An Immunoassay Utilizing DNA-Coated Cage Protein As a Signal Generator

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.