Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C

Abstract

Clostridium tetani causes a life-threatening infectious disease by production of tetanus neurotoxin (TeNT), a 150 kDa molecule composed of light (LC) and heavy chain (HC) polypeptides. The TeNT HC contains an N-terminal domain critical for LC translocation and a Cterminal toxin receptor-binding domain known as fragment C. Despite extensive investigations on epitope specificity of anti-TeNT antibodies, the immunodominant neutralizing epitopes of the toxin are poorly defined. This study describes the generation and characterization of four monoclonal antibodies (MAb) specific for TeNT. The characteristics of each MAb were explored in terms of isotype, specificity, affinity, and immuno-globulin heavy chain variable region (IGHV) gene usage using ELISA, Western blotting, and sequencing techniques. The toxin neutralizing activity of the MAbs was also investigated using the in vitro GT1b neutralizing assay. The data demonstrated that all MAbs bind to tetanus toxin and toxoid. Sub-fragments binding analysis showed that two MAbs react with fragment C, one with both fragment C and LC, and one with LC. Only the two fragment C-specific MAbs were able to neutralize the toxin. Sequencing of the expressed VH and VL genes revealed rearrangements of various VH and VL gene segments in all hybridoma clones. Clonality of the hybridomas was also confirmed by a competition assay that showed recognition of distinct epitopes by these MAbs. The results suggest the importance of TeNT fragment C in terms of immunogenicity and toxin neutralization activity.

Comparativein vitroandin vivoassessment of toxin neutralization by anti-tetanus toxin monoclonal antibodies

“…In the present study, we report characterization of seven new and four previously described anti-TeNT specific mAbs 12 all of which react with the tetanus toxin and toxoid. Further characterization of the mAbs with subfragments of the light and heavy chain of TeNT revealed that seven mAbs (1F3E3, 1F2C2, 1F1E12, 1F2C8, 2C9B6, 3B3D9, and 1E4D5) bind to fragment C, while the other four mAbs recognized either LC alone (1F3B3 and 1F3C3), both LC and fragment C (1F4E11), or none of them (5E6B10).…”

“…12 Briefly, 2000, 1000, 500, 250, 125, and 60 ng/ ml tetanus toxoid was coated on wells of a micotiter ELISA and serial concentrations of mAbs (from 30 to 2000 ng/ml) in blocking buffer were added into each coated well and incubated at 37 °C for 1.5 h. After washing step, all wells were incubated with HRP-conjugated sheep anti-mouse Ig for 1.5 h at 37 °C. Washing was repeated and ODs were measured following addition of TMB substrate solution.…”

“…The antibody concentration resulting in 50% of the maximum absorbance value ([Ab]t) at a particular antigen coating concentration was selected for the affinity calculation using the formula Kaff Assessment of epitope specificity of monoclonal antibodies by competition ELISA Epitope specificity of mAbs was determined by competition ELISA as described previously. 12 Briefly 10 µg/ml of TeNT was coated on wells of a micotiter ELISA plate and blocked with 3% skin milk-PBS/T 0.05%. Unconjugated mAbs in final concentrations of 40, 20, 10, 5, 2.5, 1.25, and 0.612 µg/ml were added together with 1 µg/ml of HRP-conjugated mAbs (prepared in our lab) and incubated at 37 °C for 1.5 h. Washing steps were repeated and ODs were measured after addition of TMB substrate solution and then stopping solution.…”

“…[9][10][11] We have recently assessed the inhibitory potential of four TeNT specific mAbs (1F3E3, 1F2C2, 1F3B3, and 1F4E11) using the in vitro GT1b binding assay. 12 However, there is no detailed information about the validity and relevance of this method to the results obtained from in vivo experiments. Here, we report characterization of four previously reported 12 and seven new anti-TeNT-specific murine mAbs recognizing different epitopes of the tetanus toxin.…”

“…12 However, there is no detailed information about the validity and relevance of this method to the results obtained from in vivo experiments. Here, we report characterization of four previously reported 12 and seven new anti-TeNT-specific murine mAbs recognizing different epitopes of the tetanus toxin. The TeNT inhibitory activity of these mAbs was determined in vitro by the GT1b binding assay and neutralization activity of these mAbs was assessed in vivo using toxin neutralization assay in mice.…”