Method Description

Cell-Binding Assay

Patient sample 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.(Package insert: IIFT: Neurology Mosaics, Instructions for the indirect immunofluorescence test. EUROIMMUN; FA_112d-1_A_UK_C13, 02/25/2019)

Ganglioside Antibodies: IgG/IgM Disialo, GD1b; IgG/IgM Monos, GM1 Enzyme-Linked Immunosorbent Assays:

Antiganglioside antibodies are detected by enzyme-linked immunosorbent assays (ELISA). Microwells are precoated with GM1 or GD1b antigen. The calibrator, controls, and diluted patient samples are added to the wells, and autoantibodies recognizing GM1 or GD1b bind during the first incubation. After washing the wells to remove all unbound proteins, purified alkaline phosphatase-conjugated antihuman IgG or IgM is added. The conjugated secondary IgG or IgM binds to the captured human autoantibody, and the excess unbound conjugated IgG or IgM is removed by a further wash step. The bound conjugated IgG or IgM is visualized with 4-nitrophenyl phosphate substrate, which gives a yellow reaction product, the intensity of which is proportional to a concentration of autoantibody in the sample. A base is added to each well to stop the reaction, and the final product is read at 405 nm. For screening assays, patient results are calculated by dividing the optical density (OD) of patient samples or controls by the average OD of the calibrator. Any sample with a ratio of patient to calibrator OD greater than 2.0 is considered positive. Any positive sample on screening is then titered. For titer assays, patient samples are diluted, and the last dilution where the ratio of patient to calibrator OD is greater than 2.0 is reported as the end-point titer.(Unpublished Mayo method)

Ganglioside GQ1b Antibody, IgG ELISA:

Microwells are precoated with GQ1b antigen. The calibrator, controls, and diluted patient samples are added to the wells, and autoantibodies recognizing GQ1b bind during the first incubation. After washing the wells to remove all unbound proteins, purified horseradish peroxidase-labeled anti-human IgG conjugate is added. The conjugated IgG binds to the captured human autoantibody, and the excess unbound conjugated IgG is removed by a further wash step. The bound conjugated IgG is visualized with 3,3',5,5'-tetramethylbenzidine substrate, which gives a blue reaction product, the intensity of which is proportional to a concentration of autoantibody in the sample. Acid is added to each well to stop the reaction. This produces a yellow end-product color, which is read at 450 nm. Patient results are calculated as a cutoff index (COI) by dividing the OD of patient samples or controls by the average OD of the calibrator. Any sample with a COI greater than or equal to 1.0 is considered positive. Any sample with a COI less than 1.0 is considered negative. Results are reported qualitatively as positive or negative.(Unpublished Mayo method)

Myelin-Associated Glycoprotein Autoantibodies, IgM ELISA:

Microwells are precoated with human myelin-associated glycoprotein (MAG) antigen. The calibrators, controls, and diluted patient samples are added to the wells, and autoantibodies recognizing the MAG antigen bind during the first incubation. After washing the wells to remove all unbound proteins, purified horseradish peroxidase-labeled anti-human IgM conjugate is added. The secondary antibody conjugate binds to captured human autoantibody, and the excess unbound conjugate is removed by a further wash step. The bound secondary antibody conjugate undergoes an enzymatic reaction with a chemical substrate to generate a colormetric reaction. Absorbance is measured on a plate reader and patient sample signal is compared to a calibration curve to generate Buhlmann titer units.(Package insert: anti-MAG Autoantibodies ELISA. Buhlmann Laboratories AG; 10/2018)

Flow Cytometry

This cell-binding assay utilizes flow cytometry to detect neurofascin 155 (NF155) IgG4 antibodies in patient sample. Briefly, a stable HEK293 cell line expressing human NF155 on the cell surface is premixed with parental HEK293 cells that do not express human NF155. The 2 cell populations are distinguished using a green fluorescent protein marker, which is only expressed in NF155 expressing cells. The mixture of cells is incubated with diluted patient sera to allow antibodies present in the sample to bind target antigens. Next the cells are incubated with a human IgG4 specific secondary antibody conjugated to AlexaFluor 647 to detect cell bound human IgG4 antibodies. The AlexaFluor 647 signal intensity of the different cell populations is measured using a flow cytometer. The IBI (IgG binding index) is then calculated as the median fluorescent intensity (MFI) of AlexaFluor 647 of the NF155 expressing cells divided by the MFI of the parental non-NF155 expressing cells. When the IBI is greater than or equal to 2.0 the result is considered positive for NF155 IgG4 antibodies.(Unpublished Mayo method)

Immunoblot

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)

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)

Western Blot

Western blot is performed on denatured full-length recombinant human collapsin response-mediator protein-5 (CRMP-5), reduced and subjected to 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)