An improved Fc function assay utilizing CMV antigen-coated red blood cells generated with synthetic function–spacer–lipid constructs
Article first published online: 12 JUL 2011
© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion
Volume 102, Issue 1, pages 72–78, January 2012
How to Cite
Georgakopoulos, T., Komarraju, S., Henry, S. and Bertolini, J. (2012), An improved Fc function assay utilizing CMV antigen-coated red blood cells generated with synthetic function–spacer–lipid constructs. Vox Sanguinis, 102: 72–78. doi: 10.1111/j.1423-0410.2011.01512.x
- Issue published online: 26 DEC 2011
- Article first published online: 12 JUL 2011
- Received: 3 March 2011, revised 5 May 2011, accepted 6 May 2011, published online 12 July 2011
- Fc function;
Background and Objectives The Fc function assessment of immunoglobulin products is performed to confirm that the biological activity of immunoglobulin products is not affected by manufacturing and storage conditions. The European Pharmacopoeia (EP) test is cumbersome and time-consuming especially with respect to the derivatization of red blood cells (RBC) with rubella antigen via tanninization. We investigated the KODE biosurface engineering technology for inserting specific antigens into red blood cell membranes to create kodecytes and evaluated their suitability for use in the Fc function assay.
Materials and Methods Human RBC were derivatized with function–spacer–lipid (FSL) constructs comprising of a peptide epitope of a cytomegalovirus (CMV) surface protein, conjugated to a spacer and lipid tail. These kodecytes were used in an Fc function assay based on the monitoring of complement-mediated haemolysis of immunoglobulin-coated red cells. Optimization of FSL construct, immunoglobulin and complement concentration was undertaken. Fc values obtained for various immunoglobulin batches were compared with those from the EP-based method. Carbohydrate-bearing FSLs Galili (Galα1-3Galβ1-4GlcNAcβ), Galα1-4GlcNAcβ and GalNAcα1-3Galβ were also evaluated.
Results Fc function indices determined with CMV peptide construct–coated red cells were comparable to those obtained with the EP-based method with respect to specificity and precision. Carbohydrate-bearing FSLs revealed Fc indices lower than expected.
Conclusion The use of CMV kodecytes was shown to be a convenient means of generating red cells for the determination of Fc function of immunoglobulin products and offers the possibility of significantly reducing the time required to perform this assay.