Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1984 Feb;73(2):489–496. doi: 10.1172/JCI111235

Reactions of immunoglobulin G-binding ligands with platelets and platelet-associated immunoglobulin G.

W F Rosse, D V Devine, R Ware
PMCID: PMC425040  PMID: 6699173

Abstract

Immunoglobulin G (IgG) bound to platelets is usually detected by one of two general methods: binding of labeled anti-IgG or consumption of anti-IgG. The latter method gives, in general, values 5-10-fold greater than the former under the same conditions. To investigate these discrepancies, we have compared the detection of platelet-bound IgG by a labeled anti-IgG binding assay and by a quantitative antiglobulin consumption test using the same antibodies. The interaction of 125I-labeled monoclonal anti-IgG or polyclonal anti-IgG with washed and IgG-coated platelets was studied. The binding of these ligands to washed normal platelets was largely (50-80%) nonspecific; the binding was not saturable and was only partially inhibitable by excess unlabeled anti-IgG. The binding of anti-IgG to platelets coated with anti-PIA1, a platelet-specific IgG antibody, appeared to be saturable and inhibitable; the dissociation constant (KD) of this IgG-anti-IgG reaction was 4.9 X 10(-9) for monoclonal and 1.4 X 10(-7) for polyclonal anti-IgG. The ratio of sites present on the membrane (determined by 131I-labeled anti-PIA1) to the number of binding sites for anti-IgG determined by Scatchard analysis was 0.53 for monoclonal anti-IgG and 1.3 for polyclonal anti-IgG. The binding of monoclonal anti-IgG to platelet-bound immune complexes or IgG aggregates appeared to be complex. 131I-Labeled IgG was affixed to platelets and was detected by three tests: direct binding of radiolabeled monoclonal anti-IgG and quantitative antiglobulin consumption (QAC) tests, which were quantitated either by measuring directly the amount of radiolabeled anti-IgG consumed from fluid phase (direct QAC), or indirectly by reference to a calibration curve relating the consumption of anti-IgG by known amounts of fluid-phase, non-immune IgG (indirect QAC). The amount of platelet-bound IgG detected by the direct binding of 125I-labeled monoclonal anti-IgG and by the direct QAC approximated that known to be bound to the platelet. The results of the indirect QAC test were 10-fold greater. The discrepancy appears to be due to the fact that there is a difference between the IgG-anti-IgG interaction when IgG is bound to a platelet and when it is in solution or bound to plastic nonspecifically or specifically. This difference results in a falsely high value for platelet-bound IgG when fluid-phase or plastic-bound IgG is used to calibrate the antiglobulin consumption test.

Full text

PDF
489

Selected References

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

  1. Cines D. B., Schreiber A. D. Immune thrombocytopenia. Use of a Coombs antiglobulin test to detect IgG and C3 on platelets. N Engl J Med. 1979 Jan 18;300(3):106–111. doi: 10.1056/NEJM197901183000302. [DOI] [PubMed] [Google Scholar]
  2. Dixon R., Rosse W., Ebbert L. Quantitative determination of antibody in idiopathic thrombocytopenic purpura. Correlation of serum and platelet-bound antibody with clinical response. N Engl J Med. 1975 Jan 30;292(5):230–236. doi: 10.1056/NEJM197501302920503. [DOI] [PubMed] [Google Scholar]
  3. Ey P. L., Prowse S. J., Jenkin C. R. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry. 1978 Jul;15(7):429–436. doi: 10.1016/0161-5890(78)90070-6. [DOI] [PubMed] [Google Scholar]
  4. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  5. Glikmann G., Svehag S. E. Detection and quantitation of circulating immune complexes by the C1q-protein A binding assay (C1q-PABA). Methods Enzymol. 1981;74(Pt 100):571–588. doi: 10.1016/0076-6879(81)74040-0. [DOI] [PubMed] [Google Scholar]
  6. Hegde U. M., Gordon-Smith E. C., Worlledge S. Platelet antibodies in thrombocytopenic patients. Br J Haematol. 1977 Jan;35(1):113–122. doi: 10.1111/j.1365-2141.1977.tb00567.x. [DOI] [PubMed] [Google Scholar]
  7. Hymes K., Shulman S., Karpatkin S. A solid-phase radioimmunoassay for bound anti-platelet antibody: studies on 45 patients with autoimmune platelet disorders. J Lab Clin Med. 1979 Oct;94(4):639–648. [PubMed] [Google Scholar]
  8. Kelton J. G., Meltzer D., Moore J., Giles A. R., Wilson W. E., Barr R., Hirsh J., Neame P. B., Powers P. J., Walker I. Drug-induced thrombocytopenia is associated with increased binding of IgG to platelets both in vivo and in vitro. Blood. 1981 Sep;58(3):524–529. [PubMed] [Google Scholar]
  9. Kelton J. G., Neame P. B., Gauldie J., Hirsh J. Elevated platelet-associated IgG in the thrombocytopenia of septicemia. N Engl J Med. 1979 Apr 5;300(14):760–764. doi: 10.1056/NEJM197904053001404. [DOI] [PubMed] [Google Scholar]
  10. Kelton J. G., Powers P. J., Carter C. J. A prospective study of the usefulness of the measurement of platelet-associated IgG for the diagnosis of idiopathic thrombocytopenic purpura. Blood. 1982 Oct;60(4):1050–1053. [PubMed] [Google Scholar]
  11. Kernoff L. M., Blake K. C., Shackleton D. Influence of the amount of platelet-bound IgG on platelet survival and site of sequestration in autoimmune thrombocytopenia. Blood. 1980 May;55(5):730–733. [PubMed] [Google Scholar]
  12. Klotz I. M. Numbers of receptor sites from Scatchard graphs: facts and fantasies. Science. 1982 Sep 24;217(4566):1247–1249. doi: 10.1126/science.6287580. [DOI] [PubMed] [Google Scholar]
  13. Kurlander R. J., Batker J. The binding of human immunoglobulin G1 monomer and small, covalently cross-linked polymers of immunoglobulin G1 to human peripheral blood monocytes and polymorphonuclear leukocytes. J Clin Invest. 1982 Jan;69(1):1–8. doi: 10.1172/JCI110419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Leporrier M., Dighiero G., Auzemery M., Binet J. L. Detection and quantification of platelet-bound antibodies with immunoperoxidase. Br J Haematol. 1979 Aug;42(4):605–611. doi: 10.1111/j.1365-2141.1979.tb01173.x. [DOI] [PubMed] [Google Scholar]
  15. Luiken G. A., McMillan R., Lightsey A. L., Gordon P., Zevely S., Schulman I., Gribble T. J., Longmire R. L. Platelet-associated IgG in immune thrombocytopenic purpura. Blood. 1977 Aug;50(2):317–325. [PubMed] [Google Scholar]
  16. NISONOFF A., WISSLER F. C., LIPMAN L. N., WOERNLEY D. L. Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of disulfide bonds. Arch Biochem Biophys. 1960 Aug;89:230–244. doi: 10.1016/0003-9861(60)90049-7. [DOI] [PubMed] [Google Scholar]
  17. Segal D. M., Hurwitz E. Dimers and trimers of immunoglobulin G covalently cross-linked with a bivalent affinity label. Biochemistry. 1976 Nov 30;15(24):5253–5258. doi: 10.1021/bi00669a009. [DOI] [PubMed] [Google Scholar]
  18. Sugiura K., Steiner M., Baldini M. G. Platelet antibody in idiopathic thrombocytopenic purpura and other thrombocytopenias. A quantitative, sensitive, and rapid assay. J Lab Clin Med. 1980 Oct;96(4):640–653. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES