Method Description

The JOINTstat 14-3-3 eta enzyme-linked immunosorbent assay is an immunoassay that uses a microtiter plate coated with a murine monoclonal antibody specific for human 14-3-3 eta, a horseradish peroxidase (HRP)-conjugated monoclonal antibody, and a chromogenic substrate. When standards, diluted controls, and diluted patient specimens are added to the coated wells, any 14-3-3 eta present binds to the immobilized antibody. After an incubation period, the wells are washed to remove material that did not bind. An HRP-conjugated anti-14-3-3 eta antibody is then added. This conjugate binds to the captured 14-3-3 eta, forming a detectable complex. Following incubation, another wash cycle removes unbound conjugate. A chromogenic substrate is introduced, and the HRP enzyme converts the substrate into a colored product that initially appears blue. When the reaction is stopped, the color changes to yellow. The absorbance at 450 nm is measured and is proportional to the concentration of 14-3-3 eta present in the standards, controls, and patient samples.(Unpublished Mayo method)