Monoclonal Antibodies Directed Against Human Immunoglobulins: Preparation and Evaluation Procedures

  • J. J. Haaijman
  • J. Coolen
  • C. Deen
  • C. J. M. Kröse
  • J. J. Zijlstra
  • J. Radl


Monoclonal antibodies (Mabs) are prepared by immunizing an animal (generally a mouse or rat) and fusing the spleen cells, after a given period of time, with a plasmacytoma cell line (Köhler and Milstein, 1975). The fusion mixture is plated out into such numbers of microcultures that the likelihood of obtaining cultures with single fusion events is reasonable (De Blas et al., 1981). Supernatants from individual cultures are tested with a suitable immunoassay for the presence of antibodies with the desired specificity.


Ascitic Fluid Human Immunoglobulin Human Bone Marrow Cell Discontinuous Epitope Epitope Presentation 
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. Arnon, R. (1973). Immunochemistry of enzymes. In Sela, M. (ed.), The antigens, Academic Press, New York, 87–159Google Scholar
  2. Atassi, M. Z. (1975). Antigenic structure of myoglobin. The complete immuno-chemical anatomy of a protein and conclusions relating to antigenic structures of proteins. Immunochemistry, 12, 423–438PubMedCrossRefGoogle Scholar
  3. Bergquist, N. R. and Nilsson, P. (1974). The conjugation of immunoglobulins with tetramethyl rhodamine isothiocyanate by utilisation of dimethylsulphoxide (DMSO) as a solvent. J. Immunol Methods, 5, 189–198PubMedCrossRefGoogle Scholar
  4. Bloemmen, F. J., Radl, J., Haaijman, J. J., Berg, P. van der, Schuit, H. R. E. and Hijmans, W. (1976). Microfluorometric evaluation of the specificity of fluorescent antisera against mouse immunoglobulins with the defined antigen substrate spheres (DASS) system. J. Immunol Methods, 10, 337–355PubMedCrossRefGoogle Scholar
  5. Boorsma, D. M. (1984). Direct immunoenzyme double staining applicable for monoclonal antibodies. Histochemistry, 80, 103–106PubMedCrossRefGoogle Scholar
  6. Bosman, F. T., Lindeman, J., Kuiper, G., Wal, A. van der and Kreunig, J. (1977). The influence of fixation on immunoperoxidase staining of plasma cells in paraffin sections of intestinal biopsy specimens. Histochemistry, 53, 57–62PubMedCrossRefGoogle Scholar
  7. Bruin, G., Musters, W. and Biewenga, J. (1983). Production and characterization of antibodies specific for domains of human IgM. J. Immunol Methods, 60, 319–328PubMedCrossRefGoogle Scholar
  8. De Blas, A. L., Ratnaparkhi, M, V. and Mosimann, J. E. (1981). Estimation of the number of monoclonal hybridomas in a cell fusion experiment. Effect of post-fusion cell dilution on hybridoma survival. J. Immunol Methods, 45, 109–115PubMedCrossRefGoogle Scholar
  9. Ehrlich, P. H., Moyle, W, R., Moustafa, Z. A. and Canfield, R. E. (1982). Mixing two monoclonal antibodies yields enhanced affinity for antigen. J. Immunol., 128, 2709–2713PubMedGoogle Scholar
  10. Ehrlich, P. H. and Moyle, W. R. (1983). Cooperative immunoassays: ultrasensitive assays with mixed monoclonal antibodies. Science, 221, 279–281PubMedCrossRefGoogle Scholar
  11. Ewijk, W. van, Coffman, R. C. and Weissman, I. L. (1980). Immunoelectron microscopy of cell surface antigens: a quantitative analysis of antibody binding after different fixation protocols. Histochemical J., 12, 349–361CrossRefGoogle Scholar
