Demonstration of Receptor Function of Membrane Proteins by Selection and Immobilization with Monoclonal Antibodies

  • Janis Jacks Weis
  • Douglas T. Fearon


Monoclonal antibodies (MAbs) that are specific for certain types of cells and for stages of ontogenetic development can be produced without prior knowledge of antigenic differences between the cell types. Once identified, however, the elucidation of the function of these interesting antigens may represent a formidable task. In this chapter, we discuss the technique of selection and immobilization of membrane proteins by MAbs bound to Staphylococcus aureus bacteria. This technique allows visualization of receptor interactions with ligands on cells or in solution. Since multivalent interactions between S. aureus bacteria-MAb-receptor complexes and ligand-coated particles can be established, the method is particularly useful for studies on receptors where univalent reactions are of low affinity. The use of this method to identify two different membrane proteins as receptors for fragments of the third component of complement (C3) and one of these additionally as an Epstein-Barr virus (EBV) receptor, is described. To introduce these studies, the biology of C3, C3 receptors, and EBV is first briefly reviewed.


Systemic Lupus Erythematosus Receptor Function Complement Receptor Ligand Binding Site Sheep Erythrocyte 
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.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Law, S. K., and Levine, R. P., 1977, Interaction between the third complement protein and cell surface macromolecules, Proc. Natl. Acad. Sci. USA 74:2701–2705.PubMedCrossRefGoogle Scholar
  2. 2.
    Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L., and Prahl, J. W., 1980, Evidence for presence of an internal thiolester bond in third component of human complement, Proc. Natl. Acad. Sci. USA 77:5764–5768.PubMedCrossRefGoogle Scholar
  3. 3.
    Pangburn, M. K., and Müller-Eberhard, H. J., 1980, Relation of a putative thiolester bond in C3 to activation of the alternative pathway and the binding of C3b to biological targets of complement, J. Exp. Med. 152:1102–1114.PubMedCrossRefGoogle Scholar
  4. 4.
    Harrison, R. A., and Lachman, P. J., 1980, Novel cleavage products of the third component of human complement, Mol. Immunol. 17:9–20.PubMedCrossRefGoogle Scholar
  5. 5.
    Lachmann, P. J., Pangburn, M. K., and Oldroyd, R. G., 1982, Breakdown of C3 after complement activation: Identification of a new fragment, C3g, using monoclonal antibodies, J. Exp. Med. 156:205–216.PubMedCrossRefGoogle Scholar
  6. 6.
    Ross, G. D., Lambris, J. D., Cain, J. A., and Newman, S., 1982, Generation of three different fragments of bound C3 with purified Factor I or serum. I. Requirements for Factor H vs. CR1 cofactor activity, J. Immunol. 129:2051–2060.PubMedGoogle Scholar
  7. 7.
    Fearon, D. T., 1980, Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte, J. Exp. Med. 152:20–30.PubMedCrossRefGoogle Scholar
  8. 8.
    Dobson, N. J., Lambris, J. D., and Ross, G. D., 1981, Characteristics of isolated erythrocyte complement receptor type one (CR1, C4b-C3b receptor) and CRl-specific antibodies, J. Immunol. 126:693–698.PubMedGoogle Scholar
  9. 9.
    Ross, G. D., Newman, S. L., Lambris, J. D., Devery-Pocius, J. E., Cain, J. A., and Lachmann, P. J., 1983, Generation of three different fragments of bound C3 with purified Factor I or serum. II. Location of binding sites in the C3 fragments for factors B and H, complement receptors, and bovine conglutinin, J. Exp. Med. 158:334–352.PubMedCrossRefGoogle Scholar
  10. 10.
    Cooper, N. R., 1969, Immune adherence by the fourth component of complement, Science 165:396–398.PubMedCrossRefGoogle Scholar
  11. 11.
    Tedder, T. F., Fearon, D. T., Gartland, G. L., and Cooper, M. D., 1983, Expression of C3b receptors on human B cells and myelomonocytic cells but not on natural killer cells, J. Immunol. 120:1668–1673.Google Scholar
  12. 12.
