Method Description

Cell-Binding Assay:

Patient specimen is applied to a composite slide containing transfected and nontransfected HEK-293 cells. After incubation and washing, fluorescein-conjugated goat-antihuman IgG is applied to detect the presence of patient IgG binding.(Package insert: IIFT: Neurology Mosaics, Instructions for the indirect immunofluorescence test. EUROIMMUN; FA_112d-1_A_UK_C13, 02/2019)

Methodology for detecting Kelch-like protein 11 (KLHL11)-IgG uses an in-house developed cell binding assay (CBA) with confirmation by a tissue indirect immunofluorescence assay (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 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 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 Images will be scored positive or negative.(Unpublished Mayo method)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:

The patient's sample is tested by a standardized IFA that uses a composite frozen section of mouse cerebellum, kidney, and gut tissues. After incubation with sample and washing, fluorescein-conjugated goat-antihuman IgG is applied. Neuron-specific autoantibodies are identified by their characteristic fluorescence staining patterns. Samples that are scored positive for any neuronal nuclear or cytoplasmic autoantibody are titrated to an endpoint. Interference by coexisting non-neuron-specific autoantibodies can usually be eliminated by serologic absorption.(Honorat JA, Komorowski L, Josephs KA, et al. IgLON5 antibody: neurological accompaniments and outcomes in 20 patients. Neruol Neruoimmunol Neruoinflamm. 2017;4(5):e385. doi:10.1212/NXI.0000000000000385)

Radioimmunoassay:

(125)I-labeled recombinant human antigens or labeled receptors are incubated with patient specimen. After incubation, anti-human IgG is added to form an immunoprecipitate. The amount of (125)I-labeled antigen in the immunoprecipitate is measured using a gamma-counter. The amount of gamma emission in the precipitate is proportional to the amount of antigen-specific IgG in the specimen. Results are reported as units of precipitated antigen (nmol) per liter of patient sample.(Griesmann GE, Kryzer TJ, Lennon VA. Autoantibody profiles of myasthenia gravis and Lambert-Eaton myasthenic syndrome. In: Rose NR, Hamilton RG, et al. eds. Manual of Clinical and Laboratory Immunology. 6th ed. ASM Press: 2002:1005-1012; Jones AL, Flanagan EP, Pittock SJ, et al. Responses to and outcomes of treatment of autoimmune cerebellar ataxia in adults. JAMA Neurol. 2015;72[11]:1304-1312. doi:10.1001/jamaneurol.2015.2378)

Western Blot:

Neuronal antigens extracted aqueously from adult rat cerebellum, full-length recombinant human collapsin response-mediator protein-5 (CRMP-5), or full-length recombinant human amphiphysin protein is denatured, reduced, and separated by electrophoresis on 10% polyacrylamide gel. IgG is detected autoradiographically by enhanced chemiluminescence.(Yu Z, Kryzer TJ, Griesmann GE, et al. CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity. Ann Neurol. 2001;49[2]:146-154; Dubey D, Jitprapaikulsan J, Bi H, et al. Amphiphysin-IgG autoimmune neuropathy: A recognizable clinicopathologic syndrome. Neurology. 2019;93(20):e1873-e1880. doi:10.1212/WNL.0000000000008472)

Immunoblot:

All steps are performed at room temperature (18-28° C) utilizing the EUROBlot One instrument. Diluted patient specimen (1:12.5) is added to test strips (strips containing recombinant antigen manufactured and purified using biochemical methods) in individual channels and incubated for 30 minutes. Positive specimens will bind to the purified recombinant antigen and negative specimens will not bind. Strips are washed to remove unbound antibodies and then incubated with anti-human IgG antibodies (alkaline phosphatase-labelled) for 30 minutes. The strips are again washed to remove unbound anti-human IgG antibodies and nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate substrate is added. Alkaline phosphatase enzyme converts the soluble substrate into a colored insoluble product on the membrane to produces a black band. Strips are digitized via picture capture on the EUROBlot One instrument and evaluated with the EUROLineScan software.(O'Connor K, Waters P, Komorowski L, et al. GABAA receptor autoimmunity: A multicenter experience. Neurol Neuroimmunol Neuroinflamm. 2019;6[3]:e552 doi:10.1212/NXI.0000000000000552)