Supplementary information

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1 Supplementary information Ultra Q-bodies : quench-based antibody probes that utilize dye-dye interaction show enhanced antigen-dependent fluorescence Authors: Ryoji Abe, Hee-Jin Jeong,, Dai Arakawa, Jinhua Dong,, Hiroyuki hashi,, Rena Kaigome, Fujio Saiki, Kyosuke Yamane,, Hiroaki Takagi, and Hiroshi Ueda,,* Ushio Inc., 9 Moto-Ishikawa-cho, Aoba-ku, Yokohama, Kanagawa -, Japan Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-- Hongo, Bunkyo-ku, Tokyo -, Japan. Chemical Resources Laboratory, Tokyo Institute of Technology, 9-R-, agatsuta-cho, Midori-ku, Yokoyama, Kanagawa -, Japan. * Correspondence should be addressed to H.U. (ueda@res.titech.ac.jp) S

2 Supplementary Table S Dye Detection H chain L chain E x / E m ormalized FL intensity at λ max EC (mg/ml) H/HTLT TAMRA TAMRA /.. H/ TAMRA R /.. H/HRLT R TAMRA /..7 H/HTLA TAMRA ATT /.. H/HALT ATT TAMRA /.. H/ TAMRA R / 7..7 S

3 Supplementary Figure Legend Figure S. Thermal shift assay. (a) Scheme of the temperature-dependent removal of a quenching effect by denaturation of Quenchbody. (b) Temperature-dependent fluorescence intensity. The temperature was raised from to 9 C at C intervals per minute, with fluorescence readings taken at each interval. (c) Melt curve obtained as a differential of (b), showing the peaks corresponding to the apparent melting temperature T m. Figure S. Antigen-dependent fluorescence enhancement of anti-bgp Fab single/double-labeled with R (a and b) or ATT (c and d). (a) Standard curves of the fluorescence intensity at nm. The Fabs labeled with R at indicated position(s) were excited at nm in the absence and presence of BGP-C7 peptide. The intensities are relative values with respect to that in the absence of BGP-C7 peptide. (b) ormalized standard curves. (c) Standard curves of the fluorescence intensity at nm. The Fab labeled with ATT was excited at nm. (d) ormalized standard curves. Figure S. Antigen-dependent fluorescence enhancement of R/TAMRA-heterolabeled Fabs. (a) Fluorescence spectra of anti-bgp Fab labeled at the -terminal region of the H chain with R and the L chain with TAMRA (HRLT), with excitation at nm in the presence of BGP-C7 peptide as indicated. (b) The same with Fab labeled with TAMRA at the H chain and with R at the L chain (). (c) Standard curves of the fluorescence intensity at nm. The intensities are relative values with respect to that in the absence of BGP-C7 peptide. (d-f) The results of anti-sa UQ-bodies for detecting human serum albumin. The conditions are the same as in (a-c), except that HSA was used as an antigen and the fluorescence intensity at nm was taken in (f). S

4 Figure S. Detection of Influenza virus HA proteins by hetero-labeled UQ-bodies. (a) Standard curves of the fluorescence intensity at nm. The intensities are relative values with respect to that in the absence of H HA (A/Vietnam/9/). Abbreviations for the labeled Fabs are the same as in other figures. (b) Fluorescence spectra of anti-ha UQ-body labeled with TAMRA at Fd and with R at L chain (), with excitation at nm in the presence of H HA protein as indicated. (c) Comparison of the standard curves for H and H (A/California//9) with -type UQ-body. (d) Fluorescence spectra as shown in (b), except that H protein was used. Figure S. Characterization of UQ-bodies. (a) Antigen-binding activity of the double-labeled UQ-bodies. Biotinylated BGP-C ( µg/ml) was immobilized through streptavidin, and varied amounts of labeled Fab fragments were added, which was probed by HRP-conjugated anti-his Ab. (b and c) SDS-PAGE showing the purified UQ-bodies. Single (s) or double (d) labeled UQ-bodies are visualized by their fluorescence (b) or after CBB-staining (c). Figure S. Detection of bead-bound antigen by double ATT-labeled UQ-body under a Fluorescence microscopy. Detection scheme (upper), pictures taken by transmission (middle) and fluorescence (lower) are shown. Figure S7. Working model of double-labeled UQ-bodies. Less hydrophobic ATT (upper left) has a net positive charge and is easily exposed to outside of the Fab after antigen binding. More hydrophobic TAMRA has higher tendency to form H dimer, which is difficult to dissolve upon antigen binding. S

