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

Area of Science:

• Biotechnology
• Immunology
• Assay Development

Background:

• High drug interference poses a challenge for neutralizing antibody (NAb) assays.
• The PEG precipitation, Acid Dissociation, and Biotin-drug as Assay Drug (PABAD) method was previously developed to address drug interference.

Purpose of the Study:

• To troubleshoot and optimize a challenging NAb assay using the PABAD method.
• To identify a suitable positive control and improve assay performance.

Main Methods:

• A PABAD-compatible positive control (PC31) was selected from various anti-idiotype antibodies.
• A three-tier Design of Experiment (DOE) approach was used for assay optimization.
• Centrifugation-based Bluewasher protocols were developed for automated handling of PEG pellets.

Main Results:

• The poor recovery of the initial positive control (PC3) was attributed to non-specific interactions with serum factors, not PABAD incompatibility.
• A sensitive NAb assay was successfully developed using PC31 and the PABAD method.
• DOE-assisted development halved assay development time and improved sensitivity.

Conclusions:

• The PABAD method is broadly applicable for NAb assay development with appropriate positive controls.
• Automation using the Bluewasher reduced assay variability and enhanced throughput.
• Optimized NAb assays are crucial for therapeutic antibody development and monitoring.