Anti-IDs clones #6 and #17 as capture and the generic a Sulfo-TAG-mouse anti-human IgG4 using assay format 4 were provided used to perform full assay optimization. Recombinant versions of these clones were generated for further testing. The modified assay format (Format 4, Figure ) of preincubation of the sample with the biotinylated capture reagent prior to transferring to the streptavidin MSD assay is not a typical format. This approach was repeated and compared to the more traditional sequential format (Format 6, Figure ). Soluble ligand that potentially could interfere with assay performance was used to test for interference. Both the reagent from the hybridoma and the scaled up recombinant anti-ID confirmed that using the modified Format 4 provided better overall assay performance. The recombinant version of the backup clone (Clone #17) provided better overall performance when compared to the recombinant version of Clone #6. This reagent pair and assay format was taken into full assay validation to support the clinical development program.