Method Description

Cell-Binding Assay:

Patient specimen is applied to a composite slide containing transfected and nontransfected EU90 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, 02/25/2019)

Indirect Immunofluorescence Assay:

The patient's specimen is tested by a standardized immunofluorescence assay that uses a composite frozen section of mouse cerebellum, kidney, and gut tissues. After incubation with the specimen and washing, fluorescein-conjugated goat-antihuman IgG is applied. Neuron-specific autoantibodies are identified by their characteristic fluorescence staining patterns. Specimens that are scored positive for any neuronal nuclear or cytoplasmic autoantibody are titrated. 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. Published 2017 Jul 18. 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 specimen.(Griesmann GE, Kryzer TJ, Lennon VA. Autoantibody profiles of myasthenia gravis and Lambert-Eaton myasthenic syndrome. In: Rose NR, Hamilton RG, eds. Manual of Clinical and Laboratory Immunology. 6th ed. ASM Press; 2002:1005-1012; Walikonis JE, Lennon VA. Radioimmunoassay for glutamic acid decarboxylase [GAD65] autoantibodies as a diagnostic aid for stiff-man syndrome and a correlate of susceptibility to type 1 diabetes mellitus. Mayo Clin Proc. 1998;73[12]:1161-1166; 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)

Enzyme-Linked Immunosorbent Assay:

Ma2 antibodies are directed against their corresponding intracellular protein (PNMA2). The Ma2 autoantibody enzyme-linked immunosorbent assay (ELISA) is based on the principle of indirect ELISA that employs the ability of Ma2 autoantibodies to bind to antigenic protein (PNMA2) coated on the well surface of an ELISA plate. Detection of this bound antibody is accomplished using an antihuman secondary antibody that specifically recognizes the Fc region of the bound autoantibody. The secondary antibody is conjugated with alkaline phosphatase that enzymatically hydrolyzes a substrate molecule to produce an end product with a yellow color measurable at a wavelength of 405 nm. Controls and diluted samples are added in duplicate to coated plate wells and incubated for 2 hours, then washed extensively before adding the conjugated secondary antibody for an additional hour. After aspiration and washing, p-nitrophenyl phosphate substrate is added to each well and incubated for an additional hour before absorbance is measured at 405 nm.(Unpublished Mayo method)

Immunoblot for AGNA-1, amphiphysin, ANNA-1 and ANNA-2:

All steps are performed at room temperature (18 to 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 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 (NBT/BCIP) 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)

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)