Case study on human alpha 1-antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate

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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 alpha 1-antitrypsin (r alpha 1AT) 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 r alpha 1AT 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.
Publisher
WILEY-V C H VERLAG GMBH
Issue Date
2016-12
Language
English
Article Type
Article
Keywords

HAMSTER OVARY CELLS; OPTICAL BIOSENSOR; N-GLYCOSYLATION; EXPRESSION; ALPHA(1)-ANTITRYPSIN; TECHNOLOGIES; PRODUCTS; ASSAY; LINE

Citation

BIOTECHNOLOGY JOURNAL, v.11, no.12, pp.1648 - 1656

ISSN
1860-6768
DOI
10.1002/biot.201600409
URI
http://hdl.handle.net/10203/219629
Appears in Collection
BS-Journal Papers(저널논문)
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