Skip to main content
SpringerLink
Log in

Biomimetic biosensor to distinguish between inhibitory and non-inhibitory factor VIII antibodies

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Patients with hereditary or acquired haemophilia A may develop inhibitory factor VIII (FVIII) antibodies. These disrupt FVIII activity predominantly by preventing the formation of the tenase complex, leading to a serious bleeding disorder. Antibodies without inhibiting activity, however, can also be found when screening patients with haemophilia A under FVIII supplementation. Therefore, the detection of only these allo- or autoantibodies from plasma is not sufficient. Rather, the characterization of the antibody-induced effects on the coagulation cascade should be considered due to its great diagnostic importance. Currently, inhibitory activities are detected by the functional Bethesda assay, which directly measures the delay in clotting time by the patient plasma. However, this assay does not provide information on the cause of the inhibition. Here, we report the development of a surface plasmon resonance (SPR) biosensor that has the potential to integrate both quantitative and functional information on patient antibody characteristics in one measurement. Recombinant FVIII protein was immobilized on the sensor surface to detect antibodies from patient plasma. The interaction of the FIX- and FXa-clotting proteins with the formed anti-FVIII/FVIII complex could be detected subsequently within the same SPR measurement cycle. Inhibitory antibodies led to the prevention of these interactions. Thus, discrimination between the clinically relevant inhibitory and non-inhibitory antibodies was enabled. In a group of 16 patients with inhibitory antibodies (both ELISA- and Bethesda-positive), 5 patients with non-inhibitory antibodies (ELISA-positive but Bethesda-negative) and 12 healthy controls, diagnostic sensitivity and specificity data of 100 % for the FIX interaction were achieved using this biomimetic biosensor approach. The new method allows for detection and quantification, as well as for evaluation of inhibitory activity of allo- and autoantibodies, using small sample volume and short analysis time.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

Anti-FVIII:

Antibody against FVIII

AUC:

Area under the curve

BDD:

B-domain-deleted

BU:

Bethesda units

CV:

Coefficient of variation

EDC:

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

FC:

Flow cell

FIX:

Factor IX

FL:

Full length

FVIII:

Factor VIII

FXa:

Activated factor Xa

NHS:

N-Hydroxysuccinimide

ROC:

Receiver operating characteristic

RU:

Resonance units

SPR:

Surface plasmon resonance

vWF:

von Willebrand factor

References

  1. Rosendaal F, Briet E (1990) The increasing prevalence of haemophilia. Thromb Haemost 63:145

    CAS  Google Scholar 

  2. Ehrenforth S, Kreuz W, Scharrer I, Kornhuber B (1992) Factor VIII inhibitors in haemophiliacs. Lancet 742–743

  3. Gilles JGG, Grailly SC, De Maeyer M, Jacquemin MG, VanderElst LP, Saint-Remy J-MR (2004) In vivo neutralization of a C2 domain-specific human anti-Factor VIII inhibitor by an anti-idiotypic antibody. Blood 103:2617–2623

    Article  CAS  Google Scholar 

  4. Jacquemin MG, Desqueper BG, Benhida A, Vander Elst L, Hoylaerts MF, Bakkus M, Thielemans K, Arnout J, Peerlinck K, Gilles JG, Vermylen J, Saint-Remy JM (1998) Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from A hemophilia a patient with inhibitor. Blood 92:496–506

    CAS  Google Scholar 

  5. Ma AD, Carrizosa D (2006) Acquired factor VIII inhibitors: pathophysiology and treatment. ASH 432–437

  6. Sharathkumar A, Lillicrap D, Blanchette VS, Kern M, Leggo J, Stain AM, Brooker L, Carcao MD (2003) Intensive exposure to factor VIII is a risk factor for inhibitor development in mild hemophilia A. Thromb Haemost 1:1228–1236

    Article  CAS  Google Scholar 

  7. Fay PJ (2006) Factor VIII structure and function. Int J Hematol 83:103–108

    Article  CAS  Google Scholar 

  8. Lenting PJ, van Mourik JA, Mertens K (1998) The life cycle of coagulation factor VIII in view of its structure and function. Blood 92:3983–3996

    CAS  Google Scholar 

  9. Terraube V, O’Donnell JS, Jenkins PV (2010) Factor VIII and von Willebrand factor interaction: biological, clinical and therapeutic importance. Haemophilia 16:3–13

    Article  CAS  Google Scholar 

  10. Hoffman M, Monroe D (2001) A cell-based model of hemostasis. Thromb Haemost 85:958–965

    CAS  Google Scholar 

  11. Kasper CK, Aledort L, Aronson D, Counts R, Edson JR, van Eys J, Fratantoni J, Green D, Hampton J, Hilgartner M, Levine P, Lazerson J, McMillan C, Penner J, Shapiro S, Shulman N (1975) Proceedings: a more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh 34:612

    CAS  Google Scholar 

  12. Verbruggen B, Novakova I (1995) The Nijmegen modification of the Bethesda assay for factor VIII: C inhibitors: improved specificity and reliability. Thromb Haemost 73:247–251

    CAS  Google Scholar 

  13. Peerschke EIB, Castellone DD, Ledford-Kraemer M, Van Cott EM, Meijer P (2009) Laboratory assessment of factor VIII inhibitor titer: the North American Specialized Coagulation Laboratory Association experience. Am J Clin Pathol 131:552–558

    Article  CAS  Google Scholar 

  14. Ling M, Duncan EM, Rodgers SE, Street AM, Lloyd JV (2003) Low detection rate of antibodies to non-functional epitopes on factor VIII in patients with hemophilia A and negative for inhibitors by Bethesda assay. Thromb Haemost 1:2548–2553

