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. 2011;6(6):e21246.
doi: 10.1371/journal.pone.0021246. Epub 2011 Jun 23.

Enrichment of sialylated IgG by lectin fractionation does not enhance the efficacy of immunoglobulin G in a murine model of immune thrombocytopenia

Theresa Guhr  1 , Judith Bloem, Ninotska I L Derksen, Manfred Wuhrer, Anky H L Koenderman, Rob C Aalberse, Theo Rispens
Affiliations

Enrichment of sialylated IgG by lectin fractionation does not enhance the efficacy of immunoglobulin G in a murine model of immune thrombocytopenia

Theresa Guhr et al. PLoS One. 2011.

Abstract

Intravenous immunoglobulin G (IVIg) is widely used against a range of clinical symptoms. For its use in immune modulating therapies such as treatment of immune thrombocytopenic purpura high doses of IVIg are required. It has been suggested that only a fraction of IVIg causes this anti immune modulating effect. Recent studies indicated that this fraction is the Fc-sialylated IgG fraction. The aim of our study was to determine the efficacy of IVIg enriched for sialylated IgG (IVIg-SA⁺) in a murine model of passive immune thrombocytopenia (PIT). We enriched IVIg for sialylated IgG by Sambucus nigra agglutinin (SNA) lectin fractionation and determined the degree of sialylation. Analysis of IVIg-SA⁺ using a lectin-based ELISA revealed that we enriched predominantly for Fab-sialylated IgG, whereas we did not find an increase in Fc-sialylated IgG. Mass spectrometric analysis confirmed that Fc sialylation did not change after SNA lectin fractionation. The efficacy of sialylated IgG was measured by administering IVIg or IVIg-SA⁺ 24 hours prior to an injection of a rat anti-mouse platelet mAb. We found an 85% decrease in platelet count after injection of an anti-platelet mAb, which was reduced to a 70% decrease by injecting IVIg (p<0.01). In contrast, IVIg-SA⁺ had no effect on the platelet count. Serum levels of IVIg and IVIg-SA⁺ were similar, ruling out enhanced IgG clearance as a possible explanation. Our results indicate that SNA lectin fractionation is not a suitable method to enrich IVIg for Fc-sialylated IgG. The use of IVIg enriched for Fab-sialylated IgG abolishes the efficacy of IVIg in the murine PIT model.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Measurement of the degree of sialylation by SNA lectin inhibition ELISA.
% SNA lectin binding after pre-incubation A) with IVIg, IVIg-SA (+) or IVIg-SA (−) B) with Fab and Fc fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−) C) with Fab or protK digested Fab fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−) D) with Fc or protK digested Fc fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−). Data represents mean ± SEM, n = 3.
Figure 2
Figure 2. Mass spectrometric IgG1 Fc-glycosylation profiles of A) IVIg and B) IVIg-SA (+).
A) IVIg and B) IVIg-SA (+) were subjected to tryptic cleavage and analyzed by nanoLC-ESI-ion trap-mass spectrometry. Sum mass spectra of the elution range of the IgG1 Fc-glycopeptides are shown. Glycopeptides were registered as proton adducts ([M+4H]4+). All the displayed glycopeptides have 1 missed tryptic cleavage site and share the peptide moiety T289KPREEQFNSTFR301 carrying a glycan at N297. *, contaminant or irrelevant peak.
Figure 3
Figure 3. Schematic representation of the passive-immune thrombocytopenia mouse model.
Figure 4
Figure 4. Measured platelet count at t = 16 hours of the control groups.
Results are depicted as mean with error bars representing standard error of mean (SEM), (n = 10–12 mice per group). At t = −24 h, platelet counts were 913±52*109, 915±28*109, and 867±38*109 cells/L, respectively.
Figure 5
Figure 5. Measured platelet count at t = 16 hours after IVIg pre-treatment.
Pre-treatment with A) a high dose (1.5 g/kg) of IVIg, IVIg-SA (−), IVIg-SA (+), or saline. B) a low dose (0.3 g/kg) of IVIg, IVIg-SA (−) IVIg-SA (+), or saline. Results are depicted as mean with error bars representing standard error of mean (SEM), (n = 10–12 mice per group). * P<0.01.
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