Skip to main content
PMC home page
Immunology logoLink to Immunology
. 1975 Oct;29(4):765–777.

Purification and quantification of T and B lymphocytes by an affinity method.

C R Barker, C P Worman, J L Smith
PMCID: PMC1446055  PMID: 810418

Abstract

Affinity surfaces were produced by coupling human immunoglobulin (HGG) to the surface of tissue culture grade plastic-ware with a water-soluble carbodiimide followed by treatment with anti-HGG antisera. Surface immunoglobulin SIg) bearing human B lymphocytes attach to these surfaces when centrifuged on to them and unattached cells could be recovered by inverting the trays or dishes. Optimal cell attachment conditions could be rapidly evaluated by counting cells attached to representative areas of multi-well trays and percentage of SIg-bearing cells quantified. Evidence was obtained for cell attachment through Fc receptors as well as SIg using unrelated antigen--antibody-coated trays. This could be prevented by using the F (ab')2 fragments of the antisera. Under these conditions specific attachment through K and lambda light chains could be achieved with normal and chronic lymphocytic leukaemic lymphocytes. Using tissue culture plastic Petri dishes and relatively small quantities of antiserum, larger numbers of lymphocytes could be processed to produce T lymphocytes containing less than 1 per cent of contaminating SIg-positive cells.

Full text

PDF
774

Selected References

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

  1. Basten A., Sprent J., Miller J. F. Receptor for antibody-antigen complexes used to separate T cells from B cells. Nat New Biol. 1972 Feb 9;235(58):178–180. doi: 10.1038/newbio235178a0. [DOI] [PubMed] [Google Scholar]
  2. Greaves M. F., Brown G. Purification of human T and B lymphocytes. J Immunol. 1974 Jan;112(1):420–423. [PubMed] [Google Scholar]
  3. Grey H. M., Rabellino E., Pirofsky B. Immunoglobulins on the surface of lymphocytes. IV. Distribution in hypogammaglobulinemia, cellular immune deficiency, and chronic lymphatic leukemia. J Clin Invest. 1971 Nov;50(11):2368–2375. doi: 10.1172/JCI106735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hunt S. V. Separation of thymus-derived and marrow-derived rat lymphocytes on glass bead columns. Immunology. 1973 Apr;24(4):699–705. [PMC free article] [PubMed] [Google Scholar]
  5. NISONOFF A., MARKUS G., WISSLER F. C. Separation of univalent fragments of rabbit antibody by reduction of a single, labile disulphide bond. Nature. 1961 Jan 28;189:293–295. doi: 10.1038/189293a0. [DOI] [PubMed] [Google Scholar]
  6. Paraskevas F., Lee S. T., Orr K. B., Israels L. G. A receptor for Fc on mouse B-lymphocytes. J Immunol. 1972 May;108(5):1319–1327. [PubMed] [Google Scholar]
  7. Parish C. R., Hayward J. A. The lymphocyte surface. II. Separation of Fc receptor, C'3 receptor and surface immunoglobulin-bearing lymphocytes. Proc R Soc Lond B Biol Sci. 1974 Aug 27;187(1086):65–81. doi: 10.1098/rspb.1974.0061. [DOI] [PubMed] [Google Scholar]
  8. Porath J., Axen R., Ernback S. Chemical coupling of proteins to agarose. Nature. 1967 Sep 30;215(5109):1491–1492. doi: 10.1038/2151491a0. [DOI] [PubMed] [Google Scholar]
  9. Schlossman S. F., Hudson L. Specific purification of lymphocyte populations on a digestible immunoabsorbent. J Immunol. 1973 Jan;110(1):313–319. [PubMed] [Google Scholar]
  10. Seiler F. R., Johannsen R., Sedlacek H. H., Zeiller K. Characterization of lymphocyte subpopulations of nonhuman primates separated by free-flow electrophoresis. Transplant Proc. 1974 Jun;6(2):173–181. [PubMed] [Google Scholar]
  11. Shortman K. The separation of different cell classes from lymphoid organs. 1. The use of glass bead columns to separate small lymphocytes, remove damaged cells and fractionate cell suspensions. Aust J Exp Biol Med Sci. 1966 Jun;44(3):271–286. [PubMed] [Google Scholar]
  12. Smith J. L., Clein G. P., Barker C. R., Collins R. D. Characterisation of malignant mediastinal lymphoid neoplasm (Sternberg sarcoma) as thymic in origin. Lancet. 1973 Jan 13;1(7794):74–77. doi: 10.1016/s0140-6736(73)90469-8. [DOI] [PubMed] [Google Scholar]
  13. Unanue E. R., Grey H. M., Rabellino E., Campbell P., Schmidtke J. Immunoglobulins on the surface of lymphocytes. II. The bone marrow as the main source of lymphocytes with detectable surface-bound immunoglobulin. J Exp Med. 1971 Jun 1;133(6):1188–1198. doi: 10.1084/jem.133.6.1188. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES