Abstract:A double-antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) method was developed for detection of a new staphylococcal enterotoxin P (SEP). The experimental conditions were evaluated through the index of sensitivity, intra-assay variation, inter-assay variation and spike recovery by optimizing the concentration of monoclonal antibody and antiserum, the horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (IgG/HRP), different types of buffer, blocking time, antigen incubation time, IgG / HRP incubation time, and TMB chromogenic time. Our results were as follows: the optimal concentration of anti-SEP monoclonal antibody 3.28 μg/mL, the concentration of anti-SEI rabbit serum 3.14 μg/mL, the dilution of enzyme-labeled secondary antibody 1: 3000,the blocking time 1 h, the antigen incubation time 90 min, the secondary antibody incubation time 30 min and the TMB chromogenic time 15 min. And 1 × phosphate buffer solution (pH 7.4) was shown as the best buffer. The regression equation of this method was y=0.0313x+0.0262 with R2of 0.9946. The sensitivity of the developed method was 1.80 μg/mL. The precision within the batch variation was less than 6 %, and inter-assay variation was less than 15 %. The recovery rates for LB broth, beef and cooked rice were more than 90%. Thus, this study successfully established a DAS-ELISA method for rapid detection of newly identified staphylococcal enterotoxin P.
