Preparation and characterization of a neutralizing murine monoclonal antibody against tetanus toxin

After Clostridium tetani infects the human body, it propagates under anaerobic conditions and produces tetanus neurotoxin (TeNT). TeNT can affect the central nervous system, inhibit the release of neurotransmitters, and result in respiratory failure, which are the root causes of death in tetanus patients. Identifying monoclonal antibodies (mAbs) targeting TeNT with neutralizing activity is urgently needed for the prevention and treatment of tetanus infection. In this study, through immunizing BALB/c mice with tetanus toxoid (TT), we obtained six positive hybridoma cell lines (1A7, 2C7, 3A7, 3H4, 4C1, and 4E12). Antibody isotyping showed that the antibodies are all of the IgG1/κ subclass. Ascites fluid was prepared by allogeneic ascites induction and the antibodies were purified through protein G affinity chromatography columns. Purities of the produced murine mAbs were all greater than 95%. All six antibodies bound to linear epitopes, among which 3A7 bound to the TeNT/L domain and the other five antibodies bound to the TeNT/Hc domain. Moreover, the affinity constants of these six antibodies against the antigen were all in the nanomolar range, and the affinity of 4E12 antibody reached the picomolar range. Results from toxin-neutralization assays in mice showed that 2C7 antibody delayed animal death, while 1A7, 3A7, 3H4, and 4E12 antibodies conferred partial protection. Additionally, 4C1 antibody offered complete protection, as 200 μg of 4C1 antibody fully protected against toxin challenge with 10 LD₅₀ of TeNT and had a window period of 1 h. Antibody epitope grouping results revealed that the binding epitopes of 4C1 antibody were different from those of the other five antibodies. When 4C1 antibody was used in combination with another antibody, the neutralizing activities of antibodies were all evidently enhanced. Specifically, 4C1 combined with 3A7 antibody led to the greatest improvement in neutralizing activities, and 20 μg antibodies total (10 + 10 μg) fully protected against toxin challenge with 10 LD₅₀. When 4E12, 3A7, and 4C1 antibodies were used in combination, 18 μg antibodies total (6 + 6 + 6 μg) completely neutralized 10 LD₅₀ toxin. The present study derived murine mAbs with neutralizing activities and laid the foundation for follow-up therapeutic drug development for TeNT poisoning as well as establishment of TeNT detection methods.