Elimination of shrimp endogenous alkaline phosphatase background and development of enzyme immunoassays for the detection of white spot syndrome virus (WSSV)

Enzyme-linked immunosorbent assay (ELISA) and immunodot blot assay, using monoclonal antibodies against white spot syndrome virus (WSSV) as primary antibody, and goat anti-mouse Ig serum conjugated with alkaline phosphatase (AP) as secondary antibody, were developed to detect WSSV in shrimp tissue samples. However, false-positive results were obtained and the cause was considered to be reaction of endogenous shrimp AP with ELISA and immunodot blot substrates. Four AP inhibitors were tested at different concentrations and treatment times to solve this problem. EDTA, NaHSO3, levamisole and HEPES-Na inhibited AP activity by 96.45%, 86.82%, 68.82% and 9%, respectively. In conclusion, sample pretreatment with 0.5 M EDTA eliminated shrimp endogenous AP background in ELISA and immunodot blot assays, where AP was used for detection. (C) 2004 Elsevier B.V. All rights reserved.