Taylor & Francis Online: Peer-reviewed Journals
supplemental materialsChemically-defined camelid antibody bioconjugate for the magnetic resonance imaging of Alzheimer’s diseaseMatthias Vandesquille,1,2,4,? Tengfei Li,3,5,6,? Chrystelle Po,1,2,4 Christelle Ganneau,1,2 Pascal Lenormand,3 Clémence Dudeffant,5 Christian Czech,7 Fiona Grueninger,7 Charles Duyckaerts,5 Beno?t Delatour,5 Marc Dhenain,4,? Pierre Lafaye,3,? Sylvie Bay1,2,*1Institut Pasteur, Unité de Chimie des Biomolécules, 75724 Paris Cedex 15, France; 2CNRS UMR 3523, 75724 Paris Cedex 15, France; 3Institut Pasteur, Plateforme d’Ingénierie des Anticorps, 75724 Paris Cedex 15, France; 4French Alternative Energies and Atomic Energy Commission, Institute of Biomedical Imaging, Molecular Imaging Research Center, 92260 Fontenay-aux-Roses, France; 5Sorbonne Universités, UPMC Univ. Paris 06 UMR S 1127, and Inserm, U 1127, and CNRS UMR 7225, and ICM, F-75013, Paris, France; 6Université Paris Descartes, Paris 5, France; 7F. Hoffmann-La Roche AG, Pharmaceutical Research and Early Development, NORD DTA, Roche Innovation Center Basel, CH-4070 Basel, Switzerland.? M.V. and T.L. contributed equally to this work; ? M.D. and P.L. contributed equally to this work.*Correspondence to: Sylvie Bay; E-mail: sylvie.bay@pasteur.frPhone: +33 (0)1 45 68 83 98. Fax: +33 (0)1 45 68 84 04.Address: Institut Pasteur, 28 rue du Dr Roux - 75724 Paris Cedex 15, France.Table of Contents:Page S2-S6: Supplementary detailed experimental proceduresPage S7-S11: Supplementary figuresFigure S1: Schematic representation of R3VQ, R3VQ-NH2 1 and R3VQ-SH 3 Figure S2: Immunostaining of amyloid plaques by R3VQ-NH2 1Figure S3: SDS-PAGE analysis of the VHHs and their conjugatesFigure S4: MS analysis of 2bFigure S5: RP-HPLC and MS analyses of the maleimide-(DOTA/Gd)3 4Figure S6: Characterization of R3VQ-SH 3 before and after mild reducing conditions, and of the nanoimaging agent 5Figure S7: Anti-His-tag control immunostaining of brain sections from PS2APP and wild-type mice Figure S8: IHC with anti-His-tag antibody on PS2APP brain paraffin sections obtained after intravenous administration of 5 and after direct incubation of an entire brain hemisphere with 5Figure S9: Detailed MR relaxometric parameters r1 and r2 Page S13: Supplementary referencesSUPPLEMENTARY DETAILED EXPERIMENTAL PROCEDURESGeneral synthesis methodsUnless otherwise specified, the amino-acid derivatives and the reagents are purchased from Novabiochem and Sigma-Aldrich, respectively. The molar equivalents of all reagents are indicated relative to reactive groups (5 NH2 per R3VQ-NH2 1 and 1 SH per R3VQ-SH 3). The concentration of the peptide and VHH solutions (net protein content) was determined by quantitative AAA using a Beckman 6300 analyser after hydrolysis of the compounds with 6N HCl at 110 °C for 20 h. The MS analyses of the proteins and conjugates were performed on an Alliance 2695 system coupled to a UV detector 2487 (220 nm) and to a Q-Tofmicro? spectrometer (MICROMASS) with an electrospray ionisation (positive mode) source (Waters). The samples were cooled to 4 °C on the autosampler. The linear gradient was performed with acetonitrile+0.025% formic acid (A) / water+0.04% TFA+0.05% formic acid (B) over 10 or 20 min. The column used was a XBRIDGE BEH300 C18 (3.5 ?m, 2.1x100 mm) (Waters) (gradient 10-100% A). The source temperature was maintained at 120 °C and the desolvation temperature at 400 °C. The cone voltage was 40 V. The samples were injected at 0.4-1 mg/mL concentration in their respective buffer added with