52. Avcnt N, Judson PA, Parsons SF, ct al. Monoclonal antibodics that recognizc diffcrcnt mcmbranc proteins that arc dcficicnt in Rh,,,, human crythrocytes. One group of antibodics reacts with a variety of cells and tissue whcrcas the other group is crythroidspecific. Biochem J 1988;251:499-505. Moorc S, Woodrow CF, McClclland DBL. lsolafion of mcmbranc components associated with human rcd ccll antigcns Rh(D), (c), (E) and Fy. Nature 1982;295:529-31. Gahmbcrg CG. Molccular idcntificdfion of thc human Rh,,(D) antigcn. FEBS Lett 1982;140:93-7. Moore S, Grecn C. The idcntification of specific Rhesus-polypeptide-blood-group-ABH-active-glycoprotcincomplexes i n the human red-ccll mcmbranc. Biochem J 1987;244:735-41. Avent ND, Ridgwcll K, Mawby WJ, Tanner MJA, Anstce DJ, Kumpcl 8. Protcin-sequcnce studies on Rh-related polypeptides suggcst the prescncc of at least two groups of protcins which associatc i n thc human red-cell membrane. Biochcm J 1988;256:1043-6. Ridgwcll K, Roberts SJ, Tanncr MJA, Anstec DJ. Absence of two protcins containing extracellular thiol groups of Rh,.,, human erythrocytcs. Biochem J 1983;213:267-9. Bloy F, Blanchard D, Dahr W, Bcyreuthcr K, Salmon C, Cartron JP. Dctcrmination of thc N-terminal sequence of human red ccll Rh(D) polypeptide and dcmonstration that the Rh(D), (c), and (E) antigens arc carried by distinct polypcptidc chains. Blood 1988;72:661-6.
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10. Dahr W, Kordowicz M, Moulds J, Giclen W, Lcbeck L, Krugcr J. Characterization of the Ss sialoglycoprotcin and its antigcns in Rh.,,, erythrocytcs. Blut 1987;54:13-24. 11. Dahr W, Moulds J. High-frequency antigcns of the human crythrocytc membrane sialoglycoprotcins, IV. Molccular propcrtics of the U antigen. Biol Chcm Hoppc-Scyler 1987;368:659-67. 12. Colledge KI, Pezzulich M, Marsh WL. Anti-Fy5, an antibody disclosing a probable association betwccn Rhcsus and Duffy blood group genes. Vox Sang 1973;24:193-9. 13. Habibi B, Fouillade MT, Duedari N, Issitt PD, Tippctt P, Salmon C. The antigen Duclos. A new high frequency rcd ccll antigen rclated to Rh and U. Vox Sang 1978;34:302-9. 14. Gabra GS, Bruce M, Watt A, Mitchell R. Anti-Rh 29 in a primagravida with rhesus null syndromc resulting in hacmolytic disease of the newborn. Vox Sang 1987;53:143-6. 15. Sonneborn HH, Utherman H, Tills D, Lomas CG, Shaw MA, Tippett P. Monoclonal anti-LWb. Biotest Bull 1984;2:145-8. 16. Tanner MJA, Anstee DJ, Mallinson G. et al. Effcct of cndoglycosidase F-pcptidyl N-glucosidase F prcparations on the surface components of the human erythrocyte. Carbohydr Rcs 1988; 178:203-12. 17. Laemmli UK. Clcavagc of structural proteins during thc asscmbly of the head of bacteriophage T4. Nature 1970;227:680-5. 18. Judson PA, Anstee DJ. Comparativc effcct of trypsin and chymotrypsin on blood group antigens. Med Lab Sci 1977;34:1-6. 19. Tokunaga E, Sasakawa S, Tamaka K, et al. Two apparently hcalthy Japanese individuals of type MkMkhave crythrocytes which lack both the blood group MN and Ss-active sialoglycoproteins. J Immunogenet 1979;6:383-90. 20. Okubo Y,Daniels GL, Parsons SF, et al. A Japancsc family with two sisters apparently homozygous for M*. Vox Sang 1988;54:10711.
