Application of a timing protocol to the reduction of inter-plate variability in the indirect enzyme immunoassay for detection of anti-Brucella antibody.

A simple timing protocol was developed to monitor chromogen conversion in an enzyme immunoassay, performed in microtiter plates, for the detection of antibody to Brucella abortus in bovine serum. Application of this protocol decreased the inter-plate coefficient of variation from 28.6% to 6.8% when optical density (OD) values, subsequent to the reaction of a standard antibody reagent, were compared to a static development time. Substantial reductions in variation were also observed for low titered seropositive and for seronegative control reagents. The timing protocol was based on the mathematical relationship of the OD value at 4 minutes of development to a predetermined target OD value (1.0) for a standard antibody reagent. Application of this relationship to the calculation of a variable, final development time eliminated the need for extensive data manipulation and assay calibration.