Model fp 8500

Each Q-body (500 nM, 7.5 μL) was diluted in 250 μL of PBST containing 1% BSA, and HEL was added by titration in a 5 × 5 mm quarts cell (Starna, Atascadero, CA, USA). After each addition, the solution was incubated for 2 min at 25 °C prior to the spectral measurements. The fluorescence intensity was measured using a fluorescence spectrophotometer Model FP-8500 (JASCO, Tokyo, Japan) with excitation at 546 nm and emission from 562 to 662 nm. The excitation and emission slit widths were set to 5.0 nm. Fluorescence intensities at the maximum emission wavelength (580 nm) and the normalized fluorescence intensity of each sample based on the fluorescence intensity at zero dose were plotted. Dose-response curves were fitted using Kaleida Graph 4.1 (Synergy Software, Reading, PA, USA) and the EC₅₀ values were calculated from the curve fitting to a 4-parameter logistic equation.

The dose–response of the yeast-displayed mini Q-body was measured using either a fluorescence spectrophotometer Model FP-8500 (Jasco, Tokyo, Japan) or a fluorescence microplate reader Clariostar (BMG Labtech Japan, Saitama, Japan). After the yeast-displayed mini Q-body was assembled as described previously, it was diluted to 0.3 OD₆₀₀, and the antigen (MTX or HSA) was added to the solution at eight concentrations (1, 3, 10, 30, 100, 300, 1000, and 3000 nM). For fluorescence spectral measurements, each solution (250 μL) was added in a 5 mm × 5 mm quartz cell (Starna Scientific, Hainault, UK), and the fluorescence spectrum was measured at 485 nm excitation and 510–650 nm emission wavelengths. The measurement temperature, excitation/emission bandwidths, and scanning speed were set to 25 °C, 5 nm, 200 nm/min, respectively. For fluorescence ratiometric measurement, each solution (80 μL) was applied to a 96-well black microplate and fluorescence was measured at 483 nm excitation and 535 nm or 585 nm emission wavelengths. Dose–response curves were drawn by fitting the fluorescence intensity of each concentration at 585 nm normalized to that at 535 nm (R/G ratio) using a curve fitting function as described.

To measure the fluorescence spectra, 100 ng of UQ-body was diluted in 250 μL of PBST containing 1% BSA, and antigen BGP-C7 peptide was added by titration in a 5 × 5 mm quarts cell (GL Sciences, Tokyo, Japan). After each addition, the solution was incubated for 5 min at 25°C prior to the spectral measurements. As a control, the same procedure was performed with the exception that PBST was added instead of BGP-C7 peptide in the cell. The fluorescence spectra were obtained at 25°C using a fluorescence spectrophotometer Model FP-8500 (JASCO, Tokyo, Japan) with the excitation at 546 and 524 nm for TAMRA- and ATTO520- UQ-body, respectively. The excitation and emission slit widths were set to 5.0 nm. The dose-response curves were fitted at the maximum emission wavelength using Kaleida Graph 4.1 (Synergy Software, Reading, PA).