Development of a Plaque Assay for the Detection of Red Blood Cells Carrying Abnormal or Mutant Hemoglobins

  • Masroor A. Baig
  • Aftab A. Ansari
Part of the Topics in Chemical Mutagenesis book series (TCM, volume 2)


The potential of immunological methods for the detection of single mammalian cells carrying an altered (mutant) form of marker protein molecule (antigen) has been recently reviewed.(1) The use of immunological methods for detection of mutations has been possible by using antibodies that can specifically recognize the amino acid difference(s) between the molecule normally present in a given cell and its variant form. Techniques like immunofluorescence(2,3) have been employed to distinguish cells carrying mutant forms of a protein against which antibody is available from the normal cells. In this chapter we describe the development of a simple method that could be used for detection and quan-titation of red blood cells (RBC) carrying a specific variant of hemoglobin (Hb).


Myeloma Cell Plaque Assay Indicator Cell Cyanuric Chloride Chromium Chloride 
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.
    A. A. Ansari and H. V. Mailing, in: Chemical Mutagens: Principles and Methods for Their Detection (A. Hollaender and F. J. deSerres, eds.), Vol. 7, pp. 37–93, Plenum Press, New York (1981).Google Scholar
  2. 2.
    A. A. Ansari, M. A. Baig, and H. V. Mailing, In vivo germinal mutation detection with “monospecific” antibody against lactate dehydrogenase-X, Proc. Natl. Acad. Sci. USA 77, 7352–7356 (1980).PubMedCrossRefGoogle Scholar
  3. 3.
    A. A. Ansari, M. A. Baig, and H. V. Mailing, Development of in vivo somatic mutation system using antibody against hemoglobin, Mutat. Res. 81, 243–255 (1981).PubMedCrossRefGoogle Scholar
  4. 4.
    N. K. Jerne and A. A. Nordin, Plaque formation in agar by single antibody-producing cells, Science 140, 405 (1963).CrossRefGoogle Scholar
  5. 5.
    E. S. Russel, Announcement of linkage between Hba and wa-2 (Chromosome 11, L. G. VII), Mouse Newsl. 49, 33 (1973).Google Scholar
  6. 6.
    R. A. Popp and W. St. Amand, Studies on the mouse hemoglobin locus, I. Identification of hemoglobin types and linkage of hemoglobin with albinism, J. Hered. 51, 141–144 (1960).Google Scholar
  7. 7.
    R. A. Popp, Hemoglobins of mice: Sequence and possible ambiguity at one position of the alpha-chain, J. Mol. Biol. 27, 9–16 (1967).PubMedCrossRefGoogle Scholar
  8. 8.
    K. Hilse and R. A. Popp, Gene duplication as the basis for amino acid ambiguity in the alpha chain Polypeptides of mouse hemoglobins, Proc. Natl. Acad. Sci. USA 61, 930–936 (1968).PubMedCrossRefGoogle Scholar
  9. 9.
    R. A. Popp. Sequence of amino acids in the β-chain of single hemoglobins from C57BL, SWR and NB mice, Biochim. Biophys. Acta 303, 52–60 (1973).PubMedGoogle Scholar
  10. 10.
    R. A. Popp and E. G. Bailiff, Sequence of amino acids in the major and minor β-chains of the diffuse hemoglobin from Balb/c mice, Biochim. Biophys. Acta 303, 61–67 (1973).PubMedGoogle Scholar
  11. 11.
    A. A. Ansari, L. M. Bahuguna, M. Jenison, and H. V. Mailing, Immunological comparison of mouse hemoglobins, Immunochemistry 15, 557–560 (1978).PubMedCrossRefGoogle Scholar
  12. 12.
    J. G. Gilman, Hemoglobin beta chain structural variation in mice: Evolutionary and functional implications, Science 178, 873–874 (1972).PubMedCrossRefGoogle Scholar
  13. 13.
