Method Description

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and the last dilution where the ratio of patient to calibrator OD greater than 2.0, is reported as the end-point titer.(Unpublished Mayo method)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 to1.0 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)