Skip to main content
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1977 Feb 1;145(2):372–389. doi: 10.1084/jem.145.2.372

Hapten-specific hemolytic plaque assays usually fail to detect most of the diversity in the anti-hapten response

PMCID: PMC2180598  PMID: 64583

Abstract

Immunization of rabbits or mice with a single, chemically defined hapten elicits populations of plaque-forming cells (PFC) detectable not only on sheep erythrocytes (SRBC) bearing the immunizing hapten, but also on SRBC bearing structural analogues of the immunizing hapten. Most of these analogue-reactive PFC preferentially lyse analogue- conjugated SRBC and cannot be detected on erythrocytes bearing the immunizing hapten. Thus, they represent heretofore largely unstudied components of the secretory B-cell response to haptenic immunization, and they have been termed alloreactive PFC. Such alloreactive PFC are detectable using either classical small haptens or tripeptide-enlarged counterparts of these classical haptens. They are present in large numbers both in direct and in indirect PFC assays, and they are elicited in response to both thymic-dependent and thymic-independent antigens. Relatively few alloreactive PFC can be attributed to cells producing hapten-carrier or "bridge area"-specific antibodies. Since the antibodies released by alloreactive PFC can also be detected by passive hemagglutination, their presence does not appear attributable to vagaries of complement activation. Numerous coexisting alloreactive PFC populations are detectable after haptenic immunization. In early direct PFC responses it is not nucommon for a single alloreactive PFC population to outnumber the population of PFC detectable on SRBC bearing the actual immunizing hapten. These alloreactive PFC may be the source of at least some of the new "nonspecific" Ig which is formed at the time of immunization but about which little is known for lack of available techniques. Some possible implications of these findings on the specificity of B precursor cell activation are discussed.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ASKONAS B. A., HUMPHREY J. H. Formation of specific antibodies and gamma-globulin in vitro; a study of the synthetic ability of various tissues from rabbits immunized by different methods. Biochem J. 1958 Feb;68(2):252–261. doi: 10.1042/bj0680252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. ATTARDI G., COHN M., HORIBATA K., LENNOX E. S. ANTIBODY FORMATION BY RABBIT LYMPH NODE CELLS. I. SINGLE CELL RESPONSES TO SEVERAL ANTIGENS. J Immunol. 1964 Mar;92:335–345. [PubMed] [Google Scholar]
  3. Bretscher P., Cohn M. A theory of self-nonself discrimination. Science. 1970 Sep 11;169(3950):1042–1049. doi: 10.1126/science.169.3950.1042. [DOI] [PubMed] [Google Scholar]
  4. Claflin J. L., Lieberman R., Davie J. M. Clonal nature of the immune response to phosphorylcholine. I. Specificity, class, and idiotype of phosphorylcholine-binding receptors on lymphoid cells. J Exp Med. 1974 Jan 1;139(1):58–73. doi: 10.1084/jem.139.1.58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Claflin L., Merchant B. Restricted maturation of antibody-binding characteristics for hapten-specific IgM-plaque-forming cells in mice. Cell Immunol. 1972 Sep;5(1):209–220. doi: 10.1016/0008-8749(72)90097-4. [DOI] [PubMed] [Google Scholar]
  6. Cosenza H., Köhler H. Specific inhibition of plaque formation to phosphorylcholine by antibody against antibody. Science. 1972 Jun 2;176(4038):1027–1029. doi: 10.1126/science.176.4038.1027. [DOI] [PubMed] [Google Scholar]
  7. Coutinho A., Möller G. B cell mitogenic properties of thymus-independent antigens. Nat New Biol. 1973 Sep 5;245(140):12–14. doi: 10.1038/newbio245012a0. [DOI] [PubMed] [Google Scholar]