    Article  CAS  Google Scholar 

  15. Lebreton A, Lapalud P, Chambost H, Biron-Andréani C, Morange PE, Combescure C, Marquès-Verdier A, Berger C, Schved JF, Granier C, Lavigne-Lissalde G (2011) Prevalence and epitope specificity of non-neutralising antibodies in a large cohort of haemophilia A patients without inhibitors. Thromb Haemost 105:954–961

    Article  CAS  Google Scholar 

  16. Buhl A, Metzger J, Heegaard NHH, von Landenberg P, Fleck M, Luppa PB (2007) Novel biosensor-based analytic device for the detection of anti-double-stranded DNA antibodies. Clin Chem 341:334–341

    Google Scholar 

  17. Metzger J, von Landenberg P, Kehrel M, Buhl A, Lackner KJ, Luppa PB (2007) Biosensor analysis of beta2-glycoprotein I-reactive autoantibodies: evidence for isotype-specific binding and differentiation of pathogenic from infection-induced antibodies. Clin Chem 53:1137–1143

    Article  CAS  Google Scholar 

  18. Buhl A, Page S, Heegaard NHH, von Landenberg P, Luppa PB (2009) Optical biosensor-based characterization of anti-double-stranded DNA monoclonal antibodies as possible new standards for laboratory tests. Biosens Bioelectron 25:198–203

    Article  CAS  Google Scholar 

  19. Richman DD, Cleveland PH, Oxman MN, Johnson KM (1982) The binding of staphylococcal protein A by the sera of different animal species. J Immunol 128:2300–2305

    CAS  Google Scholar 

  20. Kesser W (1975) Rapid isolation of antigens from cells with Staphylococcal protein A-antibody adsorbent. J Immunol 115:1617–1624

    Google Scholar 

  21. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  22. Grancha S, Ortiz AM, Marañón C, Hampel K, Moret A, Zimmermann B, Jorquera JI, Aznar JA (2012) Kinetics of the interaction between anti-FVIII antibodies and FVIII from therapeutic concentrates, with and without von Willebrand factor, assessed by surface plasmon resonance. Haemophilia 18:982–989

    Article  CAS  Google Scholar 

  23. Zhong D, Saenko EL, Shima M, Felch M, Scandella D (1998) Some human inhibitor antibodies interfere with factor VIII binding to factor IX. Blood 92:136–142

    CAS  Google Scholar 

  24. Fijnvandraat K, Celie PH, Turenhout EA, ten Cate JW, van Mourik JA, Mertens K, Peters M, Voorberg J (1998) A human alloantibody interferes with binding of factor IXa to the factor VIII light chain. Blood 91:2347–2352

    CAS  Google Scholar 

  25. Nogami K, Shima M, Hosokawa K (1999) Role of factor VIII C2 domain in factor VIII binding to factor Xa. J Biol Chem 274:31000–31007

    Article  CAS  Google Scholar 

  26. Gedig E (2008) Surface chemistry in SPR technology. Handbook Surface Plasmon Resonance. pp 173–220

  27. Lewis KB, Hughes RJ, Epstein MS, Josephson NC, Kempton CL, Kessler CM, Key NS, Howard TE, Kruse-Jarres R, Lusher JM, Walsh CE, Watts RG, Ettinger RA, Pratt KP, PATH (Personalized Alternative Therapies for Haemophilia) Study Investigators (2013) Phenotypes of allo- and autoimmune antibody responses to FVIII characterized by surface plasmon resonance. PLoS One 8:e61120

    Article  CAS  Google Scholar 

  28. Fay PJ (1999) Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and factor IXa. J Biol Chem 274:29826–29830

    Article  CAS  Google Scholar 

  29. Matsumoto T, Nogami K, Ogiwara K, Shima M (2012) A putative inhibitory mechanism in the tenase complex responsible for loss of coagulation function in acquired haemophilia A patients with anti-C2 autoantibodies. Thromb Haemost 107:288–301

    Article  CAS  Google Scholar 

  30. Jacquemin M, Benhida A (2000) A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor. Blood 95:156–163

    CAS  Google Scholar 

  31. Fulcher CA, Lechner K, de Graaf MS (1988) Immunoblot analysis shows changes in factor VIII inhibitor chain specificity in factor VIII inhibitor patients over time. Blood 72:1348–1356

    CAS  Google Scholar 

Download references

Acknowledgments

We thank Mareike Mentzel for skillful technical support. We are also greatly indebted to Dr. Evangeline Thaler and Chris Beaudoin for reviewing the manuscript.

Author information

Authors and Affiliations

  1. Klinikum rechts der Isar der TU München, Institute of Clinical Chemistry and Pathobiochemistry, Ismaninger Str. 22, 81675, Munich, Germany

    Carmen Kocot, Aline R. Schindler, Alexander Le Blanc, Michael Schmalenberg & Peter B. Luppa

  2. Haemophilia Centre, Medizinische Klinik III, Institute for Transfusion Medicine, Johann Wolf-gang Goethe-Universitätsklinikum, Theodor-Stern-Kai 7, 60596, Frankfurt/Main, Germany

    Wolfgang Miesbach

  3. Hemostaseology/Angiology, Klinikum Innenstadt der Ludwig Maximilians-Universität München, Ziemssenstr. 1, 80336, Munich, Germany

    Michael Spannagl

Authors
  1. Carmen Kocot
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aline R. Schindler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexander Le Blanc
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael Schmalenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wolfgang Miesbach
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Spannagl
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Peter B. Luppa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter B. Luppa.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 142 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kocot, C., Schindler, A.R., Le Blanc, A. et al. Biomimetic biosensor to distinguish between inhibitory and non-inhibitory factor VIII antibodies. Anal Bioanal Chem 407, 5685–5693 (2015). https://doi.org/10.1007/s00216-015-8751-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-015-8751-x

Keywords

Search

Navigation