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Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation.

Käsermann F, Boerema DJ, Rüegsegger M, Hofmann A, Wymann S, Zuercher AW, Miescher S - PLoS ONE (2012)

Bottom Line: Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region.Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent.The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

View Article: PubMed Central - PubMed

Affiliation: Research & Development, CSL Behring AG, Bern, Switzerland. fabian.kaesermann@cslbehring.com

ABSTRACT
It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

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Specific antibody concentrations and human erythrocyte binding.A: Concentrations of antibodies against a panel of antigens. Results are expressed as % of specific concentrations measured in IVIG. RV: rubella virus, MV: measles virus, TetTox: tetanus toxoid, EBV (EBNA): Epstein-Bar virus (nuclear antigen), B19V: human parvovirus B19, Hib: Haemophilius influencae, VZV: varizella zoster virus, CMV: cytomegalovirus, EBV (VCA): Epstein-Barr virus (capsid antigen). Concentrations are normalized to IVIG and shown in linear scale (left axes). ANA: anti-nuclear antibodies, absolute titres are shown in a log2 scale (right axis). Mean values and standard deviations measured from three independently produced batches are shown. B: Binding of different IVIG fractions to erythrocytes (RBC) from donors with blood group A, B or AB have been analysed by flow cytometry. MFI: mean fluorescence intensity.
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pone-0037243-g004: Specific antibody concentrations and human erythrocyte binding.A: Concentrations of antibodies against a panel of antigens. Results are expressed as % of specific concentrations measured in IVIG. RV: rubella virus, MV: measles virus, TetTox: tetanus toxoid, EBV (EBNA): Epstein-Bar virus (nuclear antigen), B19V: human parvovirus B19, Hib: Haemophilius influencae, VZV: varizella zoster virus, CMV: cytomegalovirus, EBV (VCA): Epstein-Barr virus (capsid antigen). Concentrations are normalized to IVIG and shown in linear scale (left axes). ANA: anti-nuclear antibodies, absolute titres are shown in a log2 scale (right axis). Mean values and standard deviations measured from three independently produced batches are shown. B: Binding of different IVIG fractions to erythrocytes (RBC) from donors with blood group A, B or AB have been analysed by flow cytometry. MFI: mean fluorescence intensity.

Mentions: To address whether fractionation of IVIG by SNA chromatography might skew the specific antibody pattern, IVIG, FT and sialic acid-enriched fractions E1 and E2 were tested for binding to various antigens. As shown in Figure 4, SNA lectin chromatography modified the distribution of specific antibodies in the E1 and E2 fractions. Whereas some specificities were not or only marginally affected (Fig. 4A; TetTox, EBV (NA)) others were either decreased (Fig. 4A; RV, MV, B19V, Hib, VZV) or increased (Fig. 4A; CMV, EBV (VCA)). Similarly, testing on Hep-2 cells indicated an enrichment of specific anti-nuclear antibodies (Fig. 4A; ANA) in the E2 fraction. No differences in titres were observed with IVIG pre-incubated under the same conditions as E2 (lactose/acetic acid pH 3.5). Furthermore, the anti-CMV titre was not decreased in desialylated E2 (NAase E2), indicating that the antibody-antigen interaction was not sialic acid dependent (data not shown). Binding of IgG to human blood group A and AB erythrocytes was markedly lowered in E1 and E2 compared to unfractionated IVIG and the FT fraction (Fig. 4B), while a less pronounced effect on binding to B erythrocytes was noted. Similar results were obtained with sheep erythrocytes and less pronounced with rabbit erythrocytes (data not shown). Treatment with neuraminidase also did not influence binding of IVIG to human erythrocytes (data not shown). Overall, these findings demonstrate that the composition of specific antibodies was skewed in sialic-acid enriched IgG fractions, supporting the claim that lectin affinity chromatography fractionation of IVIG is mainly driven by Fab-interactions. Subclass distribution in the different fractions was only marginally shifted, with slightly decreased IgG2 in E2 and a modest increase of IgG4 in E1 and E2 (Fig. S2).


Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation.

