Immunization of Mice with Hib Conjugate

Mice were anesthetized with isoflurane (FUJIFILM Wako Pure Chemical, Osaka, Japan) and then subcutaneously immunized with a total volume of 200 μl PBS containing either 0.01 μg of the Hib capsular polysaccharide PRP, 0.01 μg of PRP plus 0.024 μg of tetanus toxoid (TT) (EMD Millipore, Burlington, MA, United States), Haemophilus B PRP–TT conjugate vaccine (ActHIB; Sanofi, Tokyo, Japan) equivalent to 0.01 μg of PRP or 1 μg of PRP with or without 1 μg of Alcaligenes lipid A (Wang et al., 2020 (link)), or PBS only. Mice received three immunizations at 1-week intervals. One week after the final immunization, blood was harvested from the mice and kept on ice until centrifuged at 4°C, 3,000 × g for 10 min. The serum was transferred into a fresh tube and stored at −80°C.