Journal of Immunogenetics ( 1990), 17, 30 1-308.
WORKSHOP 2 MONOCLONAL ANTIBODIES AGAINST GLYCOPHORINS A N D OTHER GLYCOPROTEINS D . J. A N S T E E *AND E . LlSOWSKA? *International Blood Group Reference Laboratory, South Western Regional Blood Transfusion Centre. Bristol. U .K . and ?Department of Immunochemistry. Institute of Immunology and Experimental Therapy, Polish Academy of Sciences. Wroclaw, Poland
INTRODUCTION The Workshop panel included 39 antibodies of which 30 were submitted as specific for Glycophorins A, B or C. Four antibodies reacted with Lutheran-related antigens (CD44) and five were directed against other glycoproteins (Kell, Kidd, 12E7, CD59, band 3). Those laboratories which submitted reports on some or all of these antibodies are listed in Table 1. In contrast to the First Workshop the specificities assigned by the antibody makers were given to Workshop participants at the time they received samples for testing. The techniques used by Workshop participants to study the antibodies were generally the same as those used in the First Workshop held in Paris in 1987. The antibodies were tested most extensively by serological methods, i.e. direct or indirect agglutination of untreated and neuraminidase- or protease-treated erythrocytes. In several laboratories the antibodies were tested against a panel of erythrocytes carrying variant or defective antigens. Some laboratories carried out immunochemical studies using immunoblotting, microplate ELISA, inhibition of antibodies with modified antigens or their fragments. With one exception, the technical details of the methods used were at the discretion of individual participating laboratories. The exception was the inclusion of a standard protocol for the serological examination of M, N antibodies. The Workshop was divided into three sections: Section 2.1 : M, N Antibodies; Section 2.2: Antibodies to Glycophorins and other glycoproteins; Section 2.3: Lutheran related. Kell and Jk antibodies. Section 2. I ( M , N Antibodies) Fourteen antibodies were examined in this section (5 anti-M, 9 anti-N). All five submitted anti-Ms (1 30-1 34) were confirmed as specific for the M antigen and were considered to have potential lor use as blood typing reagents. All anti-M antibodies reacted with epitopes dependent on sialic acid residues. The specificity of four of them was based on the Correspondence : Dr D. J . Anstee, International Blood Group Reference Laboratory, South Western Regional Blood Transfusion Centre, Southmead Road, Bristol BSl0 SND, U . K . 30 I
D . J . Anstee and E. Lisowska TABLE1. Laboratories submitting Workshop reports
Blanchard, D., Dahr, W., Guimbretiere, L., Bernard, D., Guimbretiere, J. and Muller, J.Y. Centre Regional de Transfusion Sanguine, Nantes, France. Bourel, D. Centre Regional de Transfusion Sanguine, Rennes, France. Chung, A. Canadian Red Cross Society, Ottawa, Canada. Ciaffoni, S . Servizio Transfusionale e di Immunoemetalogia, Verona, Italy. Coghlan, G. Rh Laboratory, Winnipeg, Canada. Condon, J. Red Cross Blood Bank, Victoria, Melbourne, Australia. Daniels, G.L. MRC Blood Group Unit, London, United Kingdom. Duk, M., Jaskiewicz, E., Wasniowska, K., Czerwinski, M. and Lisowska, E. Institute of Immunology and Experimental Therapy, Wroclaw, Poland. Fletcher, A., Bryant, J., Yuan, F.F. New South Wales Red Cross Transfusion Service, Sydney, Australia. Fraser, R.H. Glasgow and West of Scotland Blood Transfusion Service, Carluke, Scotland, United Kingdom. James, M.F. West Midlands Blood Transfusion Centre, Birmingham, United Kingdom. King, M.J., Amphlett, N., Jones, N. and Morris, C. International Blood Group Reference Laboratory, Bristol, United Kingdom. Lin-Chu, M. Mackay Memorial Hospital, Taiwan. Macdonald, B. Red Cross Blood Transfusion Service, Brisbane, Australia. Mayr, W .R. Institut fur Transfusionsmedezin, Aachen, Federal Republic of Germany. Moores, P., Smart, E. and Green, F. Natal Blood Transfusion Service, Durban, South Africa. Moulds, J., Moulds, M.K. and Hoffman, M.C. Gamma Biologicals Inc., Houston, Texas, United States of America. Okubo, Y. Osaka Red Cross Blood Center, Osaka, Japan. Parson, S.F., Spring, F.A., Mallinson, G., Judson, P. and Anstee, D.J. South Western Regional Blood Transfusion Centre, Bristol, United Kingdom. Pinder, L. New Zealand Blood Transfusion Services, Auckland, New Zealand. Poole, J., Byrne, P., Liew, Y.W. and Banks, J. International Blood Group Reference Laboratory, Bristol, United Kingdom. Reid, M.E. International Blood Group Reference Laboratory, Bristol, United Kingdom. Sistonen, P. and Pirkola, A. Finnish Red Cross Blood Transfusion Service, Helsinki, Finland. Skov, F. Blood Bank, Centralsygehuset, Nykobing, Denmark. Sonneborn, H. Biotest AG, Offenbach, Federal Republic of Germany. Telen, M.J., Green, A.M. and Rao, N. Duke University Medical Center, Durham, North Carolina, United States of America. Tippett, P. MRC Blood Group Unit, London, United Kingdom.