  8. Davie J. M., Paul W. E. Receptors on immunocompetent cells. II. Specificity and nature of receptors on dinitrophenylated guinea pig albumin- 125 I-binding lymphocytes of normal guinea pigs. J Exp Med. 1971 Aug 1;134(2):495–516. doi: 10.1084/jem.134.2.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davie J. M., Paul W. E. Receptors on immunocompetent cells. IV. Direct measurement of avidity of cell receptors and cooperative binding of multivalent ligands. J Exp Med. 1972 Mar 1;135(3):643–659. doi: 10.1084/jem.135.3.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Diener E., Feldmann M. Relationship between antigen and antibody-induced suppression of immunity. Transplant Rev. 1972;8:76–103. doi: 10.1111/j.1600-065x.1972.tb01565.x. [DOI] [PubMed] [Google Scholar]
  11. Green I., Vassalli P., Nussenzweig V., Benacerraf B. Specificity of the antibodies produced by single cells following immunization with antigens bearing two types of antigenic determinants. J Exp Med. 1967 Mar 1;125(3):511–526. doi: 10.1084/jem.125.3.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gronowicz E., Coutinho A., Möller G. Differentiation of B cells: sequential appearance of responsiveness to polyclonal activators. Scand J Immunol. 1974;3(4):413–421. doi: 10.1111/j.1365-3083.1974.tb01274.x. [DOI] [PubMed] [Google Scholar]
  13. Hanna E. E., Merchant B. Secretion of monospecific anti-hapten IgG antibodies by rabbit immunocytes. J Immunol. 1971 Jul;107(1):172–184. [PubMed] [Google Scholar]
  14. Inman J. K., Merchant B., Claflin L., Tacey S. E. Coupling of large haptens to proteins and cell surfaces: preparation of stable, optimally sensitized erythrocytes for hapten-specific, hemolytic plaque assays. Immunochemistry. 1973 Mar;10(3):165–174. doi: 10.1016/0019-2791(73)90005-0. [DOI] [PubMed] [Google Scholar]
  15. Inman J. K., Merchant B., Tacey S. E. Synthesis of large haptenic compounds having a common functional group that permits covalent linkage to proteins, cell surfaces, and adsorbents. Immunochemistry. 1973 Mar;10(3):153–163. doi: 10.1016/0019-2791(73)90004-9. [DOI] [PubMed] [Google Scholar]
  16. Inman J. K. Thymus-independent antigens: the preparation of covalent, hapten-ficoll conjugates. J Immunol. 1975 Feb;114(2 Pt 1):704–709. [PubMed] [Google Scholar]
  17. Little J. R., Eisen H. N. Specificity of the immune response to the 2,4-dinitrophenyl and 2,4,6-trinitrophenyl groups. Ligand binding and fluorescence properties of cross-reacting antibodies. J Exp Med. 1969 Feb 1;129(2):247–265. doi: 10.1084/jem.129.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Merchant B., Petersen B. Comparative sensitivities of the hemolytic plaque-in-agar and microchamber assays for detection of rabbit immune cells. J Immunol. 1968 Nov;101(5):860–867. [PubMed] [Google Scholar]
  19. Montgomery P. C., Rockey J. H., Williamson A. R. Homogeneous antibody elicited with dinitrophenyl-gramicidin-S (rabbit-isoelectric focusing). Proc Natl Acad Sci U S A. 1972 Jan;69(1):228–232. doi: 10.1073/pnas.69.1.228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Möller G. One non-specific signal triggers b lymphocytes. Transplant Rev. 1975;23:126–137. [PubMed] [Google Scholar]
  21. NOSSAL G. J., MAKELA O. Kinetic studies on the incidence of cells appearing to form two antibodies. J Immunol. 1962 May;88:604–612. [PubMed] [Google Scholar]
  22. Rouquès R., Inman J. K., Merchant B. Detection of cells secreting antibodies more reactive with an alternate structure than with the immunizing hapten. Int Arch Allergy Appl Immunol. 1972;42(6):852–870. doi: 10.1159/000230663. [DOI] [PubMed] [Google Scholar]
  23. SILVERSTEIN A. M., THORBECKE G. J., KRANER K. L., LUKES R. J. FETAL RESPONSE TO ANTIGENIC STIMULUS. III. GAMMA-GLOBULIN PRODUCTION IN NORMAL AND STIMULATED FETAL LAMBS. J Immunol. 1963 Sep;91:384–395. [PubMed] [Google Scholar]
  24. Walters C. S., Wigzell H. Heteroclitic cells in anti-hapten systems: some studies at the cellular and serum level. Cell Immunol. 1972 Dec;5(4):570–578. doi: 10.1016/0008-8749(72)90107-4. [DOI] [PubMed] [Google Scholar]
  25. Wigzell H. Specific fractionation of immunocompetent cells. Transplant Rev. 1970;5:76–104. doi: 10.1111/j.1600-065x.1970.tb00357.x. [DOI] [PubMed] [Google Scholar]
  26. Wofsy L., Kimura J., Truffa-Bachi P. Cell separation on affinity columns: the preparation of pure populations of anti-hapten specific lymphocytes. J Immunol. 1971 Sep;107(3):725–729. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES