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J Immunol Methods. 2019 Oct;473. pii: S0022-1759(19)30176-0. doi: 10.1016/j.jim.2019.07.004. Epub 2019 Jul 12.

Pharmacodynamic assessment of cell-bound natalizumab on PBMC samples stored in liquid nitrogen.

Author information

1
Department of Immunopathology, Sanquin Research, Amsterdam, the Netherlands; Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands.
2
Department of Neurology, Amsterdam Neuroscience, VUmc MS Center Amsterdam, VU University Medical Center, Amsterdam, the Netherlands.
3
Department of Immunopathology, Sanquin Research, Amsterdam, the Netherlands; Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands. Electronic address: t.rispens@sanquin.nl.

Abstract

Natalizumab is a monoclonal IgG4 antibody used for treatment of relapsing remitting MS. Natalizumab interferes with lymphocyte migration by blocking alpha-4 integrin (CD49d). Saturation levels of alpha-4 integrin on circulating T cells by natalizumab have been associated with clinical effectiveness of therapy. However, in most cases, measurements have been carried out using freshly isolated PBMCs. The aim of this study was to set up and evaluate a method to measure relative levels of cell-bound natalizumab using frozen PBMC samples. A new method was set up to measure cell-bound natalizumab by flow cytometry on T cell subsets using fully saturated cells as a 100% reference. A comparison was made between spike samples and samples of natalizumab-treated MS patients freshly isolated and stored in liquid nitrogen. Cell-bound natalizumab could be measured (using an anti-IgG4 antibody) on cells stored in liquid nitrogen. Natalizumab was found to slowly dissociate from the cells during isolation and subsequent sample work-up. This dissociation was more pronounced for monovalent natalizumab resulting from Fab arm exchange (the predominant isoform in patients) than bivalent natalizumab straight from the vial. We established a correction factor to account for this phenomenon. The resulting method has good accuracy compared to assessing fresh cells. The inter-assay precision (%CV) is ca. 12% using frozen cells. In conclusion, we established a method to assess relative levels of cell-bound natalizumab on cells obtained from frozen PBMC samples.

KEYWORDS:

CD49d; IgG4; Integrin; Natalizumab; Receptor saturation

PMID:
31306641
DOI:
10.1016/j.jim.2019.07.004

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