Design and characterization of highly immunogenic heat-stable enterotoxin of enterotoxigenic Escherichia coli K99+

In this study, STa peptide of enterotoxigenic Escherichia coli K99+ was purified and successfully covalently cross-linked to modified bovine serum albumin after thorough evaluation of three different hapten-carrier conjugation protocols. Dimethyformamide (DMF) based STa-conjugation protocol demonstrated higher biological activity (10 × 106 STa Total Mouse Units [MU]) and 100% conjugation efficiency. A range of conjugation ratio of 4–12 STa molecules per one molecule of BSA was achieved and confirmed by matrix-assisted laser desorption ionization-time of flight/mass spectroscopy (MALDI-TOF/MS). This conjugate was used for immunization of ten rabbits for STa antibody production. A high antibody binding titer (106) against STa was obtained with a neutralization capacity of 3 × 104 STa MUs/ml serum. These levels of high STa binding and neutralizing antibodies titers propose the potential use of this conjugate for the development of immunotherapeutic reagents and/or STa-based vaccine against ETEC K99+.