Peptide-Carrier Conjugate Immunization

BALB/c mice (female, 4–6 weeks) were immunised intramuscularly on day 0, 21 and 28 with 30 μg of peptide-DT or peptide-CRM conjugates formulated in alhydrogel (Alum, Brenntag Biosector, Denmark) in 1:1 ratio (v/v), as previously described^{25 (link)}. For the combination vaccine, 30 μg of each conjugate was admixed in a ratio of 1:1 by weight and formulated in Alum as previously described^{9 (link)}. Control mice received saline with adjuvant alone. For carrier priming experiments, mice were immunised three times with DT and then rested for 4 weeks before they received three immunisations with MJ8CombiVax. A parallel cohort of naïve mice was also vaccinated with MJ8CombiVax. In all experiments, one-week post last immunisation mice were bled via submandibular vein and serum isolated. The serum samples were stored at −20 °C until used. ELISA was used to determine murine Ag-specific IgG antibody titers as previously described^{34 (link)}. Goat anti-mouse IgG-HRP (Bio-Rad laboratories) was used to determine Ab titers. Titre was defined as the highest dilution that gave an optical density (OD) reading of more than three standard deviations (SD) above the mean OD of control wells containing normal mouse sera at the same dilution^{34 (link)}.