    Wilson, J. G., Tedder, T. F., and Fearon, D. T., 1983, Characterization of human T lymphocytes that express the C3b receptor, J. Immunol. 131:684–689.PubMedGoogle Scholar
  13. 13.
    Vranian, G., Jr., Conrad, D. H., and Ruddy, S., 1981, Specificity of C3 receptors that mediate phagocytosis by rat peritoneal mast cells, J. Immunol. 126:2302–2306.PubMedGoogle Scholar
  14. 14.
    Gelfand, M. C., Frank, M. M., and Green, I., 1975, A receptor for the third component of complement in the human renal glomerulus, J. Exp. Med. 142:1029–1034.PubMedCrossRefGoogle Scholar
  15. 15.
    Kazatchkine, M. D., Fearon, D. T., Appay, M. D., Mandet, C., and Bariety, J., 1982, Immuno-histochemical study of the human glomerular C3b receptor in normal kidney and in seventy-five cases of renal diseases, J. Clin. Invest. 69:900–912.PubMedCrossRefGoogle Scholar
  16. 16.
    Emancipator, S. N., Iida, K., Nussenzweig, V., and Gallo, G. R., 1983, Monoclonal antibodies to human complement receptor (CR1) detect defects in glomerular diseases, Clin. Immunol. Immunopathol. 27:170–175.PubMedCrossRefGoogle Scholar
  17. 17.
    Daha, M. R., Bloem, A. C., and Ballieux, R. E., 1984, Immunoglobulin production by human peripheral lymphocytes induced by anti-C3 receptor antibodies, J. Immunol. 132:1197–1201.PubMedGoogle Scholar
  18. 18.
    Wong, W. W., Wilson, J. G., and Fearon, D. T., 1983, Genetic regulation of a structural polymorphism of human C3b receptor, J. Clin. Invest. 72:685–693.PubMedCrossRefGoogle Scholar
  19. 19.
    Dykman, T. R., Cole, J. L., Iida, K., and Atkinson, J. P., 1983, Polymorphism of human erythrocyte C3b/C4b receptor, Proc. Natl. Acad. Sci. USA 80:1698–1702.PubMedCrossRefGoogle Scholar
  20. 20.
    Dykman, T. R., Hatch, J. A., and Atkinson, J. P., 1984, Polymorphism of the human C3b/C4b receptor. Identification of a third allele and analysis of receptor phenotypes in families and patients with systemic lupus erythematosus, J. Exp. Med. 159:691–703.PubMedCrossRefGoogle Scholar
  21. 21.
    Dykman, T., Hatch, J., Aqua, M., and Atkinson, J., 1984, Identification of novel C3b/C4b receptor (C3bR) phenotypes in normal individuals and SLE, Fed. Proc. 43:1454 (abstract).Google Scholar
  22. 22.
    Miyakawa, Y., Yamada, A., Kosaka, K., Tsuda, F., Kosuga, E., and Mayumi, M., 1981, Defective immune adherence (C3b) receptor on erythrocytes of patients with systemic lupus erythematosus, Lancet 2:493–497.PubMedCrossRefGoogle Scholar
  23. 23.
    Wilson, J. G., Wong, W. W., Schur, P. H., and Fearon, D. T., 1982, Mode of inheritance of decreased C3b receptors on erythrocytes of patients with systemic lupus erythematosus, N. Engl. J. Med. 307:981–986.PubMedCrossRefGoogle Scholar
  24. 24.
    Iida, K., Mornaghi, R., and Nussenzweig, V., 1982, Complement receptor (CR1) deficiency in erythrocytes from patients with systemic lupus erythematosus, J. Exp. Med. 155:1427–1438.PubMedCrossRefGoogle Scholar
  25. 25.
    Ross, G. D., Walport, M. J., Parker, C. J., Lentine, A. F., Fuller, C. R., Yount, W. J., Myones, B. L., Winfield, J. B., and Lachmann, P. J., 1984, Acquired loss of erythrocyte (E) CR1 (C3b-receptor) in systemic lupus erythematosus and other diseases with autoantibodies and/or complement activation, Arthritis Rheum. 27:S28.Google Scholar
  26. 26.