    J. T. Dodge, C. Mitchell, and D. J. Hanahan, The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes, Arch. Biochem. Biophys. 100, 119–130 (1963).PubMedCrossRefGoogle Scholar
  14. 14.
    M. A. Baig and A. A. Ansari, Studies of mouse immunoglobulin allotypes by reverse plaque assay: Detection of spleen cells secreting immunoglobulins with specific allotype in C57BL/ 6 mice, Cell. Immunol. 66, 164–170 (1982).PubMedCrossRefGoogle Scholar
  15. 15.
    R. D. Schreiber, R. W. Noble, and M. Reichlin, Restriction of heterogeneity of goat antibodies specific for human hemoglobin S, J. Immunol. 114, 170–175 (1975).PubMedGoogle Scholar
  16. 16.
    A. M. Dozy, D. F. Kleihauer, and T. H. J. Huisman, Studies on the heterogeneity of hemoglobin, XIII. Chromatography of various human and animal hemoglobin types on DEAE-Sephadex, J. Chromatogr. 32, 723–727 (1968).PubMedCrossRefGoogle Scholar
  17. 17.
    E. Antonini and M. Brunori, Hemoglobin and Myoglobin in their Reactions with Ligands, North-Holland, Amsterdam (1973).Google Scholar
  18. 18.
    S. Avrameas B. Taudou and S. Chuilon Glutaraldehyde cyanuric chloride and tetraazotized O-dianisidine as coupling reagents in the passive hemagglutination test Immuchemistry 6 61–16 1969Google Scholar
  19. 19.
    P. Cuatrecasas and C. B. Anfinsen, Affinity chromatography, Annu. Rev. Biochem. 40, 259–278 (1971).PubMedCrossRefGoogle Scholar
  20. 20.
    J. H. Ludens, J. R. DeVries, and D. D. Fanestil, Criteria for affinity chromatography of steroid-binding macromolecules, J. Biol. Chem. 247, 7533–7538 (1972).PubMedGoogle Scholar
  21. 21.
    V. Sica, I. Parikh, E. Nola, G. A. Puca, and P. Cuatrecasas, Affinity chromatography and the purification of estrogen receptors, J. Biol. Chem. 248, 6543–6558 (1973).PubMedGoogle Scholar
  22. 22.
    I. Parikh, V. Sica, E. Nola, G. A. Puca, and P. Cuatrecasas, Estrogen receptors, Methods Enzymol, 34, 670–688 (1974).PubMedCrossRefGoogle Scholar
  23. 23.
    I. Parikh and P. Cuatrecasas, in: Immunochemistry of Proteins (M. Z. Attasi, ed.), Vol. 2, pp. 1–44, Plenum Press, New York (1977).CrossRefGoogle Scholar
  24. 24.
    G. Stamatoyannopoulos, W. G. Wood, Th. Papayannopoulou, and P. E. Nute, An analytical form of hereditary persistence of fetal hemoglobin in blacks and its association with sickle cell trait, Blood 46, 683–692 (1975).PubMedGoogle Scholar
  25. 25.
    W. G. Wood, G. Stamatoyannopoulos, G. Lim, and P. E. Nute, F-Cells in the adult: Normal values and levels in individuals with hereditary and acquired elevation of HbF, Blood 46, 671–682 (1975).PubMedGoogle Scholar
  26. 26.
    P. E. Nute, W. G. Wood, G. Stamatoyannopoulos, C. Olweny, and P. J. Failkow, The Kenya form of hereditary persistence of fetal hemoglobin: Structural studies and evidence for homogeneous distribution of hemoglobin F using fluorescent anti-hemoglobin F antibodies, Br. J. Haematol. 32, 55–63 (1976).PubMedCrossRefGoogle Scholar
  27. 27.
    Th. Papayannopoulou, M. Brice, and G. Stamatoyannopoulos, Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals, Proc. Natl. Acad. Sci. USA 73, 2033–2037 (1976).PubMedCrossRefGoogle Scholar
  28. 28.
    Th. Papayannopoulou, G. Lim., T. C. MacGuire, V. Ahern, P. E. Nute, and G. Stamatoyannopoulos, Use of specific fluorescent antibodies for the identification of hemoglobin C in erythrocytes, Am. J. Hematol. 2, 105–112 (1977).PubMedCrossRefGoogle Scholar
  29. 29.
    A. A. Ansari, M. A. Baig, and H. V. Mailing, Purification of fluorescein-labeled specific antihemoglobin antibody using cross-linked immunoabsorbent, J. Immunol. Methods 42, 45–51 (1981).PubMedCrossRefGoogle Scholar
  30. 30.
    G. Köhler and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256, 495–497 (1975).PubMedCrossRefGoogle Scholar
  31. 31.
    J. W. Goding, Antibody production by hybridomas, J. Immunol. Methods 39 285–308 (1980).PubMedCrossRefGoogle Scholar
  32. 32.
    S. F. St. Groth and D. Scheidegger, Production of monoclonal antibodies: Strategy and tactics, J. Immunol. Methods 35, 1–21 (1980).CrossRefGoogle Scholar
  33. 33.
    V. T. Oi, P. P. Jones, J. W. Goding, L. A. Herzenberg, and L. A. Herzenberg, Properties of monoclonal antibodies to mouse Ig allotypes, H-2 and Ia antigen, Curr. Top. Microbiol. Immunol. 81, 115–129 (1978).PubMedGoogle Scholar
  34. 34.
    M. Shulman, C. D. Wilde, and G. Köhler, A better cell line for making hybridomas secreting specific antibodies, Nature 276, 269–270 (1978).PubMedCrossRefGoogle Scholar
  35. 35.
    J. F. Kearney, A. Radbruch, B. Liesegang, and K. Rajewsky, A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibodysecreting hybrid cell lines, J. Immunol. 123, 1548–1550 (1979).PubMedGoogle Scholar
  36. 36.
    P. Coflfmo, R. Baumal, R. Laskov, and M. D. Scharf, Cloning of mouse myeloma cells and detection of rare variants, J. Cell. Physiol. 79, 429–440 (1972).CrossRefGoogle Scholar
  37. 37.
    W. Lernhardt, J. Andersson, A. Coutinho, and F. Melchers, Cloning of murine transformed cell lines in suspension culture with efficiencies near 100%, Exp. Cell Res. 111, 309–316 (1978).PubMedCrossRefGoogle Scholar
  38. 38.
    H. Hengartner, A. L. Luzzati, and M. Schreier, Fusion of in vitro immunized lymphoid cells with X63Ag8, Curr. Top. Microbiol. Immunol. 81, 92–99 (1978).PubMedGoogle Scholar
  39. 39.
    B. K. Van Weemen and A. H. W. M. Schuurs, Immunoassay using antigen-enzyme conjugates, FEBS Lett. 15, 232–236 (1971).PubMedCrossRefGoogle Scholar
  40. 40.
    E. Engvall and P. Perlamnn, Enzyme-linked immunoabsorbent assay (ELISA). Quantitative assay of immunoglobulin G, Immunochemistry 8, 871–874 (1971).PubMedCrossRefGoogle Scholar
  41. 41.
    Y. Naot and J. S. Remington, Use of enzyme-linked immunoabsorbent assays (ELISA) for detection of monoclonal antibodies: Experience with antigens of Toxoplasma gondii, J. Immunol. Methods 43, 333–341 (1981).PubMedCrossRefGoogle Scholar
  42. 42.
    A. A. Ansari and H. V. Mailing, A rapid screening method for the detection of monospecific antibodies, J. Immunol. Methods 24, 383–387 (1978).PubMedCrossRefGoogle Scholar
  43. 43.
    E. R. Gold and H. H. Fundenberg, Chromic chloride: A coupling reagent for passive hemagglutination reactions, J. Immunol. 99, 859–866 (1967).PubMedGoogle Scholar
  44. 44.
