Method Description

NMO-IgG Fluorescence-Activated Cell Sorting Assay (FACS)

Human embryonic kidney cells (HEK 293) are transfected transiently with a plasmid (pIRES2-Aequorea coerulescens green fluorescent protein [AcGFP]) encoding both green fluorescent protein (GFP) and AQP4-M1. After 36 hours, the mixed population of cells (transfected expressing AQP4 on the surface and GFP in the cytoplasm and nontransfected lacking AQP4 and GFP) are harvested with short exposure to trypsin. Patient cerebrospinal fluid (CSF) is then added to cells at a 1 in 2 screening dilution. After incubation and washing, the cells are resuspended with AlexaFluor 647-conjugated goat antihuman IgG-gamma specific secondary antibody (Southern Biotech catalog No. 2040-31, 1:2000 in LCBB), held on ice, washed, fixed with 4% paraformaldehyde, and examined with flow cytometry (BD FACSCanto; Becton, Dickinson and Co). Two populations are gated on the basis of GFP expression: positive (high AQP4 expression) and negative (low or no AQP4 expression). The median Alexafluor 647 fluorescence intensity (MFI) for the AcGFP-positive population indicates relative abundance of human IgG potentially bound to AQP4 surface epitopes; MFI for the GFP-negative population indicated nonspecifically-bound IgG. The IgG binding index was calculated as the ratio of the average MFI for duplicate aliquots of each cell population: (MFI GFP positive/MFI GFP negative). We established conservative cutoff IgG binding index values of 2.00 for M1-FACS.

If FACS assay is positive at screening dilution, FACS Titer Assay is performed at an additional charge. The patient CSF is titrated to endpoint. The dilution where the IgG-binding index is greater than or equal to 2, is considered the endpoint dilution. If a patient is positive at a 1:2 dilution, but negative at 1:4, the endpoint will be reported as 2.