Additional file 1: Table S1. of Simple, mild, one-step labelling of proteins with gallium-68 using a tris(hydroxypyridinone) bifunctional chelator: a 68Ga-THP-scFv targeting the prostate-specific membrane antigen
Creators: name: Nawaz, Saima
name: Mullen, Gregory
name: Sunassee, Kavitha
name: Jayanta Bordoloi
name: Blower, Philip
name: Ballinger, James
Publisher: name: Figshare
Year: 2017
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 60506 Virology

description: Deconvoluted electrospray mass spectra of J591c-scFv pre- and post-reduction and conjugation with THP-mal. Table S2. Ex vivo biodistribution data (%ID/g) for 68Ga-THP-mal-J591-scFv in mice bearing DU145 (PSMA-) and Du145-PSMA (PSMA+) (mean and standard deviation are shown; n = 4 in each group). Figure S1. Electrospray mass spectra of J591c-scFv pre-TCEP treatment (top, 27,923.2 corresponds to disulfide formed from J591c linked to cysteine via a disulfide bond), J591c-scFv post-TCEP treatment (middle, 27,803.9 corresponds to J591-scFv), and TCEP-treated J591-scFv after incubation with THP-mal (bottom, 28,723.7 corresponds to THP-mal-J591-scFv adduct). Figure S2. The effect of different molar excesses of TCEP on dimerisation of J591-scFv. Lanes: 1: molecular weight markers; 2: non-reduced protein; 3: reduced protein (NuPAGE reducing agent); 4: 0.4:1 M ratio TCEP to protein; 5: 1:1 M ratio; 6: 2:1 M ratio; 7: 5:1 M ratio; 8: 10:1 M ratio; 9: 15:1 M ratio; 10: 20:1 M ratio; 11: 30:1 M ratio. Figure S3. FPLC purification of THP-mal-J591-scFv conjugate on Superdex 75 HR 10/30 size-exclusion column eluted with phosphate-buffered saline at 0.5 ml/min. Conjugate elutes before free THP-mal. Free THP-mal can be seen eluting at 15–18 min, monomeric THP-conjugated protein at 10 min and dimeric protein at 8 min. Figure S4. Exemplar data showing radiolabelling efficiency (% protein bound, determined by ITLC) at different protein conjugate concentrations and times. At concentrations of 0.25 mg/ml or higher, labelling efficiency was 100% at all time points from 10 s onward (the first data point in each series represents a sample taken after 10 s incubation). Figure S5. SDS-PAGE analysis of serum stability of 68Ga-THP-mal-J591-scFv. A: radioactive gel analysed with cyclone phosphor imager. B: gel stained with Coomassie blue. Lanes: 1: molecular weight markers; 2: human serum incubated with ammonium acetate-buffered 68Ga eluate; 3: radiolabelled 68Ga-THP-mal-J591-scFv conjugate control (without serum incubation); 4: conjugate incubated with human serum for 1 min; 5: 30 min; 6: 60 min; 7: 120 min; 8: 180 min; 9: 240 min; 10: 360 min. Figure S6. Time course of 68Ga-THP-mal-J591c-scFv activity (SUVmax) in DU145-PSMA xenografts derived from serial images in a single mouse determined by PET imaging. A region of interest near the centre of each organ was drawn from which SUVmax was determined in each 15 min bin. Figure S7. Ex vivo biodistribution data for 68Ga-THP-mal-J591-scFv in mice bearing DU145 (PSMA-, blue) and DU145-PSMA (PSMA+, red), 90 min post-injection. Error bars represent standard deviation (n = 4 per group). Data are the same as those shown in the main manuscript (Fig. 5) but expanded to include kidneys. (PDF 1307 kb)