5 Figure S. Effect of linker length between TAMRA and maleimide. (a) Dose-response of double TAMRA UQ-bodies with three different linker lengths (,, and ). (b) ormalized absorption spectra of free maleimide dyes (b) and of the UQ-bodies at nm. The ratio of two absorption peaks at shorter (~ nm) and longer (~ nm) shows relative amount of H-dimer. S

6 Supplementary Figure S a Quenched Heat (+ / min) Fluoresce b Fluorescence Intensity S S, TAMRA-scFv, TAMRA-Fab,,,, 7 9 Temperature ( ) c Δ(FI) / ΔT S S - - Denaturation TAMRA-scFv Tm=7 TAMRA-Fab Tm= 7 9 Temperature ( )

7 Supplementary Figure S a c. HRLn. HnLR. HRLR BGP-C7 (M) HALn HnLA HALA BGP-C7 (M) b Response (%) d Response (%) HRLn HnLR HRLR BGP-C7 (M) HALn HnLA HALA BGP-C7 (M)

8 Supplementary Figure S 9 7 Ex= nm 9 e [HSA] M -7 M -7 M - M - M - M - M - M 9 Ex= nm d c Ex= nm / Em= nm HRLT BGP-C7 (M) - - f Ex= nm [BGP-C7] M -9 M -9 M - M - M -7 M - M - M - M HRLT Ex= nm b [HSA] M -7 M -7 M - M - M - M - M - M 9 HRLT [BGP-C7] M -9 M -9 M - M - M -7 M - M - M - M a 9 7 Ex= nm / Em= nm HRLT -7 - HSA (M) - -

9 Supplementary Figure S a b 7 HTLT HRLT HTLA HALT 7 [H HA (µg/ml)].. c 7.. H HA (µg/ml) H H d 9 7 [H HA (µg/ml)] HA (µg/ml) 9

10 Supplementary Figure S.".". +Ag +BGP".." " A)A"()) A-A (-).". ncentra>on"(ng) " " " " TAMRA Q-body conc. (nm) " TAMRA"labeled"EQ)body"Concentra>on"(ng) +Ag +BGP" +BGP" s "d." " " " s d s d k k 7 k 7 k k ATT c " " " " " " k k ATT"labeled"EQ)body"Concentra>on"(ng) k k -Ag )BGP".." " )BGP" s d. " " " b. " " " -Ag )BGP" A)A"()) A)A"()) A-A (-) a " " ATT Q-body conc. (nm) " ATT"labeled"EQ)body"Concentra>on"(ng) k TAMRA ATT TAMRA

11 Supplementary Figure S Streptavidin coated bead Biotinylated BGP peptide BGP Q-body + Ag, - Qbody + Ag, + Qbody - Ag, + Qbody

12 Supplementary Figure S7 ATT-C-maleimide H CH CH H C + + HCH CH CH Ag logp =. H Cl - TAMRA-C-maleimide (CH ) C - + (CH ) - + Ag logp =.9 H

13 Supplementary Figure S a ormalized F.I. (-)..... (n=) BGP-C7 Concentration (M) b ormalized Absorbance (-) c C (CH ) ormalized Absorbance (-).... CQ CQ CQ X axis : Y axis : ormalized absorbance (-) (CH ) C ormalized Absorbance (-) + (CH ) TAM-C-mal TAM-C-mal TAM-C-mal + (CH ). (CH ) C + (CH ) C - H C -.-fold.-fold.-fold H C -