Book Review Blood Transfusion Therapy: A Problem Oriented Approach, J.A.F. Napier. New York: John Wiley & Sons, 1987. $80.00. The date of publication does not detract from the nice organization and easy readability of this book. As the author freely concedes in his preface, some of the therapeutic suggestions offered, if controversial, are “influenced by my own prejudices and may at times appear unjustifiably dogmatic.” He hopes, nevertheless, “that in the main these will be representative of the most considered opinions available.” In this hope the reviewer believes the author has been successful. The chapters on the harmful effects of transfusion and the infections transmissible by transfusion provide useful summaries of these problems, in spite of the fact that rapid progress in the elucidation of hepatitis C makes the section on non-A, non-B hepatitis a bit dated. The clarity of these chapters would make this book useful for most hospital pathologists, anesthesiologists, and other
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21. Bloy C; Blanchard D, Huct M, Hcrmand P, Cclton JL, Cartron JP. Rcport on monoclonal R h antibodics: scrological and biochemical investigations. Rcv Fr Transfus Immunohcmatol 1988;31:208-21. 22. Dahr W, Uhlenbruck G, lssitt PD, Allen FH Jr. SDS-polyacrylamidc gel clcctrophorctic analysis of the mcmbranc glycoproteins from S -s - U - erythrocytcs. J Immunogcnct 1975;2:249-51.
Gary Mallinson, BSc, Research Scicntist, Blood Group Refcrencc Laboratory. David J. Anstec, PhD, Dircctor, Blood Group Refcrcncc Laboratory, South Wcstcrn Rcgional Blood Transfusion Centre. Bristol BSlO 5ND, UK. [Reprint requcsts] Neil D. Avcnt, PhD, Scnior Scientific Officcr, Blood Group Rcfercncc Laboratory. Kay Ridgwcll, PhD, Research Assistant, Dcpartmcnt of Biochcmistry, Univcrsity of Bristol. Michael J.A. Tanncr, PhD, Rcadcr, Dcpartmcnt of Biochemistry, University of Bristol. Gcoffrcy L. Daniels, PhD, Scicntist, Mcdical Rcscarch Council Blood Group Unit, Univcrsity Collcgc London, London, UK. Patricia Tippctt, PhD, Dircctor, Mcdical Rcscarch Council Blood Group Unit. Albcrt E.G.Kr. von dcm Bornc, MD, PhD, Scnior Invcstigator, Departmcnt of Immuno-hcmatology, Ccntral Laboratory of thc Ncthcrlands Red Cross Blood Transfusion Scrvicc, Amstcrdam, Thc Ncthcrlands.
physicians involved in transfusion committee audits, especially those who are preparing audit criteria for their hospital. The main emphases of the book are an enumeration of the products available for transfusion and a description of the use of these products in specific medical and surgical conditions. Treatment of coagulation disorders is concisely presented. Where the text, in the interest of keeping the book to a manageable size, appears to be somewhat brief, it is supported by a generous bibliography for those who require more detailed information. The book will not replace standard reference books on blood banking, but it does fill a need for the quick identification of problems encountered, especially by pathologists in small to medium-sized hospitals who only occasionally encounter blood banking and transfusion problems and who can then provide better assistance to their clinical colleagues.
