(532) ELISA-Based Neutralizing Antibody (NAb) Detection as a Fit-for-Purpose Alternative to Cell-Based Assays for Biotherapeutic Immunogenicity Assessment

Introduction/Rationale: Neutralizing antibodies (NAbs) are a critical component of the adaptive immune response to biotherapeutic agents, capable of blocking biological function, altering pharmacokinetics, and reducing clinical efficacy. Conventional cell-based assays remain the gold standard for NAb detection because they capture functional activity, yet they are inherently variable due to cell-line dependence, receptor expression, and complex assay validation requirements. To address these challenges, we evaluated ELISA-based NAb assays as fit-for-purpose alternatives that model the same ligand–receptor interactions in a controlled, reproducible format.

Methods: Competitive ligand-binding ELISAs were designed for three representative biotherapeutic classes: monoclonal antibody, peptide drug, and peptibody. Each assay quantified the inhibition of drug–target interaction by NAbs through measurable signal reduction. Reference NAb standards were spiked across defined concentration ranges, and results were benchmarked against published cell-based assay data. Analytical performance was assessed for sensitivity, precision, accuracy, and cut-point determination per ICH M10 and FDA Bioanalytical Guidelines.

Results: All three ELISA formats demonstrated strong linearity (R² > 0.99) and precision (CV < 10%) across working ranges, with sensitivity comparable to published cell-based data. Inhibition profiles correlated closely with reported cellular responses, confirming mechanistic equivalence in binding blockade. The assays offered enhanced reproducibility, reduced turnaround time, and simplified validation.

Conclusion: ELISA-based NAb detection represents a scientifically robust, cost-effective, and high-throughput approach for immunogenicity assessment of diverse biologics. By modeling immune neutralization through ligand–receptor competition, this platform provides a fit-for-purpose surrogate to traditional cell-based assays, advancing standardized and reproducible immunogenicity evaluation in translational immunology.