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0.6. Properties of monoclonal antibodies reacting with human erythrocyte antigens
51
In addition to antiPr and anti-Gd CA, several further CA specificities were found. Until now only single examples of the CA have been published. Su antigen is partially protease susceptible. It is fully expressed at birth. Anti-Sa recognizes the NeuAm2-3Galpl. . . structure on RBC membrane SGP. Fl antigen is protease resistant. It differs from Pr1,2,3, Gd1,2 and Sa antigens since it is weakly expressed on newborn (and i adult) RBC [ 51. It is, therefore a developmentally regulated antigen like I but differing from I, since NeuAc is not involved in the I antigenic determinant. The Fl CA recognizes the binary determinant consisting of NeuAc linked to the terminal Gal of the l-3 branch and of Fuc linked to the other terminal Gal of the l-6 branch of the branched polylactosaminyl core structure known to represent the I antigenic determinants [6]. Lud antigen recognized by the anti-Lud Ca is a further NeuAcdependent developmentally regulated antigen. It differs from the Fl antigen, since the Lud antigen is fully expressed on i adult RBC [ 71. The data indicate that monoclonal anti&, anti-Gd and “related” CA are excellent markers for cell surface profiles of neuraminyl structures. References 1 2 3 4 5 6
D. Roelcke, Eur. J. Zmmunol., 3 (1973) 206. D. Roelcke, W. Riesen, H.P. Geisen and W. Ebert, VOX Sang., 33 (1977) 304. S.K. Kundu, D.M. Marcus and D. Roelcke, Zmmunol. Let&, 4 (1982) 263. D. Roelcke and R. Brossmer, Prot. Biol. Fluids, 31 (1983) (in press). D. Roelcke, VOX Sang., 41 (1981) 98. R. Kannagi, D. Roelcke, K.A. Peterson, Y. Okada, S.B. Levery and S. Hakomori, Carbohyd. Res., (in press). 7 D. Roelcke, VOX Sang., 41 (1981) 316.
0.6. PROPERTIES OF MONOCLONAL HUMAN ERYTHROCYTE ANTIGENS
ANTIBODIES
REACTING
WITH
H.H. Sonnebom, H. Uthemann and A.C. Munroa (Biotest-Serum-Institut GmbH, Geleitsstrasse 103, Offenbach, F.R.G. and aGlasgow and West of Scotland Blood Transfusion Service, Lanarkshire, U.K.) Since the development of the monoclonal antibody technique by Kiihler and Milstein, this technology has had great influence in immunology, and a lot of monoclonal antibodies have been produced for the investigation and determination of soluble antigens and especially for the characterization of cells and cell surface antigens. In order to get more standardized reagents for blood group typing and to get more information on the biochemistry of blood group antigens we have immunized Balb/c mice with whole red blood cells. After fusing the spleen cells with the mouse myeloma cell line X63Ag8.653 we obtained several
52
clones secreting antibodies with blood group specificity, We found that each monoclonal antibody (m ab) has its own individual properties and they need different conditions for optimal reactions: Concerning the M/N blood group antigens we have investigated two m ab (BS 40, BS 41) reacting preferably with the N antigen, which show the strongest reaction at pH 7.0 whereas at pH 8.5 the discrimination between the M and N antigens is best and by this show a better specificity at pH 8.5. Regarding anti-M antibodies BS 38 shows optimal specificity at pH 7.0 and 37°C BS 39 at pH 8.5 and 37°C and BS 44 between pH 7.5 and 9 and over a broad temperature range, BS 44 seems to be a very useful routine reagent and first successful routine trials have been performed with this antibody. All the antibodies reacting with M/N antigens differ in their reaction with enzyme treated cells and by this are useful in investigating the nature of these antigens. Two antibodies are reacting in the Coombs test using anti-human globulin reagent (BS 45 reacting with cellano antigens and BS 46 showing specificity for D). If ascitic fluid is used in the Coombs test, unspecific reactions may be observed and in these cases supernatant has to be used. Concerning ABO blood group antigens the reaction sometimes is very much dependent on the stabilizer used, for example for BS 47, reacting preferably with A, antigen. This is also a very general phenomenon and has to be considered for each individual m ab. We obtained two antibodies reacting with AB, from which BS 53 shows at higher concentration weak reactions also with O-cells whereas BS 52 is specific and may be used as routine reagent. We also found two different pH optima a phenomenon which is still under investigation. IMPROVED METHOD 0.7. COAGULATION FACTOR XIIIA
FOR
PHENOTYPING
OF
HUMAN
S. Weidinger, F. Schwarzfischer, H.-J. Leifheit and H. Cleve (Institut fiir Anthropologie und Humangenetik der Universitlt Miinchen, RichardWagner-Str. 10/I, D-8000 Miinchen 2, F.R.G.) The inherited polymorphism of the subunit A of factor XIII (F XIIIA), the fibrin stabilizing factor of the human coagulation system, was examined with two different methods. Isoelectric focusing (IEF) on polyacrylamide gels with a pH gradient from 5 to 8 gave superior resolution when compared with agarose gel electrophoresis and subsequent immunofixation. For the classification of genetic F XIIIA phenotypes platelet preparations from ACD-blood were used. Platelets were prepared by differential centrifugation, lysed and applied with filter papq on 0.5-mm thick flat bed gels. Separation by IEF was carried out for 180 min at maximally 1600 V, 25 mA, and 18 W. The patterns were developed by immune printing with monospecific antiserum.
