I am developing an Anti-Drug Antibody assay and keep getting a negative percent inhibition (negative control sample results are higher in confirmatory than in screening format) when running individual and pooled normal human serum samples . This leads to a negative confirmatory cut point for the confirmatory test.

The assay format is a bridging ECL assay. Samples are acidified, followed by neutralization with screening maser mix (containing biotin and Sulfo-TAG drug) or confirmatory master mix (screening master mix with unconjugated drug). Transferred to a streptavidin coated MSD plate.

I have tried changing the pH, adding tween and type of neutralization buffer and also a variety of different blocking buffers but the higher confirmatory signal still persists.

Samples with Positive Control added have normal positive percent inhibition. Any advice is appreciated!