Anti-factor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do not exhibit isotypic restriction

Abstract A significant proportion of hemophilia A patients receiving transfusions of factor VIII (FVIII) develop a specific antibody response towards FVIII. These antibodies are usually detected by assays in which they inhibit the function of the molecule, such as the Bethesda clotting test. We have prepared anti-FVIII antibodies by specific immunoadsorption from the plasma of four hemophiliacs with stable inhibitor levels. The isotypic distribution of such antibodies was determined and their capacity to bind to insolubilized FVIII was compared with their inhibitory activity in two functional assays, namely, the Bethesda assay and a chromogenic assay. In addition, the FVIII epitope specificity was determined by competition with monoclonal antibodies for the binding to insolubilized FVIII. We show here that (1) anti-FVIII antibodies are not isotypically restricted; thus, a significant proportion of specific IgG2 was found; (2) antibodies are frequently directed towards epitopes of FVIII that are not directly involved in the function of the molecule and therefore escape detection in the Bethesda method or chromogenic assay; and (3) each patient shows a unique pattern of FVIII epitope recognition. We conclude that evaluation of anti-FVIII antibodies by a functional method does not provide an accurate evaluation of the specific antibody response. These findings have important implications for the comparison of the immunogenicity of FVIII molecules produced by different technologies and for the development of methods to control anti-FVIII antibody production.

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Anti-factor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do not exhibit isotypic restriction

Blood ◽

10.1182/blood.v82.8.2452.bloodjournal8282452 ◽

1993 ◽

Vol 82 (8) ◽

pp. 2452-2461 ◽

Cited By ~ 1

Author(s):

JG Gilles ◽

J Arnout ◽

J Vermylen ◽

JM Saint-Remy

Keyword(s):

Antibody Response ◽

Factor Viii ◽

Specific Antibody ◽

Hemophilia A ◽

Significant Proportion ◽

Functional Method ◽

Specific Antibody Response ◽

Functional Assays ◽

Epitope Specificity ◽

Chromogenic Assay

A significant proportion of hemophilia A patients receiving transfusions of factor VIII (FVIII) develop a specific antibody response towards FVIII. These antibodies are usually detected by assays in which they inhibit the function of the molecule, such as the Bethesda clotting test. We have prepared anti-FVIII antibodies by specific immunoadsorption from the plasma of four hemophiliacs with stable inhibitor levels. The isotypic distribution of such antibodies was determined and their capacity to bind to insolubilized FVIII was compared with their inhibitory activity in two functional assays, namely, the Bethesda assay and a chromogenic assay. In addition, the FVIII epitope specificity was determined by competition with monoclonal antibodies for the binding to insolubilized FVIII. We show here that (1) anti-FVIII antibodies are not isotypically restricted; thus, a significant proportion of specific IgG2 was found; (2) antibodies are frequently directed towards epitopes of FVIII that are not directly involved in the function of the molecule and therefore escape detection in the Bethesda method or chromogenic assay; and (3) each patient shows a unique pattern of FVIII epitope recognition. We conclude that evaluation of anti-FVIII antibodies by a functional method does not provide an accurate evaluation of the specific antibody response. These findings have important implications for the comparison of the immunogenicity of FVIII molecules produced by different technologies and for the development of methods to control anti-FVIII antibody production.

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Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response

PLoS ONE ◽

10.1371/journal.pone.0153183 ◽

2016 ◽

Vol 11 (4) ◽

pp. e0153183 ◽

Cited By ~ 4

Author(s):

Don Changsom ◽

Hatairat Lerdsamran ◽

Witthawat Wiriyarat ◽

Warunya Chakritbudsabong ◽

Bunpote Siridechadilok ◽

...

Keyword(s):

Antibody Response ◽

Specific Antibody ◽

Specific Antibody Response ◽

Functional Assays ◽

Neuraminidase Subtype

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The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies

Blood ◽

10.1182/blood.v89.10.3663 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3663-3671 ◽

Cited By ~ 123

Author(s):

Richard Prescott ◽

Hiroaki Nakai ◽

Evgueni L. Saenko ◽

Inge Scharrer ◽

Inga Marie Nilsson ◽

...

Keyword(s):

Immune Response ◽

Antibody Response ◽

Factor Viii ◽

Light Chain ◽

Hemophilia A ◽

Epitope Specificity ◽

Extensive Analysis ◽

Number Of Patients ◽

Recombinant Fviii ◽

Inhibitor Titer

Abstract Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as ≥ 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.

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The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies

Blood ◽

10.1182/blood.v89.10.3663.3663_3663_3671 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3663-3671 ◽

Cited By ~ 1

Author(s):

Richard Prescott ◽

Hiroaki Nakai ◽

Evgueni L. Saenko ◽

Inge Scharrer ◽

Inga Marie Nilsson ◽

...

Keyword(s):

Immune Response ◽

Antibody Response ◽

Factor Viii ◽

Light Chain ◽

Hemophilia A ◽

Epitope Specificity ◽

Extensive Analysis ◽

Number Of Patients ◽

Recombinant Fviii ◽

Inhibitor Titer

Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as ≥ 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.

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Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype

Hämostaseologie ◽

10.1055/s-0037-1619052 ◽

2010 ◽

Vol 30 (04) ◽

pp. 207-211 ◽

Cited By ~ 24

Author(s):

A. Pavlova ◽

J. Oldenburg

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Significant Proportion ◽

Haemophilia A ◽

Missense Mutations ◽

One Stage ◽

Chromogenic Assay ◽

Fviii Activity ◽

Mechanistic Basis

SummarySeverity of bleeding phenotype in hemophilia A (HA) depends on the underlying mutation in the F8 gene and, ultimately, on the concentration and functional integrity of the factor VIII (FVIII) protein in circulating plasma. Initial diagnosis for HA and monitoring of treatment is typically performed by measuring of FVIII activity by either one-stage assay or chromogenic assay.We review evidence for why both types of assay do not give comparable results in a significant proportion of patients with non-severe haemophilia A and why the discrepancy in results between both methods segregates with distinct subclasses of known missense mutations causing haemophilia A. The current understanding of the mechanistic basis for how FVIII:C assay discrepancies arise are discussed.We propose that both methods should be used in initial patient diagnosis along with follow-up genetic analysis to avoid potential misdiagnosis and to optimize treatment monitoring of patients with HA phenotypes.

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