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Anti-galactosyl Natural Antibodies in the Patients with Antibody Deficiency

Anti-galactosyl Natural Antibodies in the Patients with Antibody Deficiency. M. Hamanová, V. Thon, J. Litzman, J. Lokaj. University Centre for Primary Immunodeficiencies Department of Clinical Immunology and Allergology St. Anne University Hospital, Masaryk University Brno, Czech Republic.

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Anti-galactosyl Natural Antibodies in the Patients with Antibody Deficiency

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  1. Anti-galactosyl Natural Antibodies in the Patients with Antibody Deficiency M. Hamanová, V. Thon, J. Litzman, J. Lokaj University Centre for Primary Immunodeficiencies Department of Clinical Immunology and Allergology St. Anne University Hospital, Masaryk University Brno, Czech Republic

  2. Natural Antibodies • Natural Antibodies against carbohydrate antigens are thougt to be produced through life as a polyclonal response to antigenic stimulation by gastrointestinal bacteria but their origin is uncertain.

  3. New possibility:Gala1-3Galwas identified as the epitope of rabbit erythrocytes. Natural Antibodies: „Classical investigation“ - rabbit • Xenoagglutinins were • determiedby direct • HEMAGGLUTINATION test with rabbit erytrocytes.

  4. Characterization of Natural Antibodies Natural antibodies against carbohydrate antigenes: • Xenoreactive IgM Ab = 1,8 – 4,1%, resp. 3,9 – 8,0% of total IgM • Xenoreactive IgG Ab = 1 – 2,4% of total IgG McMorrow, I. M., Comrack, C. A., Sachs, D. H., DerSimonian, H. (1997). Heterogeneity of human anti-pig natural antibodies cross-reactive with the Gal(a1,3)Galactose epitope. Transplantation 64: 501-10.

  5. Development of the method - NA • ELISA with antigen Gala1-3Gal • in the isotypes IgM, IgG and IgA, • enable to detect • „classical natural antibodies against rabbit erythrocytes“. • Rieben, R., von Allmen, E., Korchagina, E. Y., Nydegger, U. E., Neethling, F. A., Kujundzic, M., Koren, E., Bovin, N. V., Cooper, D. K. C. (1995). Detection, immunoabsorption, and inhibition of cytotoxic activity of anti-aGal antibodies using newly developed substances with synthetic Gala1-3Gal disaccharide epitopes. Xenotransplantation 2: 98-106.

  6. Developement and determination of IgM, IgG and IgA anti-a-galactosyl natural antibodies (NA) by ELISA and comparison with hemagglutination.

  7. Gal b 3 Gal 1 a 4 ELISAforNatural antibodies • antigen: • PAA-b-disaccharide Gala1-3Gal • (PAA-BdiLectinity Holdings, Inc., Moscow, Russia)

  8. ELISAfor IgM-, IgG- and IgA-NA • healthy donors • CVID • neonates

  9. ELISA • AGGLUTINATION dilution OD 1:128 1:64 1:32 IgM IgG IgA n = 15 dil. 1:40 Occurrence of anti-aGalNA in healthy donors

  10. ELISA • AGGLUTINATION dilution OD 1:8 1:4 IgM IgG IgA IgM IgG IgA <4 n = 15 dil. 1:40 Occurrence of anti-aGalNA in CVID

  11. AGGLUTINATION dilution 1:128 1:64 IgM IgG IgA Occurrence of anti-aGalNA in IVIG • ELISA OD Endobulin n = 4 dil. 1:40

  12. ELISA mother OD IgM IgM IgG IgG IgA IgA n = 15 dil. 1:40 Occurrence of anti-aGalNAin cord blood • ELISA cord blood OD n = 15 dil. 1:40

  13. IgM IgM IgM IgM IgG IgG IgG IgG IgA IgA IgA IgA Occurrence of anti-aGalNAin neonates, followed in time • ELISA • ELISA • ELISA • ELISA OD OD OD OD cord blood 6. month 12. month 24. month n = 28 n = 19 n = 14 n = 25 dil. 1:40 dil. 1:40 dil. 1:40 dil. 1:40

  14. With our specific ELISA using Gala1-3Gal antigen we determine natural antibodies in different Ig class such as IgM, IgG and IgA and so specific investigate up to 95% of xenoreactive natural antibodies. With the new reproducible assay we are able to detect natural antibodies in patients with CVID, particularly in low concentrations in IgM and IgA isotypes. Investigation of genetically conserved natural antibodies may permit functional evaluation and characterization of the differentiation state of B lymfocytes. As a perspective Gala1-3Gal antigencould also be appropriate for the detection of natural antibodies at the cellular level in ELISPOT assay.

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