  12. Ey, P. L., Prowse, S. J. and Jenkin, C. R. (1978). Isolation of pure IgGl, IgG2a and IgG2b immunoglobulins from mouse serum using protein-A Sepharose. Immunochemistry, 15, 429–436PubMedCrossRefGoogle Scholar
  13. Fazekas de St. Groth, S. and Scheidegger, D. (1980). Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods, 35, 1–21CrossRefGoogle Scholar
  14. Friguet, B., Djavadi-Ohaniance, L. and Goldberg, M. E. (1984). Some monoclonal antibodies raised with a native protein bind preferentially to the denatured antigen. Molecular Immunol., 21, 673–677CrossRefGoogle Scholar
  15. Geysen, H. M., Barreling, S. J. and Meloen, R. H. (1985). Small peptides induce antibodies with sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein. Proc. Natl. Acad. Sci. USA, 82, 178–182PubMedCentralPubMedCrossRefGoogle Scholar
  16. Giessen, M. van der, Lange, B. de and Lee, B. van der (1974). The production of precipitating antiglobulin reagents specific for the subclasses of human IgG. Immunology, 27, 655–663PubMedGoogle Scholar
  17. Graham, R. C. and Karnovsky, N. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14, 291–302PubMedCrossRefGoogle Scholar
  18. Graham, R. C., Lundholm, U. and Karnovsky, N. J. (1965). Cytochemical demonstration of peroxidase activity with 3-amino-9-ethylcarbazole. J. Histochem. Cytochem., 13, 150–155PubMedCrossRefGoogle Scholar
  19. Haaijman, J. J. (1977). Quantitative immunofluorescence microscopy; methods and applications. Thesis, Leiden, The NetherlandsGoogle Scholar
  20. Haaijman, J. J. (1982). Production of monoclonal antibodies for the analysis of the ontogeny of the murine lymphoid system by flow cytometry. In Wick, G., Traill, K. N. and Schauenstein, K. (eds.), Immunofluorescence technology, Elsevier Biomedical Press, Amsterdam, 129–151Google Scholar
  21. Haaijman, J. J. and Slingerland-Teunissen, J. (1978). Equipment and preparative procedures in immunofluorescence microscopy; quantitative studies. In Knapp, W., Holubar, K. and Wick, G. (eds.), Immunofluorescence and related staining techniques, Elsevier Biomedical Press, Amsterdam, 11–29Google Scholar
  22. Haaijman,. J. J., Deen, C., Kröse, C. J. M., Zijlstra, J. J., Coolen, J. and Radi, J. (1984a). Monoclonal antibodies in immunocytology; a jungle full of pitfalls. Immunology Today, 5, 56–58PubMedCrossRefGoogle Scholar
  23. Haaijman, J. J., Bast, E. J. E. G. and Radl, J. (1984b). The evaluation of monoclonal antibodies for application in immunohistology. In De Weck, A. L. (ed.), Lymphoid cell functions in aging, Eurage Vol. III, 89–94Google Scholar
  24. Halpern, M. S. and Koshland, M. E. (1970). Novel subunit in secretory IgA. Nature, 228, 1276–1278PubMedCrossRefGoogle Scholar
  25. Hijmans, W., Haaijman, J. J. and Schuit, H. R. E. (1981). Immunofluorescence. In Adler, W. H. and Nordin, A. A. (eds.), Immunological techniques applied to aging research, CRC Press, Boca Raton, Fla, 141–163Google Scholar
  26. Hijmans, W., Schuit, H. R. E. and Hulsing-Hesselink, E. (1971). An immunofluorescence study on intracellular immunoglobulins in human bone marrow cells. Ann. NY Acad. Sci., 177, 290–305Google Scholar
  27. Hijmans, W., Schuit, H. R. E. and Klein, F. (1969). An immunofluorescence procedure for the detection of intracellular immunoglobulins. Clin. Exp. Immunol., 4, 457–472PubMedCentralPubMedGoogle Scholar
