Comparative Measurement of Anti-Factor VIII Antibody by Bethesda Assay and ELISA Reveals Restricted Isotype Profile and Epitope Specificity

Towfighi, Farzaneh; Gharagozlou, Soheila; Sharifian, Ramazan Ali; Kazemnejad, Anoushirvan; Esmailzadeh, Khadijeh; Managhchi, Mohammad Reza; Shokri, Fazel

doi:10.1159/000086580

Cited by 27 publications

(31 citation statements)

References 55 publications

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“…Inhibitor formation is a serious complication in the management of HA patients and more than thirty years of research has provided insight into humoral anti-FVIII immune responses, which often include IgG 4 immunoglobulins [7], [12], [13]. The IgG 4 subtype is frequently associated with chronic exposure to protein antigens [14].…”

Section: Discussionmentioning

confidence: 99%

Phenotypes of Allo- and Autoimmune Antibody Responses to FVIII Characterized by Surface Plasmon Resonance

et al. 2013

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Evidence of antibody isotype/subtype switching may provide prognostic value regarding the state of immune responses to therapeutic proteins, e.g. anti-factor VIII (FVIII) antibodies that develop in many hemophilia A patients, clinically termed “inhibitors”. A sensitive, high- information-content surface plasmon resonance (SPR) assay has been developed to quantify IgG subtype distributions and the domain specificity of anti-drug antibodies. Plasma samples from 22 subjects with an allo- or auto-immune reaction to FVIII were analyzed. Pre-analytical treatment protocols were developed to minimize non-specific binding and specific matrix interference due to von Willebrand factor-FVIII interactions. The dynamic range for IgG quantification was 0.2–5 µg/ml (∼1–33 nM), allowing characterization of inhibitor-positive samples. Subtype-specific monoclonal antibodies were used to quantify the IgG subtype distribution of FVIII-specific antibodies. Most samples obtained from multiply-infused inhibitor subjects contained IgG4 antibodies. Several distinct phenotypes were assigned based on the IgG subtype distribution: IgG1, IgG4, IgG1 & IgG4, and IgG1, IgG2 & IgG4. An IgG1-only response was found in mild/moderate HA subjects during early FVIII infusions, and analysis of serial samples followed antibody class switching as several subjects’ immune responses developed. Competition studies utilizing a recombinant FVIII-C2 domain indicated 40–80% of FVIII-specific antibodies in most samples were directed against this domain.

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Section: Discussionmentioning

confidence: 99%

Phenotypes of Allo- and Autoimmune Antibody Responses to FVIII Characterized by Surface Plasmon Resonance

et al. 2013

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“…Some groups have developed immunoassays that can detect both types of antibodies, I‐Ab and NI‐Ab. A number of published reports have utilized ELISA, which provides an alternative way to examine the immune response to FVIII [6–10]. Anti‐FVIII antibody detection by ELISA generally agreed with that of the Bethesda method.…”

Section: Discussionmentioning

confidence: 77%

“…A number of published reports have utilized enzymelinked immunosorbent assay (ELISA) to detect I-Ab and NI-Ab [6][7][8][9][10]. These NI-Ab may be found in haemophilia A patients without I-Ab (15-38%).…”

Section: Introductionmentioning

confidence: 99%

A flow cytometry evaluation of anti-FVIII antibodies: correlation with ELISA and Bethesda assay

et al. 2010

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In this study, we describe a flow cytometry (FC) system for detecting antibodies to factor VIII (FVIII) and compare its results with those of enzyme-linked immunosorbent assay (ELISA) that detects both inhibitory (I-Ab) and non-inhibitory (NI-Ab) antibodies and the Nijmegen modification of the Bethesda method, detecting I-Ab. FC was set up in our laboratory. Recombinant FVIII (rFVIII) was coupled to microspheres (FVIII-m) and reacted with different plasma dilutions. Microspheres without rFVIII were used as control (control-m). Captured anti-FVIII antibodies were detected using anti-human IgG. Plasma samples from the following patients with severe haemophilia A (SHA) patients were evaluated: 17 P (patients without I-Ab, <0.5 BU mL(-1)); 13 PI (patients with I-Ab, 1.1-8200 BU mL(-1)). Of these 13, two PI were referred during immune tolerance induction (ITI), and plasmas from 12 healthy donors (HD) were evaluated. Semiquantitative results were given as an index (the highest mean fluorescence intensity ratio between FVIII-m and control-m multiplied by the inverse of the corresponding plasma dilution). Both plasma and serum were suitable for the test. FC agreed with the Bethesda method (r = 0.8; P = 0.0001). FC and ELISA had 80% of coincidence. Four of 17 patients (23.5%) had NI-Ab by FC, and two of them developed high levels of I-Ab later on. This test provides a useful alternative for measuring FVIII antibodies supplementing Bethesda assay. FC is fast and easy to perform. No more than 200 μL of plasma or serum is required especially making it useful for paediatric patients.

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“…It is hard to compare existing immunoassays with the assay used in this study, because the former are non-quantitative with respect to the molar concentration of anti-FVIII antibodies. [38, 45–47]…”

Section: Discussionmentioning

confidence: 99%

Product‐dependent anti‐factor VIII antibodies

et al. 2013

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Introduction The development of anti-factor (F)VIII antibodies in hemophilia A (HA) subjects undergoing replacement therapy has been well-documented. The correlation between antibody development and the FVIII product used for replacement therapy remains a subject of discussion. Aim To evaluate the presence of anti-FVIII antibodies toward three commercial rFVIII products in 34 HA subjects’ plasmas. Methods Antibodies were quantitated by a Multiplex Fluorescence Immunoassay. Results All plasmas contained anti-FVIII antibodies at variable concentrations ranging from 50 nM to 570 μM. Eleven of 20 HA subjects treated with one (r)FVIII product contained inhibitory anti-FVIII antibodies (0.8–3584 BU). The inhibitory antibody titer and the molar concentrations of total antibody were mildly correlated (r2=0.6). Pronounced differences in antibody recognition with the three rFVIII products were observed. For the group treated with Product “A”, the titer toward this product was 2.4-fold higher than that observed with another full-length rFVIII-containing product (Product “B”) and almost 4-fold higher than that measured with a B domain-less rFVIII product (Product “C”). For the group of 14 HA subjects treated with FVIII other than Product “A”, only one showed higher antibody titer when measured with this product. Conclusions Our data suggest that the development of anti-FVIII antibodies is biased toward the product used for treatment and that a significant fraction of antibodies bind to the B domain of FVIII.

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Comparative Measurement of Anti-Factor VIII Antibody by Bethesda Assay and ELISA Reveals Restricted Isotype Profile and Epitope Specificity

Cited by 27 publications

References 55 publications

Phenotypes of Allo- and Autoimmune Antibody Responses to FVIII Characterized by Surface Plasmon Resonance

Phenotypes of Allo- and Autoimmune Antibody Responses to FVIII Characterized by Surface Plasmon Resonance

A flow cytometry evaluation of anti-FVIII antibodies: correlation with ELISA and Bethesda assay

Product‐dependent anti‐factor VIII antibodies

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