Streamlined neutralizing antibody assay development: overcoming serum interference, utilization of DOE and automation

This study detailed the troubleshooting and optimization of a challenging neutralizing antibody (NAb) assay using a previously published PEG precipitation, Acid Dissociation, and Biotin-drug as Assay Drug (PABAD) method to overcome high drug interference. A strong NAb positive control, PC3, showed poor recovery in the PABAD workflow. A PABAD-compatible PC, PC31 was selected among different anti-idiotype antibodies with varied kinetic profiles. A three-tier Design of Experiment (DOE) approach was applied to optimize assay conditions. Centrifugation-based Bluewasher protocols were also established to automate the handling of PEG pellets. The reason for the poor performance of PC3 with the PABAD method was identified, and a sensitive NAb assay was successfully developed using PC31. The poor recovery of PC3 was not due to incompatibility with the PABAD method, but rather to its non-specific interaction with serum factors. Once a specific NAb PC was identified, the NAb assay was successfully developed using PABAD, demonstrating a broad application of the method. DOE-assisted assay development cut the development time by half and meaningfully improved the assay sensitivity. Finally, the implementation of the Bluewasher in the pellet wash further reduced assay variability and enhanced automation of the PABAD method.