Thromb Haemost 1990; 64(03): 469-472
DOI: 10.1055/s-0038-1647338
Original Article
Schattauer GmbH Stuttgart

Identification of Platelet Antigens by Immunoprecipitation of Unlabelled Platelet Glycoproteins

Werner Stenziger
The Department of Internal Medicine, University of Münster, Federal Republic of Germany
,
Beate Kehrel
The Department of Internal Medicine, University of Münster, Federal Republic of Germany
,
Jürgen van de Loo
The Department of Internal Medicine, University of Münster, Federal Republic of Germany
› Author Affiliations
Further Information

Publication History

Received: 15 November 1989

Accepted after revision02 June 1990

Publication Date:
25 July 2018 (online)

Summary

A time-saving and sensitive method for the identification of antigens on unlabelled platelet glycoproteins (GP) based on immunoprecipitation has been developed. Platelet solubilisates were incubated with antibodies to form GP-antibody complexes that were precipitated with Protein G Sepharose 4 Fast Flow (PGS). Pcs-bound material was eluted, separated by SDSPAGE under reducing conditions and visualized by silver staining. The method was designed as an alternative to radioimmunoprecipitation for laboratories not equipped to work with radioactive substances. It is proposed as a complementary procedure for the characterization of platelet GP antigens by murine monoclonal antibodies and human alloantibodies.

 
  • References

  • 1 Von dem Borne AE G K. Immunological structures of the platelet membrane. In: Platelet Immunology. Curr Stud Haematol Blood Tiansf, No.55 Kaplan-Gonet SC. ed Karger; Basel: 1988: 3l-44
  • 2 Woods VL, Oh EH, Mason D, McMillan R. Autoantibodies against the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP. Blood 1984; 63: 368-75
  • 3 Woods VL, Kurata Y, Montgomery RR, Thni P, Mason D, Oh EH, McMillan R. Autoantibodies against platelet glycoprotein Ib in patients with chronic immune thrombocytopenic purpura. Blood 1984; 64: 156-160
  • 4 McGregor JL, Brochier J, Wild E, Follea G, Trzeciak MC, James E, Dechavanne M, McGregor L, Clemetson KJ. Monoclonal antibodies against platelet membrane glycoproteins - Characterization and effect on platelet function. Eur J Biochem 1983; l3l: 427-436
  • 5 Kunicki TJ, Aster RH. Isolation and immunologic characterization of the human platelet alloantigen PL(Al). Mol Immunol 1979; 16: 353-360
  • 6 Kehrel B, Kokott R, Balleiseo L, Stenzinger W, Clemetson KJ, van de Loo J. Selective staining of platelet glycoproteins using twodimensional O’Farrell gel electrophoresis and biotin-avidin-conjugated lectins. Thromb Haemostas 1987; 58: 960-963
  • 7 Kiefel V, Santoso S, Weisheit M, Mueller-Eckhardt C. Monoclonal antibody - specific immobilization of platelet antigens (MAIPA): A new tool for the identification of platelet-reactive antibodies. Blood 1987; 70: 1722-1726
  • 8 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folinphenol reagent. J Biol Chem 1951; 193: 265-75
  • 9 Hotchkiss AJ, Leissinger CA, Smith ME, Jordan JY, Kautz CA, Shulman R. Evaluation by quantitative acid elution and radioimmunoassay of multiple classes of immunoglobulins and serum albumin associated with platelets in idiopathic thrombocytopenic purpura. Blood 1986; 67: 1126-31
  • 10 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5
  • 11 Morrissey JH. Silver staining for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal Biochem 1981; 117: 307-10
  • 12 McGregor JL, Clemetson KJ. Biochemistry of the platelet membrane. In: Platelet Immunology. Curr Stud Haematol Blood Tiansf, No.55 Kaplan-Gonet SC. ed Karger; Basel: 1988: 5-31
  • 13 Akerstrom B, Brodin T, Reis K, Bjorck L. Protein G: A powerful tool for binding and detection of monoclonal and polyclonal antibodies. J Immunol 1985; 135: 2589-2592
  • 14 Symington J. Electrophoretic transfer of proteins from two-dimensional gels to sheets and their detection. In: Two-dimensional Gel Electrophoresis of Proteins Celis JE, Bravo R. eds Academic Press; New York: 1984: 128-163
  • 15 Hagen I. Electroimmunochemical characterization of platelet proteins: their subcellular location and involvement in platelet adhesion, aggregation and secretion. In: Electroimmunochemical analysis of membrane proteins Bjerrum OJ. ed Elsevier; Amsterdam: 1983: 209-222
  • 16 Okita JR, Pidard D, Newman PJ, Montgomery RR, Kunicki TJ. On the association of glycoprotein Ib and actin-binding protein in human platelets. J Cell Biol 1985; 100: 317-321
  • 17 Dawes J. Radioimmunoassay of secreted platelet proteins. In: Platelets in biology and pathology III Macintyre DE, Gordon JL. eds Elsevier; Amsterdam: 1987: 493-514
  • 18 Jennings LK, Fox JE B, Edwards HH, Phillips DR. Changes in the cytoskeletal structure of human platelets following thrombin activation. J Biol Chem 1981; 256: 6927-6932
  • 19 Beardsley DS, Spiegel JB, Jacobs MM, Handin RI, Lux SE. Platelet membrane glycoprotein IIIa contains target antigens that bind anti-platelet antibodies in immune thrombocytopenias. J Clin Invest 1984; 74: 1701-1707
  • 20 Rock G, Decary F, Tittley P, Fuller V. Electroblotting and immunohistochemical staining for identification of platelet antibodies. Br J Haematol 1987; 67: 437-441
  • 21 Santoso S, Shibata Y, Kiefel Y, Mueller-Eckhardt C. Identification of the Yuk allo-antigen on platelet glycoprotein IIIa. Vox Sang 1987; 53: 48-51
  • 22 Thorpe R, Bird RC, Spitz M. Immunoblotting with monoclonal antibodies. Loss of immrrorutivity with human immunoglobulins arises from polypeptide chain separation. J Immunol Methods 1984 73. 259-265
  • 23 Thorsen LI, Gaudernack G, Brosstad E, Pedersen TM, Solum NO. Identification of platelet antigens by monoclonal antibodies using crossed immunoelectrophoresis with immunoblotting of the monoclonal antibody. Thromb Haemostas 1987; 57: 212-216
  • 24 Kieffer N, Boizard B, Didry D, Wautier JL, Nurden AT. Immunochemical characterization of the platelet-specific alloantigen Leku. A comparative study with the PLA1 alloantigen. Blood. 1984; 64: 1212-1219
  • 25 Von dem Borne AE G K, Plas van Dalen C M. Bak(a) and Lek(a) are identical antigens. Br J Haematol 1986; 62: 404-5