    G. N. Vyas, H. H. Fundenberg, H. M. Pretty, and E. R. Gold, A new rapid method for genetic typing of human immunoglobulins, J. Immunol. 100, 274–279 (1968).PubMedGoogle Scholar
  45. 45.
    G. H. Sweet and F. L. Welborn, Use of chromium chloride as the coupling reagent in modified plaque assay, J. Immunol. 106, 1407–1410 (1971).PubMedGoogle Scholar
  46. 46.
    J. W. Goding, The chromic chloride method of coupling antigens to erythrocytes: Definition of some important parameters, J. Immunol. Methods 10, 61–66 (1976).PubMedCrossRefGoogle Scholar
  47. 47.
    G. J. V. Nossal, A. E. Bussard, H. Lewis, and J. C. Maize, In vitro stimulation of antibody formation by peritoneal cells, J. Exp. Med. 131, 894–935 (1970).PubMedCrossRefGoogle Scholar
  48. 48.
    H. M. Johnson, K. Brenner, and H. E. Hall, Use of water-soluble carbodiimide as a coupling reagent in the passive hemagglutination test, J. Immunol. 97, 791–796 (1966).PubMedGoogle Scholar
  49. 49.
    A. M. Kaplan and M. J. Freeman, Enumeration of cells synthesizing antiprotein antibodies by a modified hemolytic plaque assay, Proc. Soc. Exp. Biol. Med. 127, 574–576 (1968).PubMedGoogle Scholar
  50. 50.
    E. S. Golub, R. I. Mishell, W. O. Weigle, and R. W. Dutton, A modification of the hemolytic plaque assay for the use with protein antigens, J. Immunol. 100, 133–137 (1968).PubMedGoogle Scholar
  51. 51.
    D. Pressman, D. H. Campbell, and L. Pauling, The agglutination of intact azoerythrocytes by antisera homologous to the attached groups, J. Immunol. 44, 101–105 (1942).Google Scholar
  52. 52.
    D. W. Dresser and H. H. Wortis, in: Handbook of Experimental Immunology (D. M. Weir, ed.), 1st ed., p. 1054, Blackwell Scientific Publications, Oxford (1967).Google Scholar
  53. 53.
    J. A. Kapp and I. S. Ingraham, Anti-protein plaque-forming cells detected with high efficiency by the use of red cells coupled to bovine serum glubulin through bis-diazo-benzidine, J. Immunol. 104, 1039–1042 (1970).PubMedGoogle Scholar
  54. 54.
    E. A. Kabat and M. M. Mayer, Experimental Immunochemistry, Charles C. Thomas, Springfield (1961).Google Scholar
  55. 55.
    T. Ternynck and S. Avrameas, A new method using p-benzoquinone for coupling antigens and antibodies to marker substances, Annu. Immunol. (Inst. Pasteur) 127C, 197–208 (1976).Google Scholar
  56. 56.
    S. Lemieux, S. Avrameas, and A. E. Bussard, Local hemolysis plaque assay using a new method of coupling antigens on sheep erythrocytes by glutaraldehyde, Immunochemistry 11, 261–269 (1974).PubMedCrossRefGoogle Scholar
  57. 57.
    G. Kronvall, H. M. Grey, and R. C. Williams, Jr., Protein A reacting with mouse immunoglobulins, J. Immunol. 105, 1116–1123.Google Scholar
  58. 58.
    H. M. Grey, J. W. Hirst, and M. Cohn, A new mouse immunoglobulin: IgG3, J. Exp. Med. 133, 289–304(1971).PubMedCrossRefGoogle Scholar
  59. 59.
    E. Gronowicz, A. Coutinho, and F. Melchers, A plaque assay for all cells secreting Ig of a given type or class, Eur. J. Immunol. 6, 588–590 (1976).PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1984

Authors and Affiliations

  • Masroor A. Baig
    • 1
  • Aftab A. Ansari
    • 2
  1. 1.Laboratory of GeneticsNational Institute of Environmental Health SciencesUSA
  2. 2.Northrop Services, Inc.USA

Personalised recommendations