In addition to antiPr and anti-Gd CA, several further CA specificities were found. Until now only single examples of the CA have been published. Su antigen is partially protease susceptible. It is fully expressed at birth. Anti-Sa recognizes the NeuAm2-3Galpl. . . structure on RBC membrane SGP. Fl antigen is protease resistant. It differs from Pr1,2,3, Gd1,2 and Sa antigens since it is weakly expressed on newborn (and i adult) RBC [ 51. It is, therefore a developmentally regulated antigen like I but differing from I, since NeuAc is not involved in the I antigenic determinant. The Fl CA recognizes the binary determinant consisting of NeuAc linked to the terminal Gal of the l-3 branch and of Fuc linked to the other terminal Gal of the l-6 branch of the branched polylactosaminyl core structure known to represent the I antigenic determinants [6]. Lud antigen recognized by the anti-Lud Ca is a further NeuAcdependent developmentally regulated antigen. It differs from the Fl antigen, since the Lud antigen is fully expressed on i adult RBC [ 71. The data indicate that monoclonal anti&, anti-Gd and “related” CA are excellent markers for cell surface profiles of neuraminyl structures. References 1 2 3 4 5 6
D. Roelcke, Eur. J. Zmmunol., 3 (1973) 206. D. Roelcke, W. Riesen, H.P. Geisen and W. Ebert, VOX Sang., 33 (1977) 304. S.K. Kundu, D.M. Marcus and D. Roelcke, Zmmunol. Let&, 4 (1982) 263. D. Roelcke and R. Brossmer, Prot. Biol. Fluids, 31 (1983) (in press). D. Roelcke, VOX Sang., 41 (1981) 98. R. Kannagi, D. Roelcke, K.A. Peterson, Y. Okada, S.B. Levery and S. Hakomori, Carbohyd. Res., (in press). 7 D. Roelcke, VOX Sang., 41 (1981) 316.
0.6. PROPERTIES OF MONOCLONAL HUMAN ERYTHROCYTE ANTIGENS
ANTIBODIES
REACTING
WITH
H.H. Sonnebom, H. Uthemann and A.C. Munroa (Biotest-Serum-Institut GmbH, Geleitsstrasse 103, Offenbach, F.R.G. and aGlasgow and West of Scotland Blood Transfusion Service, Lanarkshire, U.K.) Since the development of the monoclonal antibody technique by Kiihler and Milstein, this technology has had great influence in immunology, and a lot of monoclonal antibodies have been produced for the investigation and determination of soluble antigens and especially for the characterization of cells and cell surface antigens. In order to get more standardized reagents for blood group typing and to get more information on the biochemistry of blood group antigens we have immunized Balb/c mice with whole red blood cells. After fusing the spleen cells with the mouse myeloma cell line X63Ag8.653 we obtained several
52
clones secreting antibodies with blood group specificity, We found that each monoclonal antibody (m ab) has its own individual properties and they need different conditions for optimal reactions: Concerning the M/N blood group antigens we have investigated two m ab (BS 40, BS 41) reacting preferably with the N antigen, which show the strongest reaction at pH 7.0 whereas at pH 8.5 the discrimination between the M and N antigens is best and by this show a better specificity at pH 8.5. Regarding anti-M antibodies BS 38 shows optimal specificity at pH 7.0 and 37°C BS 39 at pH 8.5 and 37°C and BS 44 between pH 7.5 and 9 and over a broad temperature range, BS 44 seems to be a very useful routine reagent and first successful routine trials have been performed with this antibody. All the antibodies reacting with M/N antigens differ in their reaction with enzyme treated cells and by this are useful in investigating the nature of these antigens. Two antibodies are reacting in the Coombs test using anti-human globulin reagent (BS 45 reacting with cellano antigens and BS 46 showing specificity for D). If ascitic fluid is used in the Coombs test, unspecific reactions may be observed and in these cases supernatant has to be used. Concerning ABO blood group antigens the reaction sometimes is very much dependent on the stabilizer used, for example for BS 47, reacting preferably with A, antigen. This is also a very general phenomenon and has to be considered for each individual m ab. We obtained two antibodies reacting with AB, from which BS 53 shows at higher concentration weak reactions also with O-cells whereas BS 52 is specific and may be used as routine reagent. We also found two different pH optima a phenomenon which is still under investigation. IMPROVED METHOD 0.7. COAGULATION FACTOR XIIIA
FOR
PHENOTYPING
OF
HUMAN
S. Weidinger, F. Schwarzfischer, H.-J. Leifheit and H. Cleve (Institut fiir Anthropologie und Humangenetik der Universitlt Miinchen, RichardWagner-Str. 10/I, D-8000 Miinchen 2, F.R.G.) The inherited polymorphism of the subunit A of factor XIII (F XIIIA), the fibrin stabilizing factor of the human coagulation system, was examined with two different methods. Isoelectric focusing (IEF) on polyacrylamide gels with a pH gradient from 5 to 8 gave superior resolution when compared with agarose gel electrophoresis and subsequent immunofixation. For the classification of genetic F XIIIA phenotypes platelet preparations from ACD-blood were used. Platelets were prepared by differential centrifugation, lysed and applied with filter papq on 0.5-mm thick flat bed gels. Separation by IEF was carried out for 180 min at maximally 1600 V, 25 mA, and 18 W. The patterns were developed by immune printing with monospecific antiserum.