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New Assay for Quantification of PEGylated Proteins During in Vitro Permeability Studies

Špela Jalen, Vanja Smilović, Katarina Fidler, Barbara Podobnik, Maja Marušić, Radovan Komel, Vladka Gaberc-Porekar, Simona Jevševar

Abstract


One of the major challenges when analyzing very low amounts of PEGylated proteins is finding a sensitive analytical method. Immunoassays are most frequently used, however, conjugation can partially or completely mask protein epitopes, which can substantially lower the response and influence the quantitation range. Here we describe a novel assay that allows quantification of low amounts of PEGylated or differently conjugated proteins. The basic principle is similar to the classic sandwich ELISA but there are no antibodies used neither for capture nor for detection. Instead, Ni2+ chelation is exploited for capture and affinity between streptavidin and biotin for the detection step. The usefulness of the assay was proven in permeation studies (Caco-2 cell model) using diversely conjugated TNF-α protein. This approach could be extended to numerous other proteins eliminating the need to develop a separate assay for each protein/project.


Keywords


Immunoassay; PEGylation; His-tag; Biotin; TNF-α; Caco-2

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