Novel chemiluminescent assay for staphylococcal enterotoxin B

An enzyme-linked immunosorbent assay, a horseradish peroxidase-catalyzed fluorogenic reaction, and chemiluminescence (CL) analysis have been combined to develop a sandwich ELISA for Staphylococcal enterotoxin B (SEB) using monoclonal antibodies for different epitopes of SEB. The enzyme catalyzed reaction of 3-(4-hydroxyphenyl propionate) with the urea complex of hydrogen peroxide produced a fluorescent dimer which was detected by chemiluminescence analysis. The CL response to SEB is linear in the range from 6.0 to 564 pg mL −1 ( r = 0.9993), and the detection limit is 3.3 pg mL −1 (S/N = 3). Intra- and interassay coefficients of variation are <7.0% at three concentrations (24, 96 and 384 pg mL −1 ). The method was applied to the analysis of SEB in serum, lake water and milk samples. The results compared well with those obtained by conventional ELISAs. Figure Procedures of the proposed method. A sandwich ELISA for Staphylococcal enterotoxin B (SEB) using a pair of monoclonal antibodies that recognizes different epitopes of SEB. After the ELISA procedure, PHPPA is reacted with Hydrogen peroxide-urea, with catalysis by HRP-conjugated anti-SEB, to produce PHPPA fluorescent Dimer, which is detected by TCPO chemiluminescence.