ROBERTB. SCHULTZ, MD Edisori Regional Blood Center Fort Myers, FL
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STUDIES ON RARE PERSONS whose red cell membranes lack all the antigens of the Rh blood group system and the LW blood group system (Rh,,,, red cells) have shown that these cells are deficient in several polypeptides found in normal red cells. The antigens of the LW blood group system are associated with a glycoprotein of M , 40,000 to 47,000.1.2 The murine monoclonal antibody (MoAb) BRIC 125 identifies a glycoprotein of M , 47,000 to 52,000.3 Immunoprecipitation experiments with D antibodies have shown that a glycoprotein of M , 45,000 to 100,000 (D5o polypeptide) coprecipitates with a polypeptide of M , 30,000 to 32,000 (D30 p ~ l y p e p t i d e ) , ~ - ~ while MoAbs of the R6A type immunoprecipitate both a glycoprotein of M,35,000 to 52,000 (R6A4, polypeptide) and a polypeptide of M , 31,000 to 34,000 (R6A-32 p ~ l y p e p t i d e ) . ~Sequence .~.~ studies have shown that the D30 and R6A32 polypeptides have the same N-terminal amino acid sequence and that the R6A45 and D,, polypeptides share another N-terminal sequence.' Rh,,,, red cells either do not react at all or react only weakly with the antibodies described above. Two glycoproteins of M , similar to that of the R6A3, and R6Ad5polypeptides are immunoprecipitated by anti-c and -E ~era.~f'Other workers using human MoAbs have concluded that c, D, and E From the Blood Group Referencc Laboratory, South Westcrn Regional Blood Transfusion Centre, Southmead, Bristol, UK; the Department of Biochemistry, School of Medical Sciences, University of Bristol; thc MRC Blood Group Unit, University College London, London, UK; and the Central Laboratory of the Netherlands Rcd Cross, Amsterdam, The Netherlands. Supported in part by a grant from the Medical Rcscarch Council. Received for publication May 25, 1989; rcvision rcccivcd Scplernber 6, 1989, and accepted Septcrnber 7, 1989.
antigens are on three related but distinct polypeptides with M , similar to that of the D30 and R6A32 polypeptide^.^ Although these results show that Rh,,,, red cells are deficient in a number of different polypeptides, the exact number of polypeptides involved is still unclear. Furthermore, there is evidence that glycophorin B (syn: sialoglycoprotein 6), which carries Ss blood group antigens, is also reduced in amount in Rh,,,, red cells and that some of the determinants recognized by Duclos serum are associated with this glycoprotein.'O.ll The glycoprotein carrying Duffy blood group antigens may also be involved because Rh,,,, red cells lack the Fy5 antigen.12 Recently, three murine MoAbs directed against a novel antigen in red cells have been described (von dem Borne, et al., submitted for publication). The antibodies failed to react with Rh,,,, cells lacking the U blood group antigen, but they did react with Rh,,,, red cells having the U blood group antigen. (Rh,,,, samples with dramatically depressed expression of U antigen that fail to react with the majority of anti-U sera in direct tests are designated Rh,,,, U- .) These antibodies, therefore, show some similarity in specificity to Duclos serum13 and provide further tools for probing the nature of the abnormalities in Rh,,,, red cells. In this article, we show that one of these antibodies (MB-2D10) reacts with a carbohydratedependent epitope found on both the R6A4, and the D5o polypeptides. Materials and Methods LW(a - b - ) red cells (donor Big.) were from B.P.L. Moore, Canadian Blood Transfusion Service, Toronto; Fy(a - b - ) red cells (donors J.B. and S.F.) were from M. Leak, South London Transfusion Centre, Tooting, and G. Poole, South Western
