title: New assay for quantification of PEGylated proteins during in vitro permeability studies.
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, Ni(2+) 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-a protein. This approach could be extended to numerous other proteins eliminating the need to develop a separate assay for each protein/project.
authors.forename: Spela
authors.initials: S
authors.lastname: Jalen
authors.forename: Vanja
authors.initials: V
authors.lastname: Smilović
authors.forename: Katarina
authors.initials: K
authors.lastname: Fidler
authors.forename: Barbara
authors.initials: B
authors.lastname: Podobnik
authors.forename: Maja
authors.initials: M
authors.lastname: Marušić
authors.forename: Radovan
authors.initials: R
authors.lastname: Komel
authors.forename: Vladka
authors.initials: V
authors.lastname: Gaberc-Porekar
authors.forename: Simona
authors.initials: S
authors.lastname: Jevševar
mesh_terms: D066298:In Vitro Techniques; D010539:Permeability; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins; D011092:Polyethylene Glycols