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Updated: Aug 10, 2026
IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Published on: December 2, 2010
Flow-cytometric method for the quantitation of the Fc function of intravenous immunoglobulin preparations
I Ramasamy1, E Tran, A Charnock
1Molecular Biology Section, Therapeutic Goods Administration Laboratories, Woden, Australia. indra.ramasamy@health.gov.au
Insights
A new flow-cytometry method accurately measures the Fc function of intravenous immunoglobulin (IVIg) preparations. This technique is crucial for predicting immunoglobulin (Ig) efficacy and is reproducible for quality control.
Area of Science:
- Immunology
- Biotechnology
- Analytical Chemistry
Background:
- Intravenous immunoglobulin (IVIg) preparations are vital therapeutics.
- Measuring the Fc function of IVIg is critical for predicting its efficacy.
- Existing methods for Fc function assessment can be complex or lack sensitivity.
Purpose of the Study:
- To develop and optimize a novel flow-cytometric assay for quantifying IVIg Fc function.
- To establish a reproducible and reliable method for IVIg quality control.
Main Methods:
- Utilized a monocytic cell line (THP-1) expressing Fc gammaRI and Fc gammaRII receptors.
- Bound immunoglobulin (Ig) to the cell line via its Fc portion.
- Detected bound Ig using FITC-conjugated F(ab)2 fragment of rabbit anti-human IgG.
Main Results:
- Validation confirmed Ig binding through the Fc portion.
- The method successfully detected changes in Fc function due to dithiothreitol treatment or storage.
- Assay reproducibility was demonstrated with a coefficient of variation (CV) below 11%.
- Preliminary comparisons showed good correlation with the European Pharmacopoeia reference method.
Conclusions:
- The developed flow-cytometric method offers a technically simple approach for quantifying IVIg Fc function.
- This assay is suitable for routine quality control of IVIg preparations.
- The method aids in predicting the effector function of immunoglobulin in therapeutic preparations.
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 Fc gammaRI and Fc gammaRII 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.