B. The expected Mr values correspond to the average mass of proteins with N-ter deleted Met and one disulfide bond. The MS analysis of 4 was recorded on the same spectrometer in the positive mode by direct infusion (source temperature and desolvation temperature were maintained at 80 °C and 250 °C, respectively). The samples were dissolved at 5 ?M concentration in water / acetonitrile (1/1) with 0.1% formic acid. The purity of 3, 4 and 5 was analyzed by RP-HPLC using an Agilent 1200 pump system with a UV detector at 220 nm. For 4, the column used was a Kromasil C18 (100 ?, 5 ?m, 4.6 x 250 mm) (AIT) and the gradient was performed with acetonitrile (VWR) (C) / water+0.1%TFA (VWR) (D) over 20 min at 20 °C. For 3 and 5, the column was an Aeris Widepore C4 column (3.6 ?m, 150 x 2.1 mm) (Phenomenex) and the gradient was performed with acetonitrile+0.1% TFA (E) and D over 20 min (25-70% E, column temperature at 80 °C and sample temperature at 40 °C).Gel filtration analyses were realized on an AKTA püre system equipped with a HILOAD 3.2/300 Superdex 75 column (GE Healthcare), with elution in PBS/NaCl.The overall yields include all the synthetic steps from the starting protein 1 or 3 in the affinity column elution buffer. They were calculated by dividing the actual amount of the final products 2a, 2b, and 5 by their expected amount (net protein contents).Synthesis of maleimido-(DOTA/Gd)3 4 The synthesis of 4 was performed stepwise on solid-phase from Fmoc-Gly-Wang resin (143 mg, 0.093 mmol). The building blocks 1,4,7,10-tetraazacyclododecane-1,4,7-tris-tbutyl-acetate-10-(N-?-Fmoc-N-?-acetamido-L-lysine) [Fmoc-Lys(DOTA(OtBu)3)-OH] (1.1 eq.) (Macrocyclics) and 6-maleimidohexanoic acid (3 eq.) were incorporated manually using 2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (1.06 and 2.9 eq., respectively) / diisopropylethylamine (DIEA) (2.2 and 6 eq., respectively) as coupling reagents and dimethylformamide (DMF) (Applied Biosystems) as solvent. Fmoc-Gly-OH (3 eq.) was incorporated with DIC (3 eq.) in DMF. The coupling steps with the Lys, Gly and maleimido derivatives were monitored by the Kaiser test (E. Kaiser et al (1980) Anal. Biochem. 34, 595-598) and were completed in, respectively, 3 h, 2 h and 1 h. Fmoc protection was removed with 20% piperidine in DMF. After the third lysine derivative, the last coupling with 6-maleimidohexanoic acid was carried out on three-quarters of the product (0.07 mmol).The peptide-resin was suspended in 10 mL of TFA (Applied Biosystems) / water / triisopropylsilane (95/2.5/2.5 v/v/v) at 4 °C and stirred for 4 h at room temperature. After filtration of the resin, the solution was concentrated and the crude product precipitated with diethyl ether. After centrifugation, the pellet was dissolved in water and lyophilized to yield 119 mg of the crude DOTA-peptide which was analyzed by NMR, MS, and RP-HPLC (gradient 10-40% C, retention time 9.2 min).1H NMR (D2O)?: ? 6.69 (s, 2H, CH Mal), 4.18 (m, 2H, 2CH?), 4.08 (m, 1H, CH?), 3.87-3.78 (m, 6H, CH2 Gly), 3.74-3.48 (b, 24H, CH2CO DOTA), 3.34 (t, 2H, CH2 6-Mal, J5,6=0,017 Hz), 3.30-2.99 (b, 48H, CH2CH2N DOTA), 3.06 (b, 6H, CH2?), 2.14 (m, 2H, CH2 2-Mal), ?1.74-1.53 (m, 6H, CH2?), 1.49-1.34 (m, 10H, CH2?, CH2 3-Mal, CH2 5-Mal), 1.29-1.18 (m, 6H, CH2?), 1.16-1.06 (m, 2H, CH2 4-Mal).13C NMR (D2O)?: ? 177.11 (1C, CONH Mal), 175.15, 174.63, 174.36 (3C, CO Lys), 173.26 (2C, CO Mal), 172.93 (1C, COOH Gly), 171.48, 171.21 (2C, CO Gly), 163.04-162.68 (4C, CONH DOTA), 134.22 (2C, CH Mal), 120.59, 117.69, 114.79, 111.90 (TFA), 55.20-53.10 (12C, CH2CO DOTA), 54.04, 53.80, 53.47 (3C, CH?), ?????