Analytical Quality-by-Design-Based Development of an ELISA for In Vitro Assessment of Human Tetanus Immunoglobulin...

An enzyme-linked immunosorbent assay (ELISA) method for the in vitro evaluation of tetanus immunoglobulin potency was developed and validated using analytical quality-by-design principles, and a quality risk assessment identified critical method parameters, including antigen coating time, washing, color development time, and antibody incubation time and temperature. These variables were systematically optimized using Design of Experiments within JMP software. Following this, the concentrations of tetanus antigen and anti-tetanus antibody were further optimized to enhance the assay performance. The method demonstrated excellent linearity (R² > 0.99), accuracy, precision, and recovery in accordance with the International Council for Harmonization Q2(R1) and United States Pharmacopeia <1033> guidelines. A comparative analysis using two commercial ELISA kits revealed no statistically significant differences. These findings support the use of an analytical quality-by-design-based ELISA as a scientifically robust, reproducible, and ethical method suitable for in vitro evaluation of tetanus immunoglobulin potency in alignment with regulatory expectations.