Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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An Immunoassay Utilizing DNA-Coated Cage Protein As a Signal Generator

  • Hoang, Hoa T. (Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology) ;
  • Le, Hoa Thi (Graduate School of East-West Medical Science, Kyung Hee University) ;
  • Lee, Taemin (Interdisciplinary School of Green Energy and KIER-UNIST Advanced Center for Energy, Ulsan National Institute of Science and Engineering (UNIST)) ;
  • Kim, Byeong-Su (Interdisciplinary School of Green Energy and KIER-UNIST Advanced Center for Energy, Ulsan National Institute of Science and Engineering (UNIST)) ;
  • Ahn, Hyung Jun (Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology) ;
  • Kim, Tae Woo (Graduate School of East-West Medical Science, Kyung Hee University) ;
  • Shin, Mee Ran (Department of Prosthodontics, Dentistry, Dongtan Sacred Heart Hospital, Graduate School of Clinical Dentistry, Hallym University) ;
  • Ahn, Dae-Ro (Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology)
  • Received : 2014.04.01
  • Accepted : 2014.04.14
  • Published : 2014.08.20
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Abstract

Keywords

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 MgCl2, 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.

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