Method Description

This assay utilizes the T-REx System (Thermo Fisher). Expression of the glycine receptor alpha-1-subunit is repressed in the absence of tetracycline or doxycycline and induced in the presence of tetracycline or doxycycline.(Yao F, Svensjo T, Winkler T, Eriksson C, Eriksson E: Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. Hum Gene Ther. 1998 Sep;9[13]:1939-1950)

HEK293 cells stably expressing the tetracycline repressor and stably transfected with a plasmid encoding the alpha-1-subunit of the glycine receptor, under control of doxycycline, are grown in wells of a chamber slide. Twenty four hours prior to the assay, the wells on the bottom half of the slide are treated with culture media including doxycycline. After 24 hours, patient CSF and/or serum will be added to the living HEK293 cells held on ice. Bound IgG will be detected using a fluorophore conjugated anti-human IgG secondary antibody. Patient samples with IgG specific for the glycine receptor will be positive on doxycycline-treated cells and negative on cells not treated with doxycycline.(Unpublished Mayo method)