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Regional Blood Transfusion Centre, Bristol, UK, respectively. Rh,,,, red cells (donors H.H., A.L., and Y.T.) were from G. Garratty, American Red Cross Blood Services, Los Angeles, CAYJ. Moulds, Gamma Biologicals, Houston, TX, and B. Macdonald, Queensland Blood Transfusion Service, Brisbane, Australia, respectively. Rh,,,, U + red cells14 (donor S.M.) were from D.G. Gabra and M. Bruce, Glasgow and West of Scotland Blood Transfusion Service, Carluke, Scotland. Red cells of Bombay phenotype (0,) were from V. Ray, Blood Bank, K.E.M. Hospital, Bombay, India. MkMkcells21 were obtained from Y. Okubo, Osaka Red Cross Blood Center, Osaka, Japan. MoAb MB-2DlO was as described by von dern Borne et al. (submitted for publication), monoclonal anti-LWb BS56 was as described by Sonneborn et al.,ls and immunoblotting with human anti-Fy’ was as described by Tanner et al.16 MoAb BRIC 198 was shown to have anti-H activity by its failure to agglutinate 0, red cells, and it was absorbed by H type 11, but not H type I, a synthetic A/B substance (Synsorb, Chembiomed, Edmonton, Alberta, Canada). BRIC 198 was cloned from a fusion that used spleen cells from a Balb/c mouse given two intraperitoneal injections of group 0 human red cells (100 pL of 50% suspension in PBS, pH 7.3) 11 days apart. Fusion was performed 3 days after the second injection. The preparation of D30,D,,, R6A,,, and R6A4, polypeptides was as described,’ as were the production and use of endo-pgalactosidase (F. kerafolyficus)and endoglycosidase F (Endo F) preparations (F. rneningosepficum).lsSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of red cell membranes was carried out by the method of Laemmli” on gels containing 10 percent acrylamide with a 3 percent overlay. Immunoblotting was as described by Mallinson et al.,’ except that 5 percent (wt/vol) bovine milk powder was used as the blocking agent. Red cells were treated with typsin and sialidase as in Mallinson et al.‘ and with chymotrypsin as in Judson and Anstee.18
Results The reactivity of MB-2DlO with whole red cell membranes We examined the component(s) reacting with MB-2D10 by immunoblotting the electrophoretically separated components of normal red cell membranes. A diffusely staining band extending from M, 30,000 to the high-molecular-weight region of the gel was obtained whether the membranes were electrophoresed under reducing or non-reducing conditions. Rh,,,, U + membranes (donor S.M., Fig. I-b) also gave the diffusely staining band, but Rh,,,, U - membranes (donors Y.T., A.L., and H.H.; Fig. I-c) did not. Trypsin treatment of normal red cells did not alter the band, but it was almost completely abolished by pretreatment of normal red cells with chymotrypsin (Fig. 1-d,-e). MB-2D10 still reacted on immunoblotting of both sialidase-treated red cells and endo-p-galactosidase-treated red cells (data not shown). When normal red cells were treated with low concentrations of an endoglycosidase F-containing preparation (Endo F), no reactive band was detected upon subsequent immunoblotting with MB-2D10 (Fig. 1-f). Both the diffuse nature of the band and its extreme sensitivity to Endo F are similar to properties shown by the Fyn-active glycoprotein.16 Therefore, Fy(a - b - ) red cell membranes were also examined by immunoblotting with MB-2Dl0, and a normal staining pattern was observed (Fig. 1-g). LW(a - b - ) red cell membranes also gave a normal staining pattern with MB-2D10 (Fig. 1-h), as did membranes from MkMkred cells (which lack
FIG. 1. Identification of red cell membrane components reacting with MB-2D10 by immunoblotring: red ccll membrane samples were solubilized under nonrcducing conditions and immunoblottcd with MB2D10. a) normal membranes, b) Rh,,,, U + mcrnbranes (donor S.M.), c) Rh,,,, U - membranes (donor H.H.), d) trypsin-treated normal membranes, e) chymotrypsin-treated normal mcrnbranes, f ) Endo Ftreated normal membranes, g) Fy(a - b -) membranes (donor S.F.), h) L W ( a - b - ) rnernbrancs (donor Big.), and i) MkMk membranes (donor K.M.). All membrane samples werc run on the same gel and immunoblotted at the samc time.
glycophorins A and B as well as the U blood group antige~~)’~.’~ (Fig. 14).