  28. Hsu, S. M., Raine, L. and Fanger, H. (1981). Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. A comparison between ABC and unlabelled anitbody (PAP) procedures. J. Histochem. Cytochem., 29, 577–580PubMedCrossRefGoogle Scholar
  29. Hsu, S. M. and Soban, E. (1982). Colour modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immuno-histochemistry. J. Histochem. Cytochem., 30, 1079–1082PubMedCrossRefGoogle Scholar
  30. Jefferis, R., Lowe, J., Ling, N. R., Porter, P. and Senior, S. (1982). Immunogenic and antigenic epitopes of immunoglobulins. I. Crossreactivity of murine monoclonal antibodies to human IgG with the immunoglobulins of certain animal species. Immunology, 45, 71–77PubMedCentralPubMedGoogle Scholar
  31. Jefferis, R., Reimer, C. B., Skvaril, F., et al. (1985). Evaluation of monoclonal antibodies having specificity for human IgG subclasses: results of an IUIS/ WHO collaborative study. Immunol Letters, 10, 223–252CrossRefGoogle Scholar
  32. Jol-van der Zijde, C. M., Vossen, J. M., Weijden-Ragas, R. van der and Radl, J. (1983). Low molecular weight IgM in sera of children following bone marrow transplantation for severe aplastic anaemia and acute leukaemia. Clin. Exp. Immunol., 53, 151–158Google Scholar
  33. Kammer, K. (1983). Monoclonal antibodies to influenza A virus FM1 (H1N1) proteins require individual conditions for optimal reactivity in binding assays. Immunology, 48, 799–808PubMedCentralPubMedGoogle Scholar
  34. Koertge, T. E. and Butler, J. E. (1985). The relationship between the binding of primary antibody to solid-phase antigen in microtitration plates and its detection by ELISA. J. Immunol. Methods, 83, 283–299PubMedCrossRefGoogle Scholar
  35. Köhler, G. and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, 495–497PubMedCrossRefGoogle Scholar
  36. Lerner, R. A. (1982). Tapping the immunological repertoire to produce antibodies of predetermined specificity. Nature, 299, 592–596CrossRefGoogle Scholar
  37. Lerner, R. A. (1984). Antibodies of predetermined specificity in biology and medicine. Adv. Immunol., 36, 1–44PubMedCrossRefGoogle Scholar
  38. Lew, A. M. (1984). The effect of epitope density and antibody affinity on the ELISA as analysed by monoclonal antibodies. J. Immunol Methods, 72, 171–176PubMedCrossRefGoogle Scholar
  39. Lobo, P. I. (1983). Double immunofluorescence staining of pokeweed mitogen differentiated plasma cells—a sensitive assay to ascertain purity of anti-human Ig reagents as each cell produces only one isotype. J. Immunol. Methods, 65, 383–387PubMedCrossRefGoogle Scholar
  40. Loghem, E. van and Biewenga, J. (1983). Allotypic and isotypic aspects of human immunoglobulin A. Molecular Immunol., 20, 1001–1007CrossRefGoogle Scholar
  41. Markwell, M. A. K. (1982). A new solid-state reagent to iodinate proteins. Anal Biochem., 125, 427–432PubMedCrossRefGoogle Scholar
  42. McCune, J. M., Lingappa, V. R., Fu, S. M., Blobel, G. and Kunkel, H. G. (1980). Biogenesis of membrane-bound and secreted immunoglobulins. I. Two distinct translation products of human u-chain with identical N-termini and different C-termini. J. Exp. Med., 152, 463–468PubMedCrossRefGoogle Scholar
