Method Description

This cell-binding assay utilizes flow cytometry to detect neurofascin 155 (NF155) IgG4 antibodies in patient sera. Briefly, a stable HEK293 cell line expressing human NF155 on the cell surface is premixed with parental HEK293 cells that do not express human NF155. The 2 cell populations are distinguished using a green fluorescent protein marker, which is only expressed in NF155 expressing cells. The mixture of cells is incubated with diluted patient sera to allow antibodies present in the sample to bind target antigens. Next the cells are incubated with a human IgG4 specific secondary antibody conjugated to AlexaFluor 647 to detect cell bound human IgG4 antibodies. The AlexaFluor 647 signal intensity of the different cell populations is measured using a flow cytometer. The IBI (IgG binding index) is then calculated as the median fluorescent intensity (MFI) of AlexaFluor 647 of the NF155 expressing cells divided by the MFI of the parental non-NF155 expressing cells. When the IBI is greater than or equal to 2.0 the result is considered positive for NF155 IgG4 antibodies.(Unpublished Mayo method)