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An Immunoassay Utilizing DNA-Coated Cage Protein As a Signal Generator
An Immunoassay Utilizing DNA-Coated Cage Protein As a Signal Generator
Bulletin of the Korean Chemical Society. 2014. Aug, 35(8): 2559-2562
Copyright © 2014, Korea Chemical Society
  • Received : April 01, 2014
  • Accepted : April 14, 2014
  • Published : August 20, 2014
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About the Authors
Hoa T. Hoang
KIST Campus, University of Science and Technology (UST), Seoul 136-791, Korea.
Hoa Thi Le
Graduate School of East-West Medical Science, Kyung Hee University, Gyeonggi-do 449-701, Korea
Taemin Lee
Interdisciplinary School of Green Energy and KIER-UNIST Advanced Center for Energy, Ulsan National Institute of Science and Engineering (UNIST), Ulsan 689-798, Korea
Byeong-Su Kim
Interdisciplinary School of Green Energy and KIER-UNIST Advanced Center for Energy, Ulsan National Institute of Science and Engineering (UNIST), Ulsan 689-798, Korea
Hyung Jun Ahn
Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Korea
Tae Woo Kim
Graduate School of East-West Medical Science, Kyung Hee University, Gyeonggi-do 449-701, Korea
Mee Ran Shin
Department of Prosthodontics, Dentistry, Dongtan Sacred Heart Hospital, Graduate School of Clinical Dentistry, Hallym University, Hwaseong 445-907, Korea
Dae-Ro Ahn
KIST Campus, University of Science and Technology (UST), Seoul 136-791, Korea.

Abstract
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Experimental
To perform CP-OLISA, black 96-well microplates (Nunc, Denmark) were coated with cAb in PBS (5 μg/mL, 100 μL/ well) by incubation for 1 h at room temperature, followed by washes with PBS (3 × 300 μL). The wells were then treated with Blocking Buffer (200 μL, PBS containing 3% (w/v) BSA and 0.1% (v/v) Tween 20) and incubated for 2 h at room temperature. After rinses with PBST (PBS containing 0.05% (v/v) Tween 20), AFP solutions (0, 0.0625, 1.25, 2.5, 5.0, 10.0 ng/mL) in Assay Buffer (100 μL, PBS containing 1% (w/v) BSA and 0.05% (v/v) Tween 20) were added to the wells, followed by incubation for 1 h at room temperature. The plate was washed with PBST before addition of biotin-dAb conjugates (2 μg/mL, 100 μL) in Assay Buffer and incubated for 1 h at room temperature. After rinsing with PBST (3 × 300 μL), the solution of streptavidin (2 μg/ mL, 100 μL) was added to the wells and incubated for 1 h at room temperature. After rising with PBST (3 × 300 μL), solutions of DNA-coated protein (125 pM, 100 μL, DNA sequence: AACCACAGTG) were added to the wells, followed by incubating for 1 h at room temperature, and the plate was rinsed with PBST (3 × 300 μL). Finally, the RNase H (100 μL) solution containing F-RNA-Q probe (200 nM, FAM-5′-CACUGUGGUU-3′-BHQ1), PRI (0.4 U, Protector RNase Inhibitor, Roche, Germany) and 20 U of RNase H in RNase H buffer (40 mM Tris-HCl, 4 mM MgCl 2 , 1 mM DTT, 0.003% BSA, pH 7.7) was added and incubated for 1 h at 37 ºC. The fluorescence intensities were measured by Apliskan (Thermo scientific, Waltham, MA, USA) with the excitation/emission filter sets of 485/535nm.
Supporting Information. Experimental procedures for preparation of DNA-coated cage protein, ELISA, and OLISA.
Acknowledgements
This study was supported by a grant funded by Korea Institute of Science and Technology, a grant of the Korea Health technology R&D project, Ministry of Health & Welfare, Republic of Korea (A121191), and a grant the Proteogenomic Research Program through the National Research Foundation of Korea funded by the Korean Ministry of Education, Science and Technology.
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