Advertisement

Immunogenetics

, Volume 2, Issue 1, pp 183–197 | Cite as

Association of HL-A antigens andβ2-microglobulin at the cellular and molecular level

  • Ralph A. Reisfeld
  • Ernest D. Sevier
  • Michele A. Pellegrino
  • Soldano Ferrone
  • Miroslav D. Poulik
Article

Abstract

Surfaces of cultured human lymphoid cells RPMI 1788, RPMI 4098, RPMI 8866, Raji, and WI-L2 were found to contain bothβ2-microglobulin (β2-μ) and HL-A determinants when tested by direct complement-dependent cytotoxicity andquantitative absorption with different cytotoxic antiβ2-μ antisera and specific HL-A alloantisera. The same antigenic specificities were found in 3M KCl extracts of these cultured cells with a sensitiveβ2-μ radioimmunoassay and an HL-A antigen blocking assay. Daudi cells provided a contrast, since noβ2-μ or HL-A determinants were found on their surfaces or in 3 M KCl extracts prepared from them. Results from specific antibody blocking tests suggest a close association betweenβ2-μ and HL-A determinants on plasma membranes of cultured human lymphoid cells. A solid state immunoadsorbent containing antiβ2-μ antibodies effectively removed all detectable HL-A antigenic activity from some 3M KCl extracts of cultured human lymphoid cells as well as from some sera. Adsorption of HL-A antigens to these immunoadsorbents was specific since it was blocked only by prior addition ofβ2-μ. Once on the antiβ2-μ immunoadsorbents, HL-A antigens still reacted specifically with HL-A alloantibodies in quantitative absorption experiments. HL-A antigens andβ2-μ could be eluted from antiβ2-μ immunoadsorbents with a variety of chaotropic reagents and detergents, but thus far potassium bromide and sodium dodecyl sulfate (SDS) appear to be the most effective. SDS-PAGE of these eluates indicated that HL-A antigens were considerably purified by adsorption to antiβ2-μ immunoadsorbents and that two major molecular size fragments were distinguishable, i.e., ∼33,000 for HL-A and ∼ 12,000 forβ2-μ.

