Fig 1.
Characterization of GFAP-specific binding protein E9 and designed molecules E9-GS-ApoE (EGA) and E9-EAK-ApoE (EEA).
(A) Predicted structures and characteristics of E9, EGA and EEA. Ribbons were colored with predicted local distance difference test score per position calculated at the same time. MW: Molecular weight, pI: Isoelectric point, N: N-terminal, C: C-terminal. (B) SDS-PAGE analysis of purified E9, EGA and EEA. L: ladder, 1: E9, 2: EGA, 3: EEA. (C) ELISA analysis for comparison of E9, EGA and EEA affinity to GFAP from rat brain homogenates. OD: Optical density.
Fig 2.
Immunohistochemical staining of rat brain sections (cortex) with general GFAP antibody GA5 and three affinity proteins.
(A) E9, (B) EGA, and (C) EEA were used for immunostaining and compared with GA5-positive GFAP stains. White arrows indicate the representative position of EGA binding that is not GFAP. Scale bars: 100 μm.
Fig 3.
Cell-free protein radiosynthesis of protein tracers.
(A) Brief description of 18F protein labeling method [41]. (B) Gel autoradiography to confirm synthesis of 18F-labeled proteins. A red arrow indicate the position of [18F]FET that typically remains at the top of the NuPAGE gel during this electrophoresis. 1: Before synthesis, 2–6: After synthesis [2: no DNA, 3: 18F-E9, 4: 18F-EGA, 5: 18F-EEA].
Fig 4.
In Vitro Autoradiography in Rat Brain Sections of Neuroinflammation Model.
(A-C) Autoradiographic images of 18F-E9 (A), 18F-EGA (B) and 18F-EEA (C) in rat brain sections of LPS-induced neuroinflammation model. Images of each row are displayed with the same contrast and obtained from the same experiment using the same lot rat brain sections and protein tracers (D-F). Quantitative analysis of images from A (D), B (E), and C (F).
Fig 5.
PET imaging of neuroinflammation rat models with 18F-EEA.
(A) Experimental flow for PET scan with 18F-EEA. (B) Time activity curve of 18F-EEA in LPS-injected and contralateral side of LPS-induced rat models. (C) Accumulated PET images of LPS-induced rat models at 2–32 min and 120–180 min post intravenous administration of 18F-EEA. SUV = standardized uptake value. (D) Uptake of 18F-EEA in the left and right brains of rat models at 180 min after 18F-EEA intravenous injection.