標題: Development of a highly sensitive enzyme-linked immunosorbent assay (ELISA) through use of polyprotein G-expressing cell-based microplates
作者: Chen, Yi-Jou
Chen, Michael
Hsieh, Yuan-Chin
Su, Yu-Cheng
Wang, Chang-Hung
Cheng, Chiu-Min
Kao, An-Pei
Wang, Kai-Hung
Cheng, Jing-Jy
Chuang, Kuo-Hsiang
生物科技學系
Department of Biological Science and Technology
公開日期: 14-Dec-2018
摘要: The sensitivity of traditional enzyme-linked immunosorbent assays (ELISAs) is limited by the low binding avidity and heterogeneous orientation of capture antibodies coated on polystyrene-based microplates. Here, we developed a highly sensitive ELISA strategy by fixing poly-protein G-expressing cells on microplates to improve the coating amount and displayed orientation of capture antibodies. One or eight repeated fragment crystallisable (Fc) binding domains of protein G are stably expressed on the surface of BALB/c 3T3 cells (termed 1pG cells or 8pG cells), which then act as highly antibody-trapping microparticles. The 8pG cells showed higher antibody-trapping ability than the 1pG cells did. The antibody-coating amount of the 8pG cell-based microplates was 1.5-23 times and 1.2-6.8 times higher than that of traditional polystyrene-based and commercial protein G-based microplates, respectively. The 8pG cell-based microplates were then applied to an anti-IFN-alpha sandwich ELISA and an anti-CTLA4 competitive ELISA, respectively, and dramatically enhanced their detection sensitivity. Importantly, direct coating unpurified capture antibody produced by mammalian cells did not impair the antigen-capturing function of 8pG cell-based microplates. The 8pG cell-based microplates exhibited a significant improvement in antibody-coating amount and preserved the homogeneous orientation of capture antibodies, making them a potential replacement for traditional microplates in various formats of ELISAs.
URI: http://dx.doi.org/10.1038/s41598-018-36192-8
http://hdl.handle.net/11536/148597
ISSN: 2045-2322
DOI: 10.1038/s41598-018-36192-8
期刊: SCIENTIFIC REPORTS
Volume: 8
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