Käsermann F, Boerema DJ, Rüegsegger M, Hofmann A, Wymann S, Zuercher AW, Miescher S - PLoS ONE (2012)

Specific antibody concentrations and human erythrocyte binding.A: Concentrations of antibodies against a panel of antigens. Results are expressed as % of specific concentrations measured in IVIG. RV: rubella virus, MV: measles virus, TetTox: tetanus toxoid, EBV (EBNA): Epstein-Bar virus (nuclear antigen), B19V: human parvovirus B19, Hib: Haemophilius influencae, VZV: varizella zoster virus, CMV: cytomegalovirus, EBV (VCA): Epstein-Barr virus (capsid antigen). Concentrations are normalized to IVIG and shown in linear scale (left axes). ANA: anti-nuclear antibodies, absolute titres are shown in a log2 scale (right axis). Mean values and standard deviations measured from three independently produced batches are shown. B: Binding of different IVIG fractions to erythrocytes (RBC) from donors with blood group A, B or AB have been analysed by flow cytometry. MFI: mean fluorescence intensity.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366990&req=5

pone-0037243-g004: Specific antibody concentrations and human erythrocyte binding.A: Concentrations of antibodies against a panel of antigens. Results are expressed as % of specific concentrations measured in IVIG. RV: rubella virus, MV: measles virus, TetTox: tetanus toxoid, EBV (EBNA): Epstein-Bar virus (nuclear antigen), B19V: human parvovirus B19, Hib: Haemophilius influencae, VZV: varizella zoster virus, CMV: cytomegalovirus, EBV (VCA): Epstein-Barr virus (capsid antigen). Concentrations are normalized to IVIG and shown in linear scale (left axes). ANA: anti-nuclear antibodies, absolute titres are shown in a log2 scale (right axis). Mean values and standard deviations measured from three independently produced batches are shown. B: Binding of different IVIG fractions to erythrocytes (RBC) from donors with blood group A, B or AB have been analysed by flow cytometry. MFI: mean fluorescence intensity.
Mentions: To address whether fractionation of IVIG by SNA chromatography might skew the specific antibody pattern, IVIG, FT and sialic acid-enriched fractions E1 and E2 were tested for binding to various antigens. As shown in Figure 4, SNA lectin chromatography modified the distribution of specific antibodies in the E1 and E2 fractions. Whereas some specificities were not or only marginally affected (Fig. 4A; TetTox, EBV (NA)) others were either decreased (Fig. 4A; RV, MV, B19V, Hib, VZV) or increased (Fig. 4A; CMV, EBV (VCA)). Similarly, testing on Hep-2 cells indicated an enrichment of specific anti-nuclear antibodies (Fig. 4A; ANA) in the E2 fraction. No differences in titres were observed with IVIG pre-incubated under the same conditions as E2 (lactose/acetic acid pH 3.5). Furthermore, the anti-CMV titre was not decreased in desialylated E2 (NAase E2), indicating that the antibody-antigen interaction was not sialic acid dependent (data not shown). Binding of IgG to human blood group A and AB erythrocytes was markedly lowered in E1 and E2 compared to unfractionated IVIG and the FT fraction (Fig. 4B), while a less pronounced effect on binding to B erythrocytes was noted. Similar results were obtained with sheep erythrocytes and less pronounced with rabbit erythrocytes (data not shown). Treatment with neuraminidase also did not influence binding of IVIG to human erythrocytes (data not shown). Overall, these findings demonstrate that the composition of specific antibodies was skewed in sialic-acid enriched IgG fractions, supporting the claim that lectin affinity chromatography fractionation of IVIG is mainly driven by Fab-interactions. Subclass distribution in the different fractions was only marginally shifted, with slightly decreased IgG2 in E2 and a modest increase of IgG4 in E1 and E2 (Fig. S2).

Bottom Line: Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region.Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent.The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

View Article: PubMed Central - PubMed

Affiliation: Research & Development, CSL Behring AG, Bern, Switzerland. fabian.kaesermann@cslbehring.com

ABSTRACT
It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

Show MeSH
Related in: MedlinePlus