-46.80 (24C, CH2CH2N DOTA), 42.38, 41.00 (3C, CH2 Gly), 39.10 (3C, CH2?), 37.37 (1C, CH2 6-Mal), 35.04 (1C, CH2 2-Mal), 30.48, 30.24, 30.23 (3C, CH2?), 27.67, 27.33, 24.67 (3C, CH2 3-Mal, CH2 5-Mal, CH2?), 25.43 (1C, CH2 4-Mal), 22.43, 22.33, 22.15 (3C, CH2?).MS: [M+H]+ 1,925.9888, [M+K]+ 1,963.9391 (C82H136N22O31 calcd [M+H]+ 1,927.1195, [M+K]+ 1,965.2098).The DOTA-peptide intermediate (99 mg) was dissolved in 0.4 M Na acetate buffer pH 5 (41 mL) and added with Gd(OAc)3.xH2O (123 mg, 2 eq. relative to DOTA). After stirring at 95 °C for 25 min, the solution was cooled and loaded on a C18 reverse-phase column (2 g, diameter 1.5 cm). The column was washed with four volumes of water and the product was eluted with three volumes of water/acetonitrile 1/1 affording 79 mg of product after lyophilisation. The crude DOTA/Gd peptide was purified by C18 reverse-phase flash chromatography (30x200 mm) using a gradient with acetonitrile+0.1%TFA / buffer D over 40 min, from 5/95 to 35/65 (20 mL/min, retention time 18 min). After lyophilization of the main fraction, 61 mg of 4 were obtained with an overall yield of 64% (the overall yield includes all the synthetic steps, it was calculated on the net peptide content of the isolated product 4 based on the first Gly residue loading on the resin). 4 was analyzed by MS and RP-HPLC (gradient 5-35% C, retention time 12.2 min, purity >95%).MS: 2,388.8889 (C82H127N22O31Gd3 calcd 2,388.7901).Biochemical analysesSDS-PAGE was performed using NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) according to manufacturer’s instructions.ELISA were realized on streptavidin-coated microtiter plates (Thermo Scientific,Denmark) coated by incubation overnight at 4 °C with 1 ?g/mL of biotinylated Aβ40 diluted in PBS. Plates were washed with buffer 0.1% Tween 20 in PBS. For the random strategy, R3VQ-NH2 1, R3VQ-N-(DOTA/Gd)0-2 2a and R3VQ-N-(DOTA/Gd)3-5 2b were diluted in buffer 0.5% gelatin 0.1% Tween 20 in PBS. After 1 h incubation at 37 °C, plates were washed again before adding respectively a rabbit anti-His-tag polyclonal antibody (eBiosciences), followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abcam), and finally revealed by OPD (o-phenylendiamine dihydrochloride, Dako) according to manufacturer's protocol. For the site-specific strategy, a freshly prepared batch of R3VQ-SH 3 (in the monomer form as analyzed by non-reducing SDS-PAGE) and the conjugate R3VQ-S-(DOTA/Gd)3 5 were diluted in the same buffer as described before, after incubation with biotinylated Aβ40, a monoclonal anti-His-tag antibody (H1029-Sigma) was added, followed by peroxidase labeled goat anti-mouse antibody (ab97265-Abcam), and revealed by the same substrate.The binding properties of 3 and 5 were determined by measuring the amount of soluble Aβ40 peptide able to give 50% inhibition of immobilized Aβ40 recognition. ADDIN EN.CITE <EndNote><Cite><Author>Friguet</Author><Year>1985</Year><RecNum>129</RecNum><DisplayText><style face="superscript">1</style></DisplayText><record><rec-number>129</rec-number><foreign-keys><key app="EN" db-id="a5sse95de0rtvgepeazvwts4wes2x5zd5x5d" timestamp="1473169549">129</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Friguet, B.</author><author>Chaffotte, A. F.</author><author>Djavadi-Ohaniance, L.</author><author>Goldberg, M. E.