An ELISA-Based Alternative to Mouse Bioassays for Quantitative Evaluation of Tetanus Toxin

Tetanus toxin evaluation has traditionally relied on mouse LD₅₀ bioassays, which require extensive animal use and time, necessitating development of alternative methods in accordance with 3R principles (Replacement, Reduction, and Refinement). We developed and validated a sandwich enzyme-linked immunosorbent assay (ELISA) as an alternative to animal testing for evaluating tetanus toxin biological activity using 18 environmental and clinical isolates of Clostridium tetani, complemented by an immunochromatographic (IC) assay for rapid screening. The ELISA demonstrated excellent analytical performance with a lower limit of quantification of 2.4 ng/mL (equivalent to 85.4 LD₅₀/mL), favorable linearity (R² = 0.999), precision (CV < 1.7-8.2%), and specificity (<1% cross-reactivity with C. septicum, C. novyi, and C. perfringens). Correlation analysis between ELISA relative potency and observed minimum lethal dose values revealed a robust positive correlation (r = 0.974). Both parallel line assay and single-point quantification methods showed strong correlations with mouse bioactivity measurements (r = 0.998). The IC assay successfully detected all isolates within 15 min. The measurement range of 2.4-45.6 ng/mL effectively covered diverse toxin-production capabilities spanning a 600-fold concentration range. This validated ELISA and IC assay combination provides a reliable, rapid alternative to animal experimentation for tetanus toxin evaluation.