Method Description

The method used to detect antibodies directed against filamentous-actin is enzyme-linked immunosorbent assay. Prediluted controls and diluted patient sera are added to separate wells, allowing any actin antibodies present to bind to the antigen. Unbound sample is washed away, and an enzyme labeled anti-human IgG is added to each well. A second incubation allows the enzyme labeled anti-human IgG to bind to any patient antibodies, which have become attached to the microwells, and any unbound conjugate is removed by another wash step. The bound conjugate is visualized with 3,3',5,5' tetramethylbenzidine (TMB) substrate, which gives a blue reaction product, the intensity of which is proportional to a concentration of autoantibody in the sample. Sulfuric acid is added to each well to stop the reaction. This produces a yellow endpoint color, which is read at 450 nm. Testing is performed on the DS2 instrument by Dynex.(Package insert: QUANTA Lite Actin IgG ELISA 708785. INOVA Diagnostics; Rev. 6, 11/2018)