</author></authors></contributors><titles><title>Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay</title><secondary-title>Journal of Immunological Methods</secondary-title><alt-title>Journal of immunological methods</alt-title></titles><periodical><full-title>Journal of Immunological Methods</full-title><abbr-1>J. Immunol. Methods</abbr-1><abbr-2>J Immunol Methods</abbr-2></periodical><alt-periodical><full-title>Journal of Immunological Methods</full-title><abbr-1>J. Immunol. Methods</abbr-1><abbr-2>J Immunol Methods</abbr-2></alt-periodical><pages>305-19</pages><volume>77</volume><number>2</number><keywords><keyword>Antibodies, Monoclonal</keyword><keyword>*Antibody Affinity</keyword><keyword>*Antigen-Antibody Complex</keyword><keyword>Enzyme-Linked Immunosorbent Assay</keyword><keyword>Solutions</keyword><keyword>Spectrometry, Fluorescence</keyword><keyword>Tryptophan Synthase/immunology</keyword></keywords><dates><year>1985</year><pub-dates><date>Mar 18</date></pub-dates></dates><isbn>0022-1759 (Print)
0022-1759 (Linking)</isbn><accession-num>3981007</accession-num><urls><related-urls><url> Briefly, various concentrations of Aβ40 were incubated overnight at 4 °C with a defined quantity of each compound until equilibrium was reached. The VHH concentration used has been deduced from preliminary ELISA calibrations. Each mixture (100 ?L) was transferred to a well of microtiter plate previously coated with the antigen and was incubated for 15 min at 4 °C. After washing with PBS containing 0,1% Tween 20, unbound VHH were detected by the addition of an anti-His-tag mAb H1029 followed by β-galactosidase goat anti-mouse Igs and 4-methylumbelliferyl β-D-galactoside. Fluorescence was read (FLUOROSKAN, Labsystem, Finland) at 460 nm, after excitation at 355 nm.Production of the irrelevant VHHsAn irrelevant VHH targeting pTau epitopes (A2-SH) was obtained as previously described.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5MaTwvQXV0aG9yPjxZZWFyPjIwMTY8L1llYXI+PFJlY051
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ADDIN EN.CITE.DATA 2 The corresponding conjugate based on Gd (A2-S-(DOTA/Gd)3) was prepared following the same procedure as the one used for R3VQ-S-(DOTA/Gd)3 (compound 5).ImmunohistochemistryImmunohistochemistry was performed on PS2APP mouse paraffin coronal brain sections (5 ?m in thickness), obtained with a microtome (Microm HM 340E). Negative control was performed in parallel in age-matched wild-type littermates. Sections were de-paraffinized in xylene (5 min, 3 times), rehydrated through ethanol (100% x 2, 90 and 70%; 5 min/step) and brought to water. They were then pretreated with 98% formic acid for 5 min and finally re-hydrated in tap water for 5 min. Endogenous peroxidases were neutralized with 3% hydrogen peroxide and 20% methanol, and nonspecific binding sites were blocked with TBS-0.05% tween pH 8 BSA 2% for 30 min. In the following stages, the sections were rinsed 3 times, 5 min/time in TBS-tween between steps. The sections were then incubated overnight at 4 °C with the primary antibody (i.e. 1, 3 or 5) diluted to 1 ?g/mL in TBS-tween. Sections preincubated with 1 were then treated with rabbit anti-His-tag antibodies (eBioscience, 1/2000) and sections preincubated with 3 and 5 were treated with mouse monoclonal anti-His-tag antibodies (H1029-Sigma 1/200) for 2 h at room temperature, and finally developed with Dako REAL system Peroxidase/DAB Kit (Glostrup, Denmark) according to manufacturer's protocol. After washing with water, sections were counter-stained with Harris hematoxylin and re-rinsed in tap water. Before being mounted, sections were dehydrated in graded ethanol solution (70, 90 and 100%) and cleared in xylene. As a “gold standard”, evaluation