recognition of the N-terminal Ser residue, since these antibodies reacted with Mc variant of Glycophorin A (Serl, Glu5), and did not react with He antigen (variant form of Glycophorin B with Gly5) or with M-active Glycophorin A after one step of Edman degradation (N-terminal Ser removed). One anti-M (130) showed exact opposite reactions which indicated that its epitope was related to the fifth Gly residue. The GlyS-dependent blood group M epitopes did not require amino group. The role of amino groups in Serl-dependent epitopes was differentiated from the essential (131, 132) to intermediate (133) or a negligible one (1 34). The results of the present Workshop extended the earlier findings on reactivity of some anti-M with Mg antigen which is a variant form of Glycophorin A nonglycosylated at the N-terminal and in terms of amino acid sequence more similar to N than M antigen. In the present panel the reaction with Mg antigen was shown reproducibly for GlyS-dependent 130 antibody, and conflicting results were reported for 133. This cross-reactivity is difficult to explain and may be an interesting model for studies on the relationship between structure and antigenic specificity or on the multispecificity of monoclonal antibodies (MABS).
D.J. Anstee and E. Lisowska
standardized monoclonal anti-N’s for use as routine blood typing reagents particularly given the high frequency of the haplotype MS in many populations (Mourant et al., 1976). However, red cells expressing Glycophorin variants (particularly St(a ), MiIII, MiVI in Orientals and Dantu in Blacks) may have higher levels of ‘N’ than homozygous MS red cells. This problem was discussed at the Workshop and the view was expressed that standardization of anti-N’s so that they fail to detect these variants is likely to be extremely difficult and in any case may not be desirable since as J. Moulds (Gamma Biologicals, Houston, Texas, U.S.A.) pointed out weak reactivity with anti-N’s is frequently a most useful indicator of the occurrence of such variants.
Section 2.2 (Antibodies to Glycophorins and other glycoproteins)
Nineteen antibodies were considered in this section. In a few cases the specificitiesdefined during the Workshop were found to be different from the specificities declared by the submitting laboratories. Anti-T and anti-Tn antibodies (080, 081, 082). These antibodies are directed against glycophorins with 0-glycosidic chains lacking sialic acid (T antigen), or sialic acid and Gal residues (Tn antigen). The anti-T specificity of 080 antibody was fully confirmed. The antibody reacted with human asialoglycophorins and did not react with native or asialoagalacto-glycophorins (agglutination, immunoblotting). The distinct crossreactivity of the 080 antibody with horse red cell asialoglycophorin (antigen with T-specific oligosaccahride chains, but with different polypeptide chain) indicated that the 0-linked disaccharide Galbl-3GalNAc is an immunodominant structure in the 080 epitope. Therefore, the 080 antibody can be used for the identification of this structure. The antibodies 08 1 and 082 were specific for the Tn-type glycophorins (asialo-agalacto-). The antibodies reacted with authentic Tn antigen (agglutination, immunoblotting) and also with chemically prepared human asialo-agalacto-glycophorin (ELISA). The antibodies did not cross-react with A or Cad antigen. Moreover, they reacted very weakly with asialoagalacto-glycophorin from horse erythrocytes and with asialo-ovine submaxillary mucin, despite the fact that these glycoproteins also carry the 0-linked GalNAc-residues. In conclusion, the antibodies 081 and 082 are good reagents for Tn antigen on human erythrocytes, but they would be less sensitive in reactions with other glycoproteins containing GalNAc-Ser/Thr residues. Most probably, due to the relatively small size of these residues, some adjacent components of the antigen play an essential role in the epitopes. Anti-S antibody (148).This antibody was interesting, since no Ss-related or Glycophorin Bspecific monoclonal antibody has been described before. Blood group S and s specificities are associated with different amino acid residues at position 29 of GPB (Meth in S and Thr in s). The 148 antibody was apparently specific for Glycophorin B of blood group S. However, in some assays (e.g. agglutination of trypsin-treated ss cells) the antibody showed a crossreactivity with s. It also reacted relatively strongly with S - Dantu erythrocytes (Green et al., 1990) but failed to react with MiIV S + red cells (Poole et al., 1990).
Anti-Glycophorin A, B Antibodies (147, 149-152; 158, 160)
These antibodies were submitted as anti-GPA (149-151, 158), anti-GPB (147, 152) or unknown (160). The results of the Workshop showed that 150 and 158 were indeed anti-GPA but 149 and 151 were anti-GPA+B. The two antibodies specific for GPA (150, 158) recognized different epitopes. The results of agglutination and immunoblotting with protease-treated red cells showed that trypsin
released both epitopes, but chymotrypsin gave different effects: the epitope 150 was evidently destroyed or released, while 158 was present in the chymotryptic fragment of Glycophorin A remaining in the membrane (amino acid residues 35-1 31). Interestingly, the epitope 150 was not detected by immunoblotting in membranes of chymotrypsin-treated red cells, i.e. it was not detected in GPA molecules resistant to chymotrypsin. It suggested that MAB 150 reacts specifically with an epitope on less glycosylated chymotrypsin-sensitive molecules of GPA. It seems to be in agreement with the finding that both epitopes, 150 and 158, are independent of sialic acid. The above results, lack of reactivity with GPB and inhibition by glycopeptide fragments of GPA suggest that the epitopes 150 and 158 are located within amino acid residues 27-235 and