The reactivity of MB-2D10 with purified Rh-related components The diffuse nature of the component(s) reacting with MB2D10 on normal red cell membranes suggested that the antibody might be reacting with the diffusely migrating, glycosylated components immunoprecipitated by anti-D (Mr45,000100,000) and MoAbs of the R6A type (Mr 35,000-52,000), designated D,, polypeptide and R6A4, polypeptide, respectively.’ Immunoblotting of the purified D,, and D,, .components showed that only the D,, component reacted with MB2D10 (Fig. 2-b,-c). Similar experiments with purified R6A,, and R6A4, polypeptides showed that only the R6Ad5 component reacted with the antibody (Fig. 2-d,-e). MoAb BS56 (antiLWb)and human anti-Fy” did not react with any of these components by immunoblotting (data not shown), which indicates that the MB-2D10-reactive material does not correspond to either the Fyn glycoprotein or the LW glycoproteins, which have M,of 38,500 to 90,000 and 37,000 to 47,000, respective1y.’J6
Discussion Serologic studies (von dem Borne et al., submitted for publication) have established that MB-2D10 reacts with a chymotrypsin-sensitive, sialidase-resistant epitope on
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FIG. 2. Reactivity of MB-2D10 with purified D and R6A polypeptides by immunoblotting. a) normal red cell membranes, b) D, polypeptide, c) D,, polypeptide, d) R6A3, polypeptide. e) R6A,, polypeptide. Tracks a, b, and c are from one gel, and tracks d and e are from another.
normal red cells. The present work using immunoblotting confirms these data and also shows that the antibody recognizes an N-glycan-dependent epitope, because the epitope is destroyed by treatment with Endo F preparations. These studies (von dem Borne et al., submitted for publication) also showed that MB-2D10 reacts with Fy(a - b -), LW(a - b -), and MkMkred cells in agglutination tests. These results were confirmed by immunoblotting in the present study. Previously reported immunoprecipitation studies with MB-2D10 gave a very complex pattern of bands.21 The immunoprecipitation contained the D,, polypeptide, a diffusely migrating, higher-molecular-weight component, and, under certain circumstances, a polypeptide of M,25,000, which, it was suggested, corresponds to glycophorin B. Our results with immunoblotting clearly show that the MB-2D10 epitope is located on the D5o and the R6A4, components, and it is therefore likely that the diffuse higher-molecular-weight components observed by Bloy et correspond to the D50 and R6A45 components. The D,, and R6A4, polypeptides are known to coprecipitate with the D30 polypeptide and the R6A-32 polypeptides, respectively, in immunoprecipitation exp e r i m e n t ~ ,which ~ . ~ accounts for the presence of the D,, polypeptides in the immunoprecipitates obtained by Bloy et It seems likely that these immunoprecipitates also contain the R6A32 polypeptide. The D50 and R6A4, preparations used in this study were derived from red cells of blood group 0 and were found to carry blood group H antigen activity on immunoblotting with murine MoAb BRIC 198 (data not shown). This observation confirms that the D50 and R6A45
polypeptides correspond to the ABH-active glycosylated components that are found in immunoprecipitates obtained using anti-D and anti-c/-E, respectively.6 The presence of the MB-2D10 epitope on both the R6A45 and D50 polypeptides is not unexpected, as they share a common N-terminal ~ e q u e n c e . ~ Our results clearly show that the R6A4, and D5o polypeptides are present in Rh,,,, U + red cells. Although the MB-2D10 epitope is absent from the Rh,,,, U- red cells, it is not known whether this represents the complete absence of the R6A4, and D5o polypeptides or whether these polypeptides are altered so as not to express the MB-2D10 epitope. In this study, we have shown that the MB-2D10 epitope is located on the D,, and R6A4, polypeptides and not on the D30 or R6A32 polypeptides or, apparently, glycophorin B. Our data also suggest that the polypeptides expressing the MB-2D10 epitope are the same as the ABH-active Rh glycoproteins described by Moore and Green.6 The results point to the possibility that the U antigen arises from the interaction between MB-2D10reactive material (D5, and R6A4,) and glycophorin By because there is a known association between the absence of U antigen and the absence of glycophorin B.22
References 1. Moorc S. Identification of rcd ccll membrane componcnts associatcd with rhcsus blood group antigcn cxprcssion. In: Cartron
2.
3.
4. 5.
6. 7.
