Method Description

Cell Binding Assay:

Methodology for detecting Kelch-like protein 11 (KLHL11)-IgG uses a cell binding assay (CBA) with confirmation by tissue immunofluorescence (IFA). The CBA utilizes HEK293 cells that are stably transfected with DNA encoding the KLHL11 protein that has been tagged with green fluorescent protein (GFP). Since KLHL11 is localized to cytoplasmic vesicles when ectopically expressed, cells will be fixed and permeabilized prior to exposure to patient sample. Patients that are positive for KLHL11-IgG will have human IgG bound to the transfected cells. Binding will colocalize with the GFP-tagged KLHL11 protein in cytoplasmic vesicles. Patient IgG will be detected using a tetramethylrhodamine (TRITC)-conjugated anti-human secondary antibody. The negative samples will not bind to KLHL11-GFP in transfected cells. Performed in a 96 well plate format, the plates are scanned, and the images saved using the ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices). Images will be scored positive or negative.(Unpublished Mayo method)

Indirect Immunofluorescence Assay:

Tissue IFA utilizes mouse composite slides including brain, kidney, and stomach tissue sections, which are commercially purchased. After tissue sections are fixed and permeabilized, patient sample is added to the well. After washing with phosphate buffer saline, bound human IgG is detected with a fluorescent conjugated secondary antibody targeting human IgG. Slides are read under a fluorescent microscope for the unique tissue-specific staining pattern characteristic of KLHL11-IgG.(Unpublished Mayo method)