Monoclonal antibodies against tetanus toxin and toxoid

Abstract

Monoclonal antibodies against tetanus toxin and its toxoid were produced by immunizing mice with toxoid or toxin. They were measured by an enzyme-linked immunosorbent assay (ELISA), by a toxin neutralization test in mice (in vivo prevention test), and by their ability to prevent binding of¹²⁵I-toxin to brain membranes or gangliosides (in vitro prevention test). Six monoclonal antibodies obtained by immunization with toxoid (anti-toxoid 1–6) were investigated in more detail. They belonged to IgG class 1. Three of them (anti-toxoid 1, 2 and 3) recognized both toxoid and toxin as well as fragment B and the light chain of toxin, but not fragment C. Two other antibodies (anti-toxoid 4 and 5) were directed against toxoid only. Neither of them prevented toxin action in vitro or in vivo. Anti-toxoid 6 recognized toxin, toxoid and fragment C, but not light chain, and prevented toxin action in vitro and in vivo. Immunization against toxin was initiated with a toxin-antitoxin complex and boosted with toxin. We studied six antibodies in more detail, all of IgG type 2. Their K_D against¹²⁵I-tetanus toxin varied from 10⁻⁹ to 10⁻¹⁰ M. Anti-toxin 2 recognized toxin, toxoid, light chain and fragment B, but not fragment C. The others reacted with toxin, toxoid and fragment C, but not with light chain or fragment B. All of them prevented toxin action in vitro and in vivo. As calculated from the maximal extinction achieved in the ELISA, tetanus toxin combined with a maximum of two different antibody molecules from our set. Gel filtration data indicate that tetanus toxin reacts with monoclonal antibodies one by one. Compared with polyclonal antiserum, monoclonal antibodies yield flatter slopes in both in vitro and in vivo prevention tests. Thus, they cannot substitute for the polyclonal antibodies in clinical situations, and cannot be calibrated in international units.