Method Description

Microwells are precoated with ganglioside 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 optical density (OD) of patient sera 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 Results are reported qualitatively as positive or negative.(Unpublished Mayo method)