Matrix interference from Fc–Fc interactions in immunoassays for detecting human IgG4 therapeutics. (October 2015)

• Record Type: Journal Article
• Authors: Partridge, Michael A; Karayusuf, Elif Kabuloglu; Dhulipala, Gangadhar; Dreyer, Robert; Daly, Thomas; Sumner, Giane; Pyles, Erica; Torri, Albert
• Is Part Of: Bioanalysis. Volume 7:Number 20(2015)
• Page Start: 2701
• Page End: 2712
• Publication Date: 2015-10
• Subjects: Drugs -- Analysis -- Periodicals; 615.19005
• DOI: 10.4155/bio.15.152
• Languages: English
• ISSNs: 1757-6180

Abstract

Background: An assay measuring an IgG4 biotherapeutic in human serum used a drug-specific monoclonal antibody (mAb) capture reagent and an antihuman IgG4 mAb as detection reagent. However, serum IgG4 binding to the capture mAb via Fc-interactions was detected by the anti-IgG4 mAb, causing high background.Results: Two approaches were developed to minimize background; incorporating a mild acid sample preparation step or using the Fab of the capture antibody. Either strategy improved signal:noise dramatically, increasing assay sensitivity >20-fold. Biophysical analyses of antibody domains indicated that noncovalent Fc oligomers could inhibit the background.Conclusion: Matrix interference from human IgG4 binding to the capture mAb was reduced with a Fab fragment of the drug-specific capture antibody or by incorporating a mild acid sample treatment into the assay.