Keywords

Sodium Dodecyl Sulfate Sodium Dodecyl Sulfate Antigenic Specificity Size Fragment Absorption Experiment 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Berggard, I. and Bearn, A.G.: Isolation and Properties of a low molecular weight β2 globulin occurring in human biological fluids.J. Biol. Chem. 243: 4095–4103, 1968Google Scholar
  2. Cresswell, P., Turner, M.J., and Strominger, J.L.: Papain-solubilized HL-A antigens from cultured human lymphocytes contain two peptide fragments.Proc. Natl. Acad. Sci. USA 70: 1603–1607, 1973Google Scholar
  3. Cuatrecasas, P.: Protein purification of affinity chromatography. Derivatizations of agarose and polyacrylamide beads.J. Biol. Chem. 245:3059–3065, 1970Google Scholar
  4. David, G.S. and Reisfeld, R.A.: Protein iodination with solid state lactoperoxidase.Biochemistry 13: 1014–1021, 1974Google Scholar
  5. Evrin, P.E. and Pertoft, A.:β 2-microglobulin in human blood cells.J. Immunol. 111:1147–1154, 1973Google Scholar
  6. Evrin, P.E. and Nilsson, K.:β 2 microglobulin production in vitro by human hematopoietic, mesenchymal and epithelial cells.J. Immunol. 112:137–144, 1974Google Scholar
  7. Ferrone, S., Tosi, R.M., and Centis, D.: Anticomplementary factors affecting the lymphocytotoxicity test.In E.D. Curtini, P.L. Mattiuz, and R.M. Tozzi (eds.):Histocompatibility Testing, 1967; pp. 357–363, Munksgaard, Copenhagen, 1967Google Scholar
  8. Ferrone, S., Pellegrino, M.A., and Reisfeld, R.A.: A rapid method for direct HL-A typing of cultured lymphoid cells.J. Immunol. 107:613–615, 1971Google Scholar
  9. Ferrone, S., Pellegrino, M.A., Billing, R., Terasaki, P.I., and Reisfeld, R.A.: Production of monospecific W24 xenoantisera.Tissue Antigens, in press, 1975Google Scholar
  10. Grey, H.M., Kubo, R.T., Colon, S.M., Poulik, M.D., Cresswell, P., Springer, T., Turner, M., and Strominger, J.L.: The small subunit of HL-A antigens isβ 2-microglobulin.J. Exp. Med. 138:1608–1612, 1973Google Scholar
  11. Kennel, S.J. and Lerner, R.A.: Isolation and characterization of plasma membrane associated immunoglobulin from cultured human diploid lymphocytes.J. Mol. Biol. 76: 485–502, 1973Google Scholar
  12. Klein, E., Klein, G., Nadkarni, J.S., Nadkarni, J.J., Wigzell, H., and Clifford, P.: Surface IgM-Kappa specificity on a Burkitt lymphoma cell in vivo and in derived culture lines.Cancer Res. 28:1300–1310, 1968Google Scholar
  13. Lowry, O.H., Rosebrough, N., Farr, A., and Randall, R.: Protein measurement with the folin phenol reagent.J. Biol. Chem. 193: 265–275, 1951Google Scholar
  14. Mittal, K.K., Mickey, M.R., Singal, D.P., and Terasaki, P.I.: Serotyping for homotransplantation. XVIII. Refinement of microdroplet lymphocyte cytotoxicity test.Transplantation 6:913–927, 1968Google Scholar
  15. Nakamuro, K., Tanigaki, N., and Pressman, D.: Multiple common properties of humanβ 2-microglobulin and the common portion fragment derived from HL-A antigen molecules.Proc. Natl. Acad. Sci. USA 70: 2863–2865, 1973Google Scholar
  16. Neauport-Sautes, C., Bismuth, A., Kourilsky, F.M., and Manuel, Y.: Relationship between HL-A antigens andβ 2-microglobulin as studied by immunofluorescence on the lymphocyte membrane.J. Exp. Med. 139: 957–968, 1974Google Scholar
  17. Oh, S.K., Pellegrino, M.D., and Reisfeld, R.A.: Hypertonic salt extraction of HL-A antigens: assessment of protease activity.Proc. Soc. Exp. Biol. Med. 145:1272–1277, 1974Google Scholar
  18. Pellegrino, M.A., Ferrone, S., and Pellegrino, A.: A simple microabsorption technique for HL-A typing.Proc. Soc. Exp. Biol. Med. 139:484–188, 1972aGoogle Scholar
  19. Pellegrino, M.A., Ferrone, S., and Pellegrino, A.: Serological detection of soluble HL-A antigens.In B.D. Kahan and R.A. Reisfeld (eds.):Transplantation Antigens; pp. 433–452, Academic Press, New York, 1972bGoogle Scholar
  20. Pellegrino, M.A., Ferrone, S., Pellegrino, A., Oh, S.K., and Reisfeld, R.A.: Evaluation of two sources of HL-A antigens: platelets and serum.Eur. J. Immunol. 4:246–250, 1974aGoogle Scholar
  21. Pellegrino, M.A., Ferrone, S., Cooper, N.R., Dierich, M.P., and Reisfeld, R.A.: Variation in susceptibility of a human lymphoid cell line to immune lysis during the cell cycle.J. Exp. Med. 140:578–590, 1974 bGoogle Scholar
  22. Peterson, P.A., Rask, L., and Lindblom, J.B.: Highly purified papain-solubilized HL-A antigens containβ 2-microglobulin.Proc. Natl. Acad. Sci. USA 71:35–39, 1974Google Scholar
  23. Poulik, M.D., Bernoco, M., Bernoco, D., and Ceppellini, R.: Aggregation of HL-A antigens at the lymphocyte surface induced by antiserum toβ 2-microglobulin.Science 182:1352–1355, 1973Google Scholar
  24. Poulik, M.D. and Bloom, A.D.:β 2-microglobulin production and secretion by lymphocytes in culture.J. Immunol. 110:1430–1433, 1973Google Scholar
  25. Preud'homme, J.L., Neauport-Sautes, C., Piat, S., Silvestre, D., and Kourilsky, F.M.: Idependence of HL-A antigens and immunoglobulin determinants on the surface of human lymphoid cells.Eur. J. Immunol. 2:297–300, 1972Google Scholar
  26. Reisfeld, R.A., Pellegrino, M.A., and Kahan, B.D.: Salt extraction of soluble HL-A antigens.Science 172: 1134–1136, 1971Google Scholar
  27. Rodbard, D. and Chrambach, A.: Estimation of molecular radius, free mobility and valence using polyacrylamide gel electrophoresis.Anal Biochem. 40:95–134, 1971Google Scholar
  28. Solheim, B.G. and Thorsby, E.:β 2-microglobulin. Part of the HL-A molecule in the cell membrane.Tissue Antigens 4:83–94, 1974Google Scholar
  29. Weber, K. and Osborne, M.: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis.J. Biol. Chem. 244:4406–4412, 1969Google Scholar

Copyright information

© Springer-Verlag New York Inc 1975

Authors and Affiliations

  • Ralph A. Reisfeld
    • 3
  • Ernest D. Sevier
    • 3
  • Michele A. Pellegrino
    • 3
  • Soldano Ferrone
    • 3
  • Miroslav D. Poulik
    • 1
    • 2
    • 3
  1. 1.Department of ImmunochemistryWilliam Beaumont HospitalRoyal Oak
  2. 2.Department of Immunology and MicrobiologyWayne State University School of MedicineDetroit
  3. 3.Department of Molecular ImmunologyScripps Clinic and Research FoundationLa Jolla

Personalised recommendations