Ganglioside Binding Assay: A Complementary Approach for Enhanced Tetanus Toxoid Quality Control

Vaccine quality control has long relied on animal testing, which involves time, cost, and ethical constraints. This study introduces a ganglioside binding (GB) assay as a complementary in vitro screening tool for tetanus toxoid quality control, which was validated in a single-laboratory environment as a foundational proof-of-concept. The assay reproduces tetanus toxin binding to gangliosides on microplates using simplified procedures. Validation with samples at different inactivation stages showed excellent linearity (0.0002-0.0156 Lf/mL), reproducibility, and a strong correlation with Ramon's flocculation (R² = 0.999). The assay clearly distinguished between toxins and toxoids, with the toxoid results remaining at control levels. The time-course inactivation samples were consistent with the animal tests: partially inactivated samples (days 1-3) showed significant GB activity (p < 0.001) and caused 100% mortality, whereas samples from day 4 onward showed no activity and zero mortality. These findings demonstrate that the GB assay reliably differentiates active toxins from toxoids, which aligns with in vivo outcomes. The practical advantages include a simplified protocol, reduced complexity, and improved efficiency for routine testing of samples. As a complementary screening approach, this single-laboratory validation supports the 3Rs principle by demonstrating the potential for reducing animal use while ensuring quality assurance. Broader applicability requires multicenter validation and cross-reactivity, and multicenter validation is ongoing.