of Aβ deposition was performed on adjacent brain sections using the reference anti-Aβ 4G8 (biotinylated 4G8, Covance Signet Antibodies, Debham, MA, USA, 1:10000) as a positive control in parallel. Immunostained sections were then numerized with a NanoZoomer 2.0-RS slide scanner (Hamamatsu Photonics, Hamamatsu, Japan).Evaluation of the MRI properties of the contrast agentsEvaluation of relaxivitiesT1 and T2 relaxivities of the contrast agents were evaluated at 1.4, 7 and 11.7 Tesla. Measures at 1.4 Tesla were performed on a MINISPEC mq60 (Bruker, Billerica, MA) at 37 °C. T1 values were measured using an inversion recovery sequence with 10?15 data points. T2 values were measured using a Carr–Purcell–Meiboom–Gill sequence with τ = 1 ms, and 200 data points. Each solution was incubated at 37 °C for 10 min before measurement to reach thermal equilibrium. The longitudinal relaxivity (r1) and the transverse relaxivity (r2) were determined as the slope of the line plotting 1/T1 or 1/T2 against Gd concentration (correlation coefficient greater than 0.99).Measures at 7 and 11.7 Tesla were performed on a 7T spectrometer (Agilent, USA) interfaced with a console running VnmrJ 2.3 and an 11.7T (Bruker, Germany) spectrometer interfaced with a console running PARAVISION 6. The 7T spectrometer was equipped with a rodent gradient insert of 700 mT/m, and a quadrature birdcage coil (diameter: 23 mm) was used for emission and reception. The 11.7T spectrometer was equipped with a rodent gradient insert of 760 mT/m used for emission, and a cryoprobe (Bruker, Germany) was used for reception. The samples were imaged in 60 ?L hematocrit tubes. Measures were realized in triplicate at 25 °C and expressed as the mean +/- SEM. T1 and T2 were quantified with slightly different parameters on the 7T and 11.7T spectrometers. For the 7T spectrometer, T1 calculation was based on six successive 2D multi-slice spin echo images with five Repetition Time (TR) values (400, 750, 1500, 3000, 5000 msec), Echo Time (TE)=14 msec, number of averages (Nex)=1, bandwidth=50 kHz, field of view (FOV)=25x25 mm2, matrix (Mtx)=128x128, slice thickness=1 mm. Parametric maps of relaxation times were calculated from exponential regression curves (S=1-exp(-TR/T1)) where S is the signal intensity, TR is the repetition time and T1 is the longitudinal relaxation time (ImageJ, MRI Analysis Calculator, Karl Schmidt). T2 calculation was based on 2D multi-echo multi-slice spin echo images with TR=3300 ms, sixteen TE (TE=10 to 160 msec), Nex=1, bandwidth=100 kHz, FOV=25x125 mm2, Mtx=128x128, 20 slices, slice thickness=0.5 mm. Parametric maps of relaxation times were calculated from exponential regression curves (S= exp(-TE/T2)) where S is the signal intensity, TE is the echo time and T2 is the longitudinal relaxation time (ImageJ, MRI Analysis Calculator, Karl Schmidt).For the 11.7T measures, T1 fast spin echo (RARE) sequence was used with the following parameters: six TR values (TR=300, 500, 800, 1500, 3000, 5000 msec), TE=7 msec, Nex=1, RARE factor=2, bandwidth=85 kHz, FOV=15x15 mm2, Mtx=256x256, slice thickness=1 mm. T2 calculation was based on the T2 multi slice multi echo (MSME) sequence used with the following parameters: TR=2500 msec, thirty TE values (TE=7.5 to 223 msec), Nex=1, RARE factor=2, bandwidth=96 kHz, FOV=15x15 mm2, Mtx=256x256, slice thickness=1mm. Parametric maps of relaxation times were calculated using the TopSpin software (Bruker, Germany).Amyloid plaques quantificationMagnetic resonance images were filtered with a kernel defined in matrix form