    Eden, A., Miller, G. W., and Nussenzweig, V., 1973, Human lymphocytes bear membrane receptors for C3b and C3d, J. Clin. Invest. 52:3239–3242.PubMedCrossRefGoogle Scholar
  27. 27.
    Ross, G. D., Polley, M. J., Rabellino, E. M., and Grey, H. M., 1973, Two different complement receptors on human lymphocytes; one specific for C3b and one specific for C3b inactivator-cleaved C3b, J. Exp. Med. 138:798–811.PubMedCrossRefGoogle Scholar
  28. 28.
    Nadler, L. M., Stashenko, P., Hardy, R., van Agthoven, A., Terhorst, C., and Schlossman, S. F., 1981, Characterization of a human B cell-specific antigen (B2) distinct from B1, J. Immunol. 126:1941–1947.PubMedGoogle Scholar
  29. 29.
    Tedder, T. F., Clement, L. T., and Cooper, M. D., 1984, Expression of C3d receptors during human B cell differentiation: Immunofluorescence analysis with the HB-5 monoclonal antibody, J. Immunol. 133:678–683.PubMedGoogle Scholar
  30. 30.
    Iida, K., Nadler, L., and Nussenzweig, V., 1983, Identification of the membrane receptor for the complement fragment C3d by means of a monoclonal antibody, J. Exp. Med. 158:1021–1033.PubMedCrossRefGoogle Scholar
  31. 31.
    Weis, J. J., Tedder, T. F., and Fearon, D. T., 1984, Identification of a 145,000 Mr membrane protein as the C3d receptor (CR2) of human B lymphocytes, Proc. Natl. Acad. Sci. USA 81:881–885.PubMedCrossRefGoogle Scholar
  32. 32.
    Ault, K. A., and Springer, T. A., 1981, Cross-reaction of a rat anti-mouse phagocyte-specific monoclonal antibody (anti-Mac-1) with human monocytes and natural killer cells, J. Immunol. 126:359–364.PubMedGoogle Scholar
  33. 33.
    Beller, D. I., Springer, T. A., and Schreiber, R. D., 1982, Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor, J. Exp. Med. 156:1000–1009.PubMedCrossRefGoogle Scholar
  34. 34.
    Wright, S. D., Rao, P. E., Van Voorhis, W. C., Craigmyle, L. S., Iida, K., Talle, M. A., Westberg, E. F., Goldstein, G., and Silverstein, S. C., 1983, Identification of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies, Proc. Natl. Acad. Sci. USA 80:5699–5703.PubMedCrossRefGoogle Scholar
  35. 35.
    Arnaout, M. A., Todd, R. F., III, Dana, N., Melamed, J., Schlossman, S. F., and Colten, H. R., 1983, Inhibition of phagocytosis of complement C3- or immunoglobulin G-coated particles and of C3bi binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (Mol), J. Clin. Invest. 72:171–179.PubMedCrossRefGoogle Scholar
  36. 36.
    Jondal, M., and Klein, G., 1973, Surface markers on human B and T lymphocytes. II. Presence of Epstein-Barr virus receptors on B lymphocytes, J. Exp. Med. 138:1365–1378.PubMedCrossRefGoogle Scholar
  37. 37.
    Sixbey, J. W., Vesterinen, E. H., Nedrud, J. G., Raab-Traub, N., Walton, L. A., and Pagano, J. S., 1983, Replication of Epstein-Barr virus in human epithelial cells infected in vitro, Nature 306:480–483.PubMedCrossRefGoogle Scholar
  38. 38.
    Sixbey, J. W., Nedrud, J. G., Raab-Traub, N., Hanes, R. A., and Pagano, J. S., 1984, Epstein-Barr virus replication in oropharyngeal epithelial cells, N. Engl. J. Med. 310:1225–1230.PubMedCrossRefGoogle Scholar
  39. 39.
    Jondal, M., Klein, G., Oldstone, M. B. A., Bokish, V., and Yefenof, E., 1976, Surface markers on human B and T lymphocytes. VIII. Association between complement and Epstein-Barr virus receptors on human lymphoid cells, Scand. J. Immunol. 5:401–410.Google Scholar
  40. 40.
