Development of a radioimmunoassay for Escherichia coli heat-stable enterotoxin: comparison with the suckling mouse bioassay

Abstract

Escherichia coli strains which produce heat-stable enterotoxin (ST) are usually identified by demonstrating the production of ST. At present, ST can be detected only by bioassay methods. Recently, we purified E. coli ST, which enabled us to develop a radioimmunoassay for ST. Radioiodination of ST was performed by the lactoperoxidase method, which resulted in a high specific activity and retained the biological activity of St. Anti-ST antisera were raised in goats by injecting the goats with pure ST coupled to bovine immunoglobin G. Antibody titers ranged from 1:8,000 to 1:40,000. Using these reagents, we examined assay conditions thoroughly and found that a 14- to 18-h incubation at 4 degrees C in sodium acetate buffer with an ionic strength of 120 mM (pH 6.2) gave maximal sensitivity and reproducibility. Free ST was separated from antibody-bound ST by dextran-coated charcoal. This radioimmunoassay accurately and reproducibly measured ST in the range from 50 to 500 pg of ST per tube and could quantitate ST accurately in complex bacteriological media. This assay was specific for STa, measured human and porcine STa equally well, and did not cross-react with STb, with several other enterotoxins, or with various gastrointestinal peptides. Intact disulfide bridges in the ST molecule were required for immunoreactive activity.