as 1 1 1; 1 8 1; 1 1 1 with ImageJ freeware. ADDIN EN.CITE <EndNote><Cite><Author>Schindelin</Author><Year>2012</Year><RecNum>127</RecNum><DisplayText><style face="superscript">3</style></DisplayText><record><rec-number>127</rec-number><foreign-keys><key app="EN" db-id="a5sse95de0rtvgepeazvwts4wes2x5zd5x5d" timestamp="1466779278">127</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Schindelin, Johannes</author><author>Arganda-Carreras, Ignacio</author><author>Frise, Erwin</author><author>Kaynig, Verena</author><author>Longair, Mark</author><author>Pietzsch, Tobias</author><author>Preibisch, Stephan</author><author>Rueden, Curtis</author><author>Saalfeld, Stephan</author><author>Schmid, Benjamin</author><author>Tinevez, Jean-Yves</author><author>White, Daniel James</author><author>Hartenstein, Volker</author><author>Eliceiri, Kevin</author><author>Tomancak, Pavel</author><author>Cardona, Albert</author></authors></contributors><titles><title>Fiji: an open-source platform for biological-image analysis</title><secondary-title>Nature Methods</secondary-title></titles><periodical><full-title>Nature Methods</full-title><abbr-1>Nat. Methods</abbr-1><abbr-2>Nat Methods</abbr-2></periodical><pages>676-682</pages><volume>9</volume><number>7</number><dates><year>2012</year><pub-dates><date>Jul</date></pub-dates></dates><isbn>1548-7091</isbn><accession-num>WOS:000305942200021</accession-num><urls><related-urls><url><Go to ISI>://WOS:000305942200021</url><url> Then number of hypointense spots per mm2 was calculated by using a method similar to that reported by Jack et al.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5KYWNrPC9BdXRob3I+PFllYXI+MjAwNTwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA 5 Briefly, six coronal slices (antero-posterior direction), evenly spaced by 300 ?m, were selected and four circular ROIs (surface ~1 mm2 each) were drawn on each of these slices. The six slices were positioned so that the third slice was localized at the level of the anterior commissure. Hypointense spots were automatically detected using ImageJ freeware. The threshold was set to avoid nonspecific hypointense spots detection (i.e. MR noise), and to exclude elements that could be followed over more than two adjacent slices, or that had a tube-like shape, suggesting the presence of a blood vessel. SUPPLEMENTARY FIGURESFigure S1: Schematic representation of R3VQ, R3VQ-NH2 1 and R3VQ-SH 3. The R3VQ sequence (blue construct) includes 3 CDRs, 2 cysteines linked by a canonical disulfide bond (represented in red), and 4 lysines (A). R3VQ-NH2 1 bears a 6-Histidine tag (B). R3VQ-SH 3 contains from the N to the C terminus a 6-Histidine tag, a thrombin cleavage site (THR), R3VQ sequence followed by the heptapeptide G3SCSA containing the -SH function (C).Figure S2: Immunostaining of amyloid plaques by R3VQ-NH2 1.Brain slices of PS2APP mice incubated with 1 and revealed by an anti-His-tag antibody showed specific labeling of amyloid plaques.Figure S3: SDS-PAGE analysis of the VHHs and their conjugates.R3VQ-NH2 1, R3VQ-N-(DOTA/Gd)0-2 2a, R3VQ-N-(DOTA/Gd)3-5 2b, R3VQ-SH 3 and R3VQ-S-(DOTA/Gd)3 5. Molecular weights (kDa) are indicated on the left.The faint band observed for 2b can be due to the polydispersity of the compound, the higher density of the DOTA/Gd, and/or a partial precipitation during the sample treatment. Figure S4: MS analysis of 2b.Analysis (deconvoluted spectrum) of 2b (expected Mr=16,833.6735 (DOTA/Gd)2, 17,374.3049 (DOTA/Gd)3, 17,914.9363 (DOTA/Gd)4, 18,455.5677 (DOTA/Gd)5). Results demonstrated the