Article Figures & Data
Figures
- Figure 1.
Expression and binding properties of anti-Aβ scFvs. HEK293T cells were transiently transfected with scFv9, scFv40.1, and scFv42.2 in pSecTag. A, The sequence alignment of the anti-Aβ scFvs. B, Western blot of a 1% Triton lysate and conditioned media, detected with anti-His primary antibody and anti-rabbit-HRP secondary antibody, showing expression of the anti-Aβ scFvs. C, Western blot of a pull down of conditioned media with fAβ, detected with anti-His primary antibody and anti-rabbit-HRP secondary antibody, showing that the anti-Aβ scFvs maintain the binding selectivity of the parent antibodies. D, Conditioned media from scFv9-, scFv40.1-, and scFv42.2-transfected cells was tested in an ELISA with Aβ40 or Aβ42 as capture and anti-c-myc–HRP as detection. *p < 0.01 versus control. E, Paraffin sections of Tg2576 mice brains were stained with conditioned media from scFv-transfected cells (bottom) and anti-His primary antibody or with a corresponding parent anti-Aβ mAb (top). Representative plaque staining is shown. Magnification, 200×.
- Figure 2.
Expression of an anti-Aβ scFv in the neonatal mouse brain using AAV1. A, P0 Swiss-Webster pups were injected intracerebroventricularly with AