Protocol to measure the impact of Clostridioides difficile toxins on antibody responses using ELISA, ELISPOT, and toxin-neutralization assays

To determine the effects of bacterial toxins on the humoral immune response, accurate measurement of antibody quantity, specificity, and function is critical. Here, we present a murine injection protocol for bacterial toxins and vaccine antigens and collection of plasma via retro-orbital eye bleeds. We describe steps for measuring antigen-specific antibody titers, antibody-secreting plasma cell abundance, and in vitro function of those antibodies. This protocol may be adapted to measure the impact of several bacterial toxins on humoral immunity. For complete details on the use and execution of this protocol, please refer to Norman et al.¹.