8. 9.
JP, Rougcr P, Salmon C, cds. Red ccll membranc glycoconjugates and rclatcd gcnctic markers. Paris: Librairic Arncttc, 1983:97106. Mallinson G, Martin PG, Anstee DJ, et al. ldcntification and partial charactcrization of thc human crythrocyte mcmbranc componcnt(s) that cxpress the antigcns of thc LW blood-group systcm. Biochcm J 1986;234:649-52. Avcnt N, Judson PA, Parsons SF, ct al. Monoclonal antibodics that recognizc diffcrcnt mcmbranc proteins that arc dcficicnt in Rh,,,, human crythrocytes. One group of antibodics reacts with a variety of cells and tissue whcrcas the other group is crythroidspecific. Biochem J 1988;251:499-505. Moorc S, Woodrow CF, McClclland DBL. lsolafion of mcmbranc components associated with human rcd ccll antigcns Rh(D), (c), (E) and Fy. Nature 1982;295:529-31. Gahmbcrg CG. Molccular idcntificdfion of thc human Rh,,(D) antigcn. FEBS Lett 1982;140:93-7. Moore S, Grecn C. The idcntification of specific Rhesus-polypeptide-blood-group-ABH-active-glycoprotcincomplexes i n the human red-ccll mcmbranc. Biochem J 1987;244:735-41. Avent ND, Ridgwcll K, Mawby WJ, Tanner MJA, Anstce DJ, Kumpcl 8. Protcin-sequcnce studies on Rh-related polypeptides suggcst the prescncc of at least two groups of protcins which associatc i n thc human red-cell membrane. Biochcm J 1988;256:1043-6. Ridgwcll K, Roberts SJ, Tanncr MJA, Anstec DJ. Absence of two protcins containing extracellular thiol groups of Rh,.,, human erythrocytcs. Biochem J 1983;213:267-9. Bloy F, Blanchard D, Dahr W, Bcyreuthcr K, Salmon C, Cartron JP. Dctcrmination of thc N-terminal sequence of human red ccll Rh(D) polypeptide and dcmonstration that the Rh(D), (c), and (E) antigens arc carried by distinct polypcptidc chains. Blood 1988;72:661-6.
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MB-2D10 RECOGNIZES RH-RELATED GLYCOPROTEINS
10. Dahr W, Kordowicz M, Moulds J, Giclen W, Lcbeck L, Krugcr J. Characterization of the Ss sialoglycoprotcin and its antigcns in Rh.,,, erythrocytcs. Blut 1987;54:13-24. 11. Dahr W, Moulds J. High-frequency antigcns of the human crythrocytc membrane sialoglycoprotcins, IV. Molccular propcrtics of the U antigen. Biol Chcm Hoppc-Scyler 1987;368:659-67. 12. Colledge KI, Pezzulich M, Marsh WL. Anti-Fy5, an antibody disclosing a probable association betwccn Rhcsus and Duffy blood group genes. Vox Sang 1973;24:193-9. 13. Habibi B, Fouillade MT, Duedari N, Issitt PD, Tippctt P, Salmon C. The antigen Duclos. A new high frequency rcd ccll antigen rclated to Rh and U. Vox Sang 1978;34:302-9. 14. Gabra GS, Bruce M, Watt A, Mitchell R. Anti-Rh 29 in a primagravida with rhesus null syndromc resulting in hacmolytic disease of the newborn. Vox Sang 1987;53:143-6. 15. Sonneborn HH, Utherman H, Tills D, Lomas CG, Shaw MA, Tippett P. Monoclonal anti-LWb. Biotest Bull 1984;2:145-8. 16. Tanner MJA, Anstee DJ, Mallinson G. et al. Effcct of cndoglycosidase F-pcptidyl N-glucosidase F prcparations on the surface components of the human erythrocyte. Carbohydr Rcs 1988; 178:203-12. 17. Laemmli UK. Clcavagc of structural proteins during thc asscmbly of the head of bacteriophage T4. Nature 1970;227:680-5. 18. Judson PA, Anstee DJ. Comparativc effcct of trypsin and chymotrypsin on blood group antigens. Med Lab Sci 1977;34:1-6. 19. Tokunaga E, Sasakawa S, Tamaka K, et al. Two apparently hcalthy Japanese individuals of type MkMkhave crythrocytes which lack both the blood group MN and Ss-active sialoglycoproteins. J Immunogenet 1979;6:383-90. 20. Okubo Y,Daniels GL, Parsons SF, et al. A Japancsc family with two sisters apparently homozygous for M*. Vox Sang 1988;54:10711.