    Einhorn, L., Steinitz, M., Yefenof, E., Ernberg, I., Bakacs, T., and Klein, G., 1978, Epstein-Barr virus (EBV) receptors, complement receptors and EBV infedibility of different lymphocyte fractions of human peripheral blood. II. Epstein-Barr virus studies, Cell Immunol. 35:43–58.PubMedCrossRefGoogle Scholar
  41. 41.
    Yefenof, E., Klein, G., and Kvarnung, K., 1977, Relationships between complement activation, complement binding, and EBV absorption by human hematopoietic cell lines, Cell. Immunol. 31:225–233.PubMedCrossRefGoogle Scholar
  42. 42.
    Yefenof, E., Klein, G., Jondal, M., and Oldstone, M. B. A., 1976, Surface markers on human B-and T-lymphocytes. IX. Two-color immunofluorescence studies on the association between EBV receptors and complement receptors on the surface of lymphoid cell lines, Int. J. Cancer 17:693–700.PubMedCrossRefGoogle Scholar
  43. 43.
    Yefenof, E., and Klein, G., 1977, Membrane receptor stripping confirms the association between EBV receptors and complement receptors on the surface of human B lymphoma lines, Int. J. Cancer 20:347–352.PubMedCrossRefGoogle Scholar
  44. 44.
    Jonsson, V., Wells, A., and Klein, G., 1982, Receptors for the complement C3d component and the Epstein-Barr virus are quantitatively coexpressed on a series of B-cell lines and their derived somatic cell hybrids, Cell Immunol. 72:263–276.PubMedCrossRefGoogle Scholar
  45. 45.
    Magrath, I., Freeman, C., Santaella, M., Gadek, J., Frank, M., Spiegel, R., and Novikovs, L., 1981, Induction of complement receptor expression in cell lines derived from human undifferentiated lymphomas. II. Characterization of the induced complement receptors and demonstration of simultaneous induction of EBV receptor, J. Immunol. 127:1039–1043.PubMedGoogle Scholar
  46. 46.
    Schwaber, J. F., Klein, G., Ernberg, I., Rosen, A., Lazarus, H., and Rosen, F. S., 1980, Deficiency of Epstein-Barr virus (EBV) receptors on B lymphocytes from certain patients with common varied agammaglobulinemia, J. Immunol. 124:2191–2196.PubMedGoogle Scholar
  47. 47.
    Hutt-Fletcher, L. M., Fowler, E., Lambris, J. D., Feighny, J., Simmons, J. G., and Ross, G. D., 1983, Studies of the Epstein-Barr virus receptor found on Raji cells. II. A comparison of lymphocyte binding sites for Epstein-Barr virus and C3d, J. Immunol. 130:1309–1312.PubMedGoogle Scholar
  48. 48.
    Fingeroth, J. D., Weis, J. J., Tedder, T. F., Strominger, J. L., Biro, P. A., and Fearon, D. T., 1984, Epstein-Barr virus receptor of human B lymphocytes is the C3d receptor CR2, Proc. Natl. Acad. Sci. USA 81:4510–4514.PubMedCrossRefGoogle Scholar
  49. 49.
    Lemieux, S., Avrameas, S., and Bussard, A. E., 1974, Local haemolysis plaque assay using a new method of coupling antigens on sheep erythrocytes by glutaraldehyde, Immunochemistry 11:261–269.PubMedCrossRefGoogle Scholar
  50. 50.
    Poston, R. N., 1974, A buffered chromic chloride method of attaching antigens to red cells: Use in haemagglutination, J. Immunol. Meth. 5:91–102.CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1985

Authors and Affiliations

  • Janis Jacks Weis
    • 1
    • 2
  • Douglas T. Fearon
    • 1
    • 2
  1. 1.Department of MedicineHarvard Medical SchoolBostonUSA
  2. 2.Department of Rheumatology and ImmunologyBrigham and Women’s HospitalBostonUSA

Personalised recommendations