  43. Mestecky, J., Zikan, J. and Butler, W. J. (1971). Immunoglobulin M and secretory immunoglobulin A: presence of a common polypeptide different from light chains. Science, 171, 1163–1165Google Scholar
  44. Mestecky, J., Zikan, J., Butler, W. T. and Kulhavy, R. (1972). Studies on human secretory immunoglobulin A. III. J chain. Immunochemistry, 9, 883–900PubMedCrossRefGoogle Scholar
  45. Mierendorf, R. C. and Dimond, R. L. (1983). Functional heterogeneity of monoclonal antibodies obtained using different screening assays. Anal Biochem., 135, 221–229PubMedCrossRefGoogle Scholar
  46. Milstein, C., Wright, B. and Cuello, A. C. (1983). The discrepancy between the cross-reactivity of a monoclonal antibody to serotonin and its immunohistochemical specificity. Molecular Immunol., 20, 113–123CrossRefGoogle Scholar
  47. Moudallal, Z. A., Atlschuh, D., Briand, J. P. and Regenmortel, M. H. V. van (1984). Comparative sensitivity of different ELISA procedures for detecting monoclonal antibodies. J. Immunol. Methods, 68, 35–43CrossRefGoogle Scholar
  48. Naiem, M., Gerdes, J., Abdulaziz, Z., Sunderland, C. A., Allington, M. J., Stein, H. and Mason, D. Y. (1982). The value of immunohistological screening in the production of monoclonal antibodies. J. Immunol Methods, 50, 145–160PubMedCrossRefGoogle Scholar
  49. Nimmo, G. R., Lew, A. M., Stanley, C. M. and Steward, M. W. (1984). Influence of antibody affinity on the performance of different antibody assays. J. Immunol. Methods, 72, 177–187PubMedCrossRefGoogle Scholar
  50. Oi, V. T., Bryan, V. M., Herzenberg, L. A. and Herzenberg, L. A. (1980). Lymphocyte membrane IgG and secreted IgG are structurally and allotypically distinct. J. Exp. Med., 151, 1260–1274PubMedCrossRefGoogle Scholar
  51. Oi, V. T. and Herzenberg, L. A. (1981). Immunoglobulin producing hybrid cell lines. In Mishell, B. B. and Shiigi, S. M. (eds.), Selected methods in cellular immunology, W. H. Freeman and Co., San Francisco, 371–372Google Scholar
  52. Partridge, L. J., Lowe, J., Hardie, D. L., Ling, N. R. and Jefferis, R. (1982). Immunogenic and antigenic epitopes of immunoglobulins. II. Antigenic differences between secreted and membrane IgG demonstrated using monoclonal antibodies. J. Immunol., 128, 1–6PubMedGoogle Scholar
  53. Péterfy, F., Kuusela, P. and Mäkelä, O. (1983). Affinity requirements for antibody assays mapped by monoclonal antibodies. J. Immunol., 130, 1809–1813PubMedGoogle Scholar
  54. Reimer, C. B., Phillips, D. J., Aloisio, C. H., Moore, B. D., Galland, G. G., Wells, T. W., Black, C. M. and McDougal, J. S. (1984). Evaluation of thirty-one mouse monoclonal antibodies to human IgG epitopes. Hybridoma, 3, 263–275PubMedCrossRefGoogle Scholar
  55. Schönherr, O. T. and Roelofs, H. (1982). Monoclonal antibodies for diagnostic tests and affinity chromatography: a first step to antibody engineering. Develop. Biol. Standard, 50, 235–242Google Scholar
  56. Schuit, H. R. E., Hijmans, W. and Asma, G. E. M. (1980). Identification of mononuclear cells in human blood. I. Qualitative and quantitative data on surface markers after formaldehyde fixation of the cells. Clin. Exp. Immunol., 41, 559–566PubMedCentralPubMedGoogle Scholar
  57. Schuit, H. R. E., Hijmans, W. and Jansen, J. (1984). Surface bound or cytoplasmic immunoglobulins: interpretation of the immunofluorescence observed in cytocentrifuge slides of human lymphocytes. Clin. Exp. Immunol., 56, 694–700PubMedCentralPubMedGoogle Scholar
