Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay.

To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC). Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target. A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.