Download PDF
Thromb Haemost 1992; 67(06): 631-638
DOI: 10.1055/s-0038-1648514
Original Articles
Schattauer GmbH Stuttgart

Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assays (ELISA) for the Measurement of Vitamin K-Dependent Protein S: The Effect of Antibody Immunoreactivity on Plasma Protein S Antigen Determinations

Silvana Vigano D’Angelo
The Coagulation Service and Section of Immunochemistry and Hybridoma, Scientific Institute IRCCS, H. S. Raffaele, Milano
,
Sandra Tombesi
*   Institute of Chemistry, School of Medicine, University of Brescia, Italy
,
Santica Marcovina
The Coagulation Service and Section of Immunochemistry and Hybridoma, Scientific Institute IRCCS, H. S. Raffaele, Milano
,
Alberto Albertini
*   Institute of Chemistry, School of Medicine, University of Brescia, Italy
,
Patrizia Della Valle
The Coagulation Service and Section of Immunochemistry and Hybridoma, Scientific Institute IRCCS, H. S. Raffaele, Milano
,
Armando D’Angelo
The Coagulation Service and Section of Immunochemistry and Hybridoma, Scientific Institute IRCCS, H. S. Raffaele, Milano
› Author Affiliations
Further Information

Publication History

Received 13 October 1989

Accepted after revision 18 December 1991

Publication Date:
03 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

Two monoclonal antibodies (Mabs) specifically directed to human protein S (PS) - named 5E9E9 and 3B10.25 - were produced and their properties compared to those of 2 previously characterized anti-PS-Mabs (HPS-2 and S10). 3B10.25, similar to S10, was directed to the calcium-free conformation of PS and had virtually identical affinity for free and C4b-binding protein (C4b-BP)-bound PS; 5E9E9 similar to HPS-2, had no calcium-dependency and was selectively directed to free PS. All Mabs were equally reactive to freshly purified and thrombin-cleaved PS. To evaluate the influence of C4b-BP bound PS on PS antigen determinations, ELISA systems employing the four Mabs individually as capture antibody (Ab) and peroxidase-conjugated polyclonal anti-PS IgG as detecting Ab were developed and compared to immunoelectrophoresis (EIA) and to an ELISA employing polyclonal anti-PS IgG as capture and detecting Ab, in the determination of PS in purified systems and in plasma. With all the ELISAs there was parallelism of dilution cuiyes obtained with normal plasma and purified PS; however, supplementation of plasma with purified C4b-BP resulted in loss of parallelism when employing the Mabs directed to free PS as capture Ab. Influence of high C4b-BP on PS antigen determinations was confirmed in a series of plasma samples from patients with C4b-BP levels ranging from 70% to over 200%. Compared to the values obtained with the S10- or 3B10.25 - based ELISAs - which were similar despite a 10-fold difference in sample dilution -plasma PS was underestimated by the ELISAs employing 5E9E9 or HPS-2 while it was overestimated by EIA. In addition, plasma PS and C4b-BP levels were significantly correlated only when it was measured by EIA or the ELISAs employing S10, 3B10.25 or polyclonal IgG. These results highlight the potential influence of high C4b-BP on plasma PS antigen determination. Accurate measurement of PS by ELISA requires selection of antibodies with identical affinity for all plasma PS forms.

 

  • References

  • 1 Walker FJ. The regulation of activated protein C by a new protein: A possible function for bovine protein S. J Biol Chem 1980; 225: 5521-5524
    PubMedSearch in Google Scholar
  • 2 D’Angelo A, Lockhart MS, Vigano’D’Angelo S, Taylor Jr FB. Protein S is a cofactor for activated protein C neutralization of an inhibitor of plasminogen activation (PAI) released from platelets. Blood 1987; 69: 232-237
    PubMedSearch in Google Scholar
  • 3 Di Scipio RG, Hermodson MA, Yates SG, Davie EW. A comparison of human prothrombin, factor IX (Christmas Factor), factor X (Stuart Factor) and protein S. Biochemistry 1977; 16: 698-706
    CrossrefPubMedSearch in Google Scholar
  • 4 Lundwall A, Backowski W, Cohen E, Shaffer M, Mahr A, Dahlback B, Stenflo J, Wydro R. Isolation and sequence of cDNA for human protein S, a regulator of blood coagulation. Proc Natl Acad Sci USA 1986; 83: 6716-6720
    CrossrefPubMedSearch in Google Scholar
  • 5 Walker FJ. Protein S and the regulation of activated protein C. Semin Thromb Hemostas 1984; 10: 131-138
    Thieme ConnectPubMedSearch in Google Scholar
  • 6 Suzuki K, Stenflo J, Dahlback B, Teodorsson B. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914-1920
    PubMedSearch in Google Scholar
  • 7 Fulchner CA, Gardiner JE, Griffin JH, Zimmermann TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated protein C and its homology with factor V. Blood 1984; 63: 486-489
    PubMedSearch in Google Scholar
  • 8 Dahlback B, Stenflo J. High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein. Proc Natl Acad Sci USA 1981; 78: 2512-2516
    CrossrefPubMedSearch in Google Scholar
  • 9 Dahlback B. Purification of human C4b-binding protein and formation of its complex with vitamin K dependent protein S. Biochem J 1983; 209: 847-856
    CrossrefPubMedSearch in Google Scholar
  • 10 Dahlback B, Frohm B, Nelsestuen G. High affinity interaction between C4b-binding protein and vitamin K-dependent protein S in the presence of calcium. Suggestion of a third component in blood regulating the interaction. J Biol Chem 1990; 265: 16082-16087
    PubMedSearch in Google Scholar
  • 11 Dahlback B. Inhibition of protein Ca cofactor function of human and bovine protein S by C4bBP. J Biol Chem 1986; 261: 12022-12027
    PubMedSearch in Google Scholar
  • 12 Comp PC, Nixon RR, Cooper MR, Esmon CT. Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 1984; 74: 1082-1088
    PubMedSearch in Google Scholar
  • 13 Comp PC, Esmon CT. Recurrent venous thromboembolism in patients with partial deficiency of protein S. N Engl J Med 1984; 311: 1525-1528
    CrossrefPubMedSearch in Google Scholar
  • 14 Schwarz HP, Fischer M, Hopmeier P, Batard MA, Griffin JH. Plasma protein S deficiency in familial thrombotic disease. Blood 1984; 64: 1297-1300
    PubMedSearch in Google Scholar
  • 15 Broekmans AW, Bertina RM, Reinalda-Poot J, Engesser L, Muller HP, Leeuw JA, Michiels JJ, Brommer EJP, Briet E. Hereditary protein S deficiency and venous thromboembolism. A study in three dutch families. Thromb Haemostas 1985; 53: 273-277
    PubMedSearch in Google Scholar
  • 16 Kamiya T, Sugihara T, Ogata K, Saito H, Suzuki K, Nishioka J, Hashimoto S, Yamagata K. Inherited deficiency of protein S in a japanese family with recurrent venous thrombosis: a study of three generations. Blood 1986; 67: 406-410
    PubMedSearch in Google Scholar
  • 17 Boyer-Neumann C, Wolf M, Amiral J, Guyader AM, Meyer D, Larrieu MJ. Familial type I protein S deficiency associated with severe venous thrombosis. A study of five cases. Thromb Haemostas 1988; 60: 128
    PubMedSearch in Google Scholar
  • 18 Mannucci PM, Tripodi A, Bertina RM. Protein S deficiency associated with “juvenile” arterial and venous thromboses. Thromb Haemostas 1986; 55: 440
    PubMedSearch in Google Scholar
  • 19 Coller BS, Owen J, Jetsi J, Horowitz D, Reitman MJ, Spear J, Yeh T, Comp PC. Deficiency of plasma protein S, protein C, or antithrombin III and arterial thrombosis. Arteriosclerosis 1987; 7: 456-462
    CrossrefPubMedSearch in Google Scholar
  • 20 Israels SJ, Seshia SS. Childhood stroke associated with protein C or protein S deficiency. J Pediatr 1987; 111: 562-564
    CrossrefPubMedSearch in Google Scholar
  • 21 Girolami A, Simioni P, Lazzaro AR, Cordiano I. Severe arterial cerebral thrombosis in a patient with protein S deficiency (moderately reduced total and markedly reduced free protein S): a family study. Thromb Haemostas 1989; 61: 144-147
    PubMedSearch in Google Scholar
  • 22 Comp PC, Doray D, Patton D, Esmon CT. An abnormal distribution of protein S occurs in functional protein S deficiency. Blood 1986; 67: 504-508
    PubMedSearch in Google Scholar
  • 23 Rose PG, Essig GF, Vaccaro PS, Brandt JT. Protein S deficiency in pregnancy. Am J Obstet Gynecol 1986; 155: 140-141
