Flow–Cytometric Method for the Quantitation of the Fc Function of Intravenous Immunoglobulin Preparations

Abstract

Objectives: We have developed and optimised a new flow–cytometric method for the measurement of the Fc function of intravenous immunoglobulin (IVIg) preparations, which is important in predicting the effector function of immunoglobulin (Ig) in such preparations. Materials and Methods: Ig was bound to a monocytic cell line, THP–1 with FcγRI and FcγRII cell surface receptors, and the bound Ig detected by FITC–conjugated F(ab)₂ fragment of rabbit anti–human IgG. Results: Validation studies showed that Ig bound to the cell line through the Fc portion. The method detected alterations in Fc function caused by reduction with dithiothreitol or by storage. The method was reproducible (CV<11%) and a limited comparison study showed that it correlated with the European Pharmacopoeia reference method. Conclusions: This technically simple method is suitable for the quantitation of the Fc function of Ig preparations.

Effect of Methylene Blue Pathogen Inactivation on the Integrity of Immunoglobulin M and G

“…Beside antigen binding, the integrity of the Fc part of IgG is important for its biological efficacy, especially when antibodies are needed for defense against pathogens. To test the integrity of the Fc part of IgG we used binding of IgG to THP-1 cells expressing Fc receptors [14]. This method has been shown to be equivalent to the European Pharmacopoeia method 2.7.9 (test for Fc binding of immunoglobulin) [16], and has been used for quality control of IVIG preparations.…”

“…The influence of MB treatment on IgG-Fc binding to Fc receptors on THP-1 cells was assessed by flow cytometry (Cytomics FC 500, Beckmann Coulter Inc., Miami Lakes, USA). IVIG (Privigen ® , CSL Behring GmbH, Hattersheim am Main, Germany) was used as positive control and buffer as negative control, as previously described [14]. In brief, 100 µL (1 × 10 5 ) of THP-1 cells were incubated (30 min; 4 ° C) with diluted (1: 1,000 to 1: 64,000) plasma samples or controls, washed and stained with 100 µL of FITC-rabbit anti-Human IgG-F(ab')2 fragment (Agilent, Santa Clara, USA, 1: 75 in PBS/BSA; 30 min), washed twice and resuspended in 500 µL of PBS/BSA and assessed by forward-sideward scatter gating.…”

Fc function of a new intravenous immunoglobulin product: IGIV 10% triple virally inactivated solution

“…Ramasamy et al . [6] published a flow‐cytometric method for the quantification of the Fc‐binding function of IgG preparations. The investigators measured the binding of IgG to Fc receptors of THP‐1 cells and compared the results of their flow‐cytometric method with the results obtained with the EP reference method.…”

“…Recently, Ramasamy et al . published a flow‐cytometric method for the quantification of the Fc‐binding function of IVIG preparations that measures the binding of IVIG to Fc receptors of the human monocytic cell line THP‐1 [6]. This binding assay has the advantage that it detects the biological activity of the whole population of IgG molecules present in IVIG independently of their antigen specificity.…”