Detection and IgG subclass analysis of antibodies to factor VIII in multitransfused haemophiliacs and healthy individuals

Abstract

Using a binding assay to immobilized factor VIII (F VIII) (ELISA) we measured the amount of IgG with binding capacity to FVIII, in the plasma of patients with an inhibitor to F VIII, in multitransfused haemophiliacs without inhibitor and in a control group of blood donors. It was shown that the amount of IgG bound to VIII was elevated in patients with an inhibitor although a weak correlation could be established between the inhibitor titre (BU) and the amount of bound IgG. In all haemophiliacs without inhibitor, IgG bound to F VIII were present. Although the mean value of IgG bound to F VIII was significantly lower than the amount detected in patients with F VIII inhibitors, a group of patients developed an equal amount of IgG recognizing the F VIII molecules to the amount of IgG measured in inhibitor patients. These results indicate that the presence of an inhibitor is not related to the amount of specific IgG bound to F VIII but more likely to the position of epitopes recognized by specific IgG. The presence of IgG bound to F VIII was detected in 92% of control blood donors and an inhibitor to F VIII ranging from 0.5 to 1.3 BU mL(-1) in 17% of them. The isotypes of bound immunoglobins were identified in patients and controls: IgG4 subclass was predominant only in patients with an inhibitor and usually associated with antibodies of one or more of the other subclasses. In noninhibitor patients, very few had antibodies of IgG4 subclass with binding capacity to F VIII. These results raised the question of the clinical significance of these antibodies in multitransfused patients. The study indicates that binding assay is a complementary test to be used in multitransfused patients but cannot be used instead of the coagulation tests for detection of inhibitors.

Characterization of inhibitors to FVIII with an ELISA in congenital and acquired haemophilia A

“…It is generally agreed that IgG4 is the predominant anti ‐ FVIII antibody immunoglobulin subclass, often occurring with at least one other heavy chain subclass, usually IgG1 [1,10,24–26]. Similarly, in our study we found that IgG was the predominant immunoglobulin class in both acquired and congenital haemophilia A, but three patients with acquired haemophilia A also had antiFVIII IgM antibodies.…”

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

“…We and others have demonstrated, at the clonal level, a restricted specificity of B cell clones isolated from hemophilia A patients with inhibitor [19,20,21]. It also suggests that the inhibitory antibodies measured by Bethesda assay belong to the IgG isotype, a finding already reported by other investigators [13,22,23,24,25]. We have also demonstrated elevated levels of IgA in a small number of inhibitor-positive patients.…”

“…None of our samples was found to be of the IgG1 or IgG3 isotype. Predominance of IgG4 has been reported by almost all investigators [23,24,25, 27]. Conflicting results, however, have been observed for other IgG subclasses.…”

Effects of pre‐analytical heat treatment in factor VIII (FVIII) inhibitor assays on FVIII antibody levels

“…Several studies have shown anti-FVIII IgG 4 to be the best immunologic indicator of the presence of a functional inhibitor to FVIII. [8][9][10][11] In addition, we examined the stability of rFVIII under the various PHT conditions tested.…”