  58. Shulman, M., Wilde, C. D. and Köhler, G. (1978). A better cell line for making hybridomas secreting specific antibodies. Nature, 276, 269–270PubMedCrossRefGoogle Scholar
  59. Singer, P. A. and Williamson, A. R. (1980). Cell surface immunoglobulins u and chains of human lymphoid cells are of higher apparent molecular weight than their secreted counterparts. Eur. J. Immunol., 10, 180–186PubMedCrossRefGoogle Scholar
  60. Sinigaglia, F., Scheidegger, D., Talmadge, K. and Garotta, G. (1985). A sensitive and quantitative micro assay for the detection of mycoplasma contamination: inhibition of IL-2 dependent cell line proliferation. J. Immunol Methods, 76, 85–92PubMedCrossRefGoogle Scholar
  61. Skvaril, F. and Schilt, U. (1984). Characterization of the subclasses and light chain types of IgG antibodies to rubella. Clin. Exp. Immunol., 55, 671–676PubMedCentralPubMedGoogle Scholar
  62. Steward, M. W. and Lew, A. M. (1985). The importance of antibody affinity in the performance of immunoassays for antibody. J. Immunol Methods 78, 173–190PubMedCrossRefGoogle Scholar
  63. Swaab, D. F., Pool, C. W. and Leeuwen, F. W. van (1977). Can specificity ever be proved in immunocytochemical staining? J. Histochem Cytochem., 25, 388–396Google Scholar
  64. Tainer, J. A., Getzoff, E. D., Alexander, H., Houghten, R. A., Olson, A. J. and Lerner, R. A. (1984). The reactivity of anti-peptide antibodies is a function of the atomic mobility of sites in a protein. Nature, 312, 127–134PubMedCrossRefGoogle Scholar
  65. Tsu, T. T. and Herzenberg, L. A. (1980). Solid-phase radioimmune assays. In Mishell, B. B. and Shiigi, S. M. (eds.), Selected methods in cellular immunology, W. H. Freeman & Co., San Francisco, 373–397Google Scholar
  66. Vassalli, P., Tedghi, R., Lisowska-Bernstein, B., Tartakoff, A. and Jaton, J. C. (1979). Evidence for hydrophobic region within heavy chains of mouse B lymphocyte membrane-bound IgM. Proc. Natl. Acad. Sci. USA, 76, 5515–5519PubMedCentralPubMedCrossRefGoogle Scholar
  67. Walker, W. S., Beelen, R. H. J., Buckley, P. J., Melvin, S. L. and Shing-Erh, Y. (1984). Some fixation reagents reduce or abolish the detectability of laantigen and HLA-DR on cells. J. Immunol. Methods, 67, 89–99PubMedCrossRefGoogle Scholar
  68. Westhof, E., Altschuh, D., Moras, D., Bloomer, A. C., Mondragon, A., Klug, A. and Regenwortel, M. H. V. van (1984). Correlation between segmental mobility and the location of antigenic determinants in proteins. Nature, 311, 123–126PubMedCrossRefGoogle Scholar
  69. Wilchek, M. and Bayer, E. A. (1984). The avidin-biotin complex in immunology. Immunol. Today, 5, 39–43PubMedCrossRefGoogle Scholar
  70. Wofsy, L., Baker, P. C., Thompson, K., Goodman, J., Kimura, J. and Henry, C. (1974). Hapten-sandwich labelling. I. A general procedure for simultaneous labelling of multiple cell surface antigens for fluorescence and electromicroscopy. J. Exp. Med., 140, 523–537PubMedCentralCrossRefGoogle Scholar

Copyright information

© S. B. Pal and the Contributors 1988

Authors and Affiliations

  • J. J. Haaijman
  • J. Coolen
  • C. Deen
  • C. J. M. Kröse
  • J. J. Zijlstra
  • J. Radl

There are no affiliations available

Personalised recommendations