    CrossrefPubMedSearch in Google Scholar
  • 24 Huisveld IA, Hospers JEH, Meijers JCM, Starkenburg AE, Erich WBM, Bouma BN. Oral contraceptives reduce total protein S, but not free protein S. Thromb Res 1987; 45: 109-114
    CrossrefPubMedSearch in Google Scholar
  • 25 Melissari E, Kakkar W. The effects of oestrogen administration on the plasma free protein S and C4b-binding protein. Thromb Res 1988; 49: 489-495
    CrossrefPubMedSearch in Google Scholar
  • 26 Goualt-Heilmann M, Gadelha-Parente T, Levent M, Intrator L, Rostoker G, Lagrue G. Total and free proteinS in nephrotic syndrome. Thromb Res 1988; 49: 37-42
    CrossrefPubMedSearch in Google Scholar
  • 27 Wyshock E, Caldwell M, Crowley JP. Deep venous thrombosis, inflammatory bowel disease, and proteinS deficiency. Am J Clin Pathol 1988; 90: 633-635
    CrossrefPubMedSearch in Google Scholar
  • 28 Moalic P, Gruel Y, Body G, Foloppe P, Dalahousse B, Leroy J. Levels and plasma distribution of free and C4b-BP-bound protein S in human fetuses and full-term newborns. Thromb Res 1988; 49: 471-480
    CrossrefPubMedSearch in Google Scholar
  • 29 Fernandez JA, Estelles A, Gilalbert J, Espana F, Aznar J. Functional and immunologic protein S in normal pregnant women and in full-term newborns. Thromb Haemostas 1989; 61: 474-478
    PubMedSearch in Google Scholar
  • 30 Takahashi H, Tatewaki W, Nakamura T, Hanano M, Wada K, Shibata A. Coagulation studies in thrombotic thrombocytopenic purpura, with special reference to von Willebrand factor and protein S. Am J Hematol 1989; 30: 14-21
    CrossrefPubMedSearch in Google Scholar
  • 31 Allon M, Soffer O, Evatt BL, Hixon G, Wideman CS. Protein S and C antigen levels in proteinuric patients: dependence on type of glomerular pathology. Am J Hematol 1989; 31: 96-101
    CrossrefPubMedSearch in Google Scholar
  • 32 Conlan MG, Haire WD. Low protein S in essential thrombocythemia with thrombosis. Am J Hematol 1989; 32: 88-93
    CrossrefPubMedSearch in Google Scholar
  • 33 Bertina RM, van Wijngaarden A, Reinalda-Poot J, Poort SR, Bom VJJ. Determination of plasma protein S - the protein cofactor of activated protein C. Thromb Haemostas 1985; 53: 268-272
    PubMedSearch in Google Scholar
  • 34 Fair DS, Revak DJ. Quantitation of human protein S in the plasma of normal and warfarin-treated individuals by radioimmunoassay. Thromb Res 1984; 36: 527-533
    CrossrefPubMedSearch in Google Scholar
  • 35 Comp PC, Thurnau GR, Welsh J, Esmon CT. Functional and immunologic protein S levels are decreased during pregnancy. Blood 1986; 68: 881-885
    PubMedSearch in Google Scholar
  • 36 Malm J, Laurell M, Dahlback B. Changes in the plasma levels of vitamin K-dependent protein C and S and of C4b-binding protein during pregnancy and oral anticoagulation. Br J Haematol 1988; 68: 437-443
    CrossrefPubMedSearch in Google Scholar
  • 37 Woodhams BJ. The simultaneous measurement of total and free protein S by ELISA. Thromb Res 1988; 50: 213-220
    CrossrefPubMedSearch in Google Scholar
  • 38 Litwiller RD, Jenny RJ, Katzmann JA, Miller RS, Mann KG. Monoclonal antibodies to human vitamin K-dependent protein S. Blood 1986; 67: 1583-1590
    PubMedSearch in Google Scholar
  • 39 Marcovina S, Coppola R, Valsecchi C, Zoppo A, Gelfi C, Mannucci PM. Monoclonal antibodies directed to the calcium-free conformation of human protein S. Thromb Haemostas 1989; 62: 709-714
    PubMedSearch in Google Scholar
  • 40 D’Angelo A, Vigano’ D’Angelo S, Esmon CT, Comp PC. Acquired deficiencies of protein S. Protein S activity during oral anticoagulation, in liver disease and in disseminated intravascular coagulation. J Clin Invest 1988; 81: 1445-1454
    CrossrefPubMedSearch in Google Scholar
  • 41 Villiers MB, Reboul A, Thielens NM, Colomb MG. Purification and characterization of C4-Binding Protein from human serum. FEBS Lett 1981; 132: 49-54
    CrossrefPubMedSearch in Google Scholar
  • 42 Vigano’ D’Angelo S, Gilardoni F, D’Angelo A. Evaluation of coagulometric assays in the assessment of protein C anticoagulant activity; variable sensitivity of commercial APTT reagents to the cofactor effect of protein S. Thromb Haemostas 1989; 62: 861-867
    PubMedSearch in Google Scholar
  • 43 Vigano’ D’Angelo S, Comp PC, Esmon CT, D’Angelo A. Relationship between protein C antigen and anticoagulant activity during oral anticoagulation and in selected disease states. J Clin Invest 1986; 77: 416-425
    CrossrefPubMedSearch in Google Scholar
  • 44 Nakane PK, Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem 1974; 22: 1084-1091
    CrossrefPubMedSearch in Google Scholar
  • 45 Vigano’ D’Angelo S, D’Angelo A, Kaufman C, Sholer C, Esmon CT, Comp PC. Protein S deficiency occurs in the nephrotic syndrome. Ann Intern Med 1987; 107: 42-47
    CrossrefPubMedSearch in Google Scholar