Premium
Case study on human α1‐antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate
Author(s) -
Hansen Henning Gram,
Kildegaard Helene Faustrup,
Lee Gyun Min,
Kol Stefan
Publication year - 2016
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201600409
Subject(s) - titer , recombinant dna , coomassie brilliant blue , microbiology and biotechnology , chemistry , chromatography , biology , antibody , biochemistry , immunology , staining , gene , genetics
Accurate titer determination of recombinant proteins is crucial for evaluating protein production cell lines and processes. Even though enzyme‐linked immunosorbent assay (ELISA) is the most widely used assay for determining protein titer, little is known about the accuracy of commercially available ELISA kits. We observed that estimations of recombinant human ø1‐antitrypsin ( r ø1AT) titer by Coomassie‐stained SDS‐PAGE gels did not correspond to previously obtained titers obtained by a commercially available ELISA kit. This prompted us to develop two independent quantification assays based on biolayer interferometry and reversed‐phase high‐performance liquid chromatography. We compared the rø1AT titer obtained by these assays with three different off‐the‐shelf ELISA kits and found that the ELISA kits led to inconsistent results. The data presented here show that recombinant protein titers determined by ELISA kits cannot be trusted per se . Consequently, any ELISA kit to be used for determining recombinant protein titer must be validated by a different, preferably orthogonal method.