Journal
SCIENTIFIC REPORTS
Volume 8, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41598-018-36192-8
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Funding
- Ministry of Science and Technology, Taiwan [MOST 105-2314-B-038-081-MY3, MOST 105-2622-B-038-003-CC2, MOST 106-2320-B-038-047, MOST 107-2320-B-038-005, MOST 106-2632-B-038-001]
- National Health Research Institutes, Taiwan [NHRI-EX105-10238SC]
- Health and Welfare Surcharge of Tobacco Products Grant, Taiwan [MOHW107-TDU-B-212-114014]
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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.
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