Development of a competitive enzyme-linked immunosorbent assay (ELISA) for Escherichia coli heat-stable enterotoxin (STa)

A sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the low molecular weight heat-stable enterotoxin (ST_a) in culture supernatant fluids of enterotoxigenic Escherichia coli (ETEC). Competitive inhibition was observed between ST_a in solution and a glutaraldehyde-coupled ST_a-human serum albumin (HSA) conjugate bound to microtiter wells when antiserum raised against a glutaraldehyde-coupled ST_a-bovine serum albumin (BSA) conjugate was used as detecting antibody. No competition was observed with conjugates prepared using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or dimethyl suberimidate and antisera raised against each conjugate. A biotin/avidin system increased the sensitivity of the assay such that 133 pg/ml of purified ST_a can be detected in less than 4 h. The assay was used to detect and quantify ST_a in culture supernatant fluids from human, porcine, and bovine ETEC isolates. No cross-reactivity was observed with the heat-labile enterotoxin (LT) or the form of ST with biological activity only in piglets (ST_a). Results from the quantitative ST_a ELISA showed good correlation (0.87) with the suckling mouse bioassay and a previously described radioimmunoassay. The quantitative assay was modified to reduce the total incubation time to less than 2 h. The qualitative ST_a ELISA provides a rapid and sensitive assay for clinical isolates of ETEC and should facilitate epidemiological studies on the incidence of ST_a-producing ETEC.