Assessment of Dextran Antigenicity of Intravenous Iron Preparations with Enzyme-Linked Immunosorbent Assay (ELISA)

Abstract

Intravenous iron preparations are typically classified as non-dextran-based or dextran/dextran-based complexes. The carbohydrate shell for each of these preparations is unique and is key in determining the various physicochemical properties, the metabolic pathway, and the immunogenicity of the iron-carbohydrate complex. As intravenous dextran can cause severe, antibody-mediated dextran-induced anaphylactic reactions (DIAR), the purpose of this study was to explore the potential of various intravenous iron preparations, non-dextran-based or dextran/dextran-based, to induce these reactions. An IgG-isotype mouse monoclonal anti-dextran antibody (5E7H3) and an enzyme-linked immunosorbent assay (ELISA) were developed to investigate the dextran antigenicity of low molecular weight iron dextran, ferumoxytol, iron isomaltoside 1000, ferric gluconate, iron sucrose and ferric carboxymaltose, as well as isomaltoside 1000, the isolated carbohydrate component of iron isomaltoside 1000. Low molecular weight iron dextran, as well as dextran-based ferumoxytol and iron isomaltoside 1000, reacted with 5E7H3, whereas ferric carboxymaltose, iron sucrose, sodium ferric gluconate, and isolated isomaltoside 1000 did not. Consistent results were obtained with reverse single radial immunodiffusion assay. The results strongly support the hypothesis that, while the carbohydrate alone (isomaltoside 1000) does not form immune complexes with anti-dextran antibodies, iron isomaltoside 1000 complex reacts with anti-dextran antibodies by forming multivalent immune complexes. Moreover, non-dextran based preparations, such as iron sucrose and ferric carboxymaltose, do not react with anti-dextran antibodies. This assay allows to assess the theoretical possibility of a substance to induce antibody-mediated DIARs. Nevertheless, as this is only one possible mechanism that may cause a hypersensitivity reaction, a broader set of assays will be required to get an understanding of the mechanisms that may lead to intravenous iron-induced hypersensitivity reactions.

Keywords: anaphylaxis; antidextran; intravenous iron.

Figure 1

Reactivity of different IV iron preparations with 5E7H3 by reverse single radial immunodiffusion. Positive antigen/antibody reaction, indicated by circular turbidity around the well, is observed in the upper row for the positive control (dextran 5000) and the dextran/dextran-based preparations (LMWID, FMX, and IIM). In the lower row, the negative control (dextran 1000) as well as the non-dextran-based preparations (SFG, IS, and FCM) do not show any reaction. FCM, ferric carboxymaltose; FMX, ferumoxytol; IIM, iron isomaltoside 1000; IS, iron sucrose; LMWID, low molecular weight iron dextran; SFG, sodium ferric gluconate.

Figure 2

Schematic illustration of immune complex formation and inhibition. (A) Immune complex formation with high molecular weight (HMW) dextran and anti-dextran antibody. A large insoluble complex is generated, which may trigger DIARs; (B) Inhibition of immune complex formation by dextran 1000, i.e., hapten prophylaxis. Dextran 1000 binds to the anti-dextran antibody without formation of an immune complex and thus does not induce any immune response. The anti-dextran antibody is no longer available for binding to the HMW dextran, which thus is less likely to induce DIARs; (C) Proposed mechanism of the observed in vitro multivalent immune complex formation with iron isomaltoside 1000 (IIM) and anti-dextran antibody. In IIM, a number of isomaltoside 1000 (reduced dextran 1000) units are attached to the polynuclear iron core. The presented in vitro results suggest that IIM can act as a polyvalent higher molecular weight dextran and lead to immune complex formation. As in (A), a large insoluble complex is formed, which may trigger DIARs. (A,B) Based on Richter et al. [40].