high heterogeneity obtained with the random method on both DOTA and Gd levels. Peaks marked with an asterix correspond to incomplete Gd chelation (delta mass -154). The Mr correspond to the proteins with N-ter deleted Met and one disulfide bond. Figure S5: RP-HPLC and MS analyses of the maleimide-(DOTA/Gd)3 4.A) The elution profile is shown at 230 nm (milli-absorption units) and the purity is indicated in % area-under-the-curve. B) Positive ESI-MS m/z spectrum, acquired by direct infusion (CH3CN/H2O:50/50 + 0.1% FA), shows a mass and a typical isotopic profile (top) in agreement with the expected ones (bottom). The charge state of 3 is shown as an example. Each peak is labeled with the mass/charge (m/z) value in daltons.Figure S6: Characterization of R3VQ-SH 3 before and after mild reducing conditions, and of the nanoimaging agent 5.Analyses of R3VQ-SH 3 before (blue) and after mild reducing conditions (5 equivalent of TCEP; red), and of R3VQ-S-(DOTA/Gd)3 5 (green). A) RP-HPLC analyses. The elution profile are shown at 230 nm (milli-absorption units). B) SEC analyses. The elution profiles are shown at 280 nm (milli-absorption units). C) MS analyses (deconvoluted spectra; expected Mr=15,724.2820 for monomer, Mr=31,446.5640 for dimer and Mr=18,113.0720). The Mr correspond to the proteins with N-ter deleted Met and one disulfide bond.Figure S7: A-C. Anti-His-tag immunostaining of brain sections from PS2APP mice without R3VQ-based compounds. Brain sections were incubated with PBS (A), or with an irrelevant VHH targeting pTau epitopes: A2-S-(DOTA/Gd)3 (1 ?g/mL) (B) or A2-SH (1 ?g/mL) (C). No specific staining was observed. D-F. Anti-His-tag immunostaining of brain sections from wild-type amyloid-free mice incubated with 5 (1 ?g/mL) (D), or with the irrelevant VHH A2-S-(DOTA/Gd)3 (1 ?g/mL) (E) or A2-SH (1 ?g/mL) (F). No specific staining was observed. Scale bars: 250 ?m.Figure S8: IHC with anti-His-tag antibody on PS2APP brain paraffin sections obtained after intravenous administration of 5 (50 mg/kg, A) and after direct incubation of an entire brain fixed hemisphere with 5 (B). The detection of amyloid deposits (arrows) was similar in both conditions. DG: Dentate gyrus; hf: hippocampal fissure. Scale bars: main frame: 250 ?m; inset: 50 ?m.Figure S9: Detailed MR relaxometric parameters r1 and r2 (plotted in Figure 4).Measure of r1 and r2 (normalized per mM of Gd) were realized at 1.4, 7 and 11.7 Tesla for compounds 4 and 5 compared to a reference contrast agent (DOTA/Gd). Triplicate measures of r1 and r2 (in mM-1.s-1) are expressed as mean.REFERENCES ADDIN EN.REFLIST 1.Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods 1985; 77:305-19.2.Li T, Vandesquille M, Koukouli F, Dudeffant C, Youssef I, Lenormand P, Ganneau C, Maskos U, Czech C, Grueninger F et al. Camelid single-domain antibodies: A versatile tool for in vivo imaging of extracellular and intracellular brain targets. J Control Release 2016; 243:1-10.3.Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012; 9:676-82.4.Jack CR, Jr., Wengenack TM, Reyes DA, Garwood M, Curran GL, Borowski BJ, Lin J, Preboske GM, Holasek SS, Adriany G et al. In vivo magnetic resonance microimaging of individual amyloid plaques in Alzheimer's transgenic mice. J Neurosci 2005; 25:10041-8.5.Santin MD, Debeir T, Bridal SL, Rooney T, Dhenain M. Fast in vivo imaging of amyloid plaques using mu-MRI Gd-staining combined with ultrasound-induced blood-brain barrier opening. NeuroImage 2013; 79:288-94. ................
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