Book Review Blood Transfusion Therapy: A Problem Oriented Approach, J.A.F. Napier. New York: John Wiley & Sons, 1987. $80.00. The date of publication does not detract from the nice organization and easy readability of this book. As the author freely concedes in his preface, some of the therapeutic suggestions offered, if controversial, are “influenced by my own prejudices and may at times appear unjustifiably dogmatic.” He hopes, nevertheless, “that in the main these will be representative of the most considered opinions available.” In this hope the reviewer believes the author has been successful. The chapters on the harmful effects of transfusion and the infections transmissible by transfusion provide useful summaries of these problems, in spite of the fact that rapid progress in the elucidation of hepatitis C makes the section on non-A, non-B hepatitis a bit dated. The clarity of these chapters would make this book useful for most hospital pathologists, anesthesiologists, and other
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21. Bloy C; Blanchard D, Huct M, Hcrmand P, Cclton JL, Cartron JP. Rcport on monoclonal R h antibodics: scrological and biochemical investigations. Rcv Fr Transfus Immunohcmatol 1988;31:208-21. 22. Dahr W, Uhlenbruck G, lssitt PD, Allen FH Jr. SDS-polyacrylamidc gel clcctrophorctic analysis of the mcmbranc glycoproteins from S -s - U - erythrocytcs. J Immunogcnct 1975;2:249-51.
Gary Mallinson, BSc, Research Scicntist, Blood Group Refcrencc Laboratory. David J. Anstec, PhD, Dircctor, Blood Group Refcrcncc Laboratory, South Wcstcrn Rcgional Blood Transfusion Centre. Bristol BSlO 5ND, UK. [Reprint requcsts] Neil D. Avcnt, PhD, Scnior Scientific Officcr, Blood Group Rcfercncc Laboratory. Kay Ridgwcll, PhD, Research Assistant, Dcpartmcnt of Biochcmistry, Univcrsity of Bristol. Michael J.A. Tanncr, PhD, Rcadcr, Dcpartmcnt of Biochemistry, University of Bristol. Gcoffrcy L. Daniels, PhD, Scicntist, Mcdical Rcscarch Council Blood Group Unit, Univcrsity Collcgc London, London, UK. Patricia Tippctt, PhD, Dircctor, Mcdical Rcscarch Council Blood Group Unit. Albcrt E.G.Kr. von dcm Bornc, MD, PhD, Scnior Invcstigator, Departmcnt of Immuno-hcmatology, Ccntral Laboratory of thc Ncthcrlands Red Cross Blood Transfusion Scrvicc, Amstcrdam, Thc Ncthcrlands.
physicians involved in transfusion committee audits, especially those who are preparing audit criteria for their hospital. The main emphases of the book are an enumeration of the products available for transfusion and a description of the use of these products in specific medical and surgical conditions. Treatment of coagulation disorders is concisely presented. Where the text, in the interest of keeping the book to a manageable size, appears to be somewhat brief, it is supported by a generous bibliography for those who require more detailed information. The book will not replace standard reference books on blood banking, but it does fill a need for the quick identification of problems encountered, especially by pathologists in small to medium-sized hospitals who only occasionally encounter blood banking and transfusion problems and who can then provide better assistance to their clinical colleagues.
ROBERTB. SCHULTZ, MD Edisori Regional Blood Center Fort Myers, FL
