Method Description

Indirect Immunofluorescence Assay:

The patient's sample is tested by a standardized immunofluorescence assay 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. Neurol Neuroimmunol Neuroinflamm. 2017;4[5]:e385. doi:10.1212/NXI.0000000000000385)

Flow Cytometry:

This assay is a live cell-based binding assay using flow cytometry to detect ganglionic acetylcholine receptor (AChR) binding antibodies in patient serum. Human embryonic kidney cells (HEK293) transiently expressing human alpha-3 beta-4 ganglionic AChR subunits, along with accessory proteins required for receptor expression, are used as the antigen source. Receptor expression is enhanced prior to analysis under controlled culture conditions. Cells are incubated with diluted patient serum to allow binding of AChR-specific antibodies to surface-expressed receptors. Bound human IgG is detected using a fluorophore-conjugated anti-human IgG secondary antibody, with a viability dye included to exclude non-viable cells. Fluorescence intensity is measured by flow cytometry, and antibody binding is assessed by comparing signal from AChR-expressing cells to control cells lacking receptor expression. A humanized AChR-specific monoclonal antibody is used as an assay calibrator. The IgG Binding Index (IBI) of patient samples is compared to the assay calibrator to calculate a Cut-off Index (COI). Patient samples with a COI = 1.0 are reported as positive. Positive samples are further titrated to determine the end-point dilution, defined as the highest serum dilution that remains above the calibrator.(Unpublished Mayo method)

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 for ANNA-1 and ANNA-2:

All steps are performed at ambient temperature (18-28° C) utilizing the EUROBlot One instrument. Diluted patient sample is added to test strips (strips containing recombinant antigen manufactured and purified using biochemical methods) in individual channels and incubated for 30 minutes. Positive samples will bind to the purified recombinant antigen and negative samples will not bind. Strips are washed to remove unbound antibodies and then incubated with antihuman IgG antibodies (alkaline phosphatase-labeled) for 30 minutes. The strips are again washed to remove unbound antihuman IgG antibodies and nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate is added. Alkaline phosphatase enzyme converts the soluble substrate into a colored insoluble product on the membrane to produce 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)

Immunoblot for MAP1B:

Test strips are incubated with appropriately diluted sample, prepared to meet the assay's required end-dilution for each specimen type. Samples and controls are added directly to the strips, ensuring they are fully covered with dilution buffer, then mixed gently and incubated for 60 minutes at room temperature on a rocking platform. After incubation, the strips undergo a series of wash steps using diluted wash buffer, followed by a 30-minute incubation with a ready-to-use alkaline phosphatase-labeled IgG conjugate. The strips are washed again using the same procedure, then incubated with a ready-to-use substrate solution for 20 minutes at room temperature. The reaction is stopped by rinsing with distilled water through several wash cycles. Strips are then placed on absorbent material and allowed to dry fully, generally requiring at least two hours. Results are interpreted only after the strips have dried completely, and dried strips are stored protected from light.(Package insert: PCA-2 line assay. ravo Diagnostika GmbH; 05/2023

Cell-Binding Assay:

Patient sample is applied to a composite slide containing transfected and nontransfected cells. After incubation and washing, fluorescein-conjugated goat-antihuman IgG is applied to detect the presence of patient IgG binding.(Klein CJ, Lennon VA, Aston PA, et al. Insights From LGI1 and CASPR2 potassium channel complex autoantibody subtyping. JAMA Neurol 2013;70[2]:229-234; package insert: IIFT: Neurology Mosaics, Instructions for the indirect immunofluorescence test. EUROIMMUN; FA_112d-1_A_UK_C13, 02/2019)