Identification of Escherichia coli that produces heat-stable enterotoxin STA by a commercially available enzyme-linked immunoassay and comparison of the assay with infant mouse and DNA probe tests

Abstract

By using the infant mouse test and a commercially available competitive enzyme-linked immunosorbent assay (ELISA), 100 strains of Escherichia coli carrying STAI or STA,2 genes were shown to produce the heat-stable enterotoxin STA. An additional 100 strains were negative in both tests. The ELISA was easy to perform, and results were available within 24 h. Testing strains with an enzyme-linked DNA probe kit that incorporated both STAland STA2-specific oligonucleotides showed that the 100 strains positive in the mouse test and ELISA also hybridized with the mixed probe. Two strains carrying STAl genes but negative in the mouse test and ELISA also hybridized with the mixed probe.

Comparison of a competitive enzyme immunoassay kit and the infant mouse assay for detecting Escherichia coli heat-stable enterotoxin

“…The EIA did not detect STa in any of the infant mousenegative strains, thus indicating the specifity of this assay. A similar observation on the specificity of the EIA kit was obtained with 200 E. coli strains, half of which were STa producers (Scotland et al 1989). In our study, the EIA detected STa in all mouse-positive strains, but identical titres were obtained for two strains only (both of low titre), while for 29 strains titres were 2to 64-fold lower.…”

Differentiation of pathogenic Escherichia coli strains in Brazilian children by PCR

“…The commercially available competitive EIA for the detection of STa did not detect STa production by any of our STa-PCR-positive strains. This may reflect an increased sensitivity of the PCR compared with that of the EIA or may indicate that the toxin was not expressed under in vitro conditions (1,9,15,20,22). This is supported by the observation that 7 of 11 STa-PCR-positive strains were also detected by DNA hybridization (three strains were not viable).…”

Contribution of defined amino acid residues to the immunogenicity of recombinant Escherichia coli heat-stable enterotoxin fusion proteins

“…Detection, antigenicity and quanti¢cation of STh in the fusions were determined by means of a competitive enzyme-linked immunosorbent assay (ELISA) using the commercially available assay kit for E. coli STa (COLI ST EIA) produced by Denka Seiken Co., Ltd., Tokyo, Japan [16] including microtiter plate-attached STa as solid-phase antigen and peroxidase-conjugated STa-mono-speci¢c antibody as secondary antibody [12,13], and by means of a double antibody sandwich ELISA using mouse anti-STa monoclonal antibody as coating agent, rabbit anti-ClpG polyclonal antibody as secondary antibody and goat anti-rabbit peroxidase-conjugated IgG as tertiary antibody [13]. Detection and antigenicity were also determined by Western immunoblotting [13].…”

“…Anti-STa and anti-ClpG antibody titers in animal antisera were determined using ELISA with microtiter plates precoated with 1 Wg of puri¢ed synthetic STa [16] or with 1 Wg of puri¢ed ClpG protein [12], respectively. Sera from immunized rabbits and mice were serially diluted in PBS (pH 7.2) containing 2% skim milk and 0.5% fetal calf serum added to the wells and incubated for 2 h at 37³C.…”