Neutralization of Tetanus Toxin by Human Monoclonal Antibodies Directed Against Tetanus Toxin Fragment C

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Abstract
Two hybridomas (designated 143 and 147) producing human monoclonal antibodies (IgG1) directed against tetanus toxin were established by fusion of Epstein-Barr virus transformed human peripheral B lymphocytes with the heteromyeloma SPAM-8. The hybridomas produced antibodies in concentrations of approx. 3.5 μg/ml (hybridoma 143) and 6.4 μg/ml (hybridoma 147) using conventional flask cultures and 33.9 μg/ml and 36.2 μg/ml, respectively, in dialysis cultures. The antibodies were shown to react with tetanus toxin, toxoid and fragment C in ELISA, and reactivity with tetanus toxin and fragment C was confirmed in SDS-polyacrylamide gel electrophoresis followed by Western blots. The antibody binding sites were located to two different epitopes of fragment C as shown in a competition assay using biotinylated antibodies. Furthermore, binding of both antibodies to fragment C was inhibited by the addition of the receptor-associated ganglioside GT1b. Neutralization of tetanus toxin in concentrations equivalent to 100-120 IU per mg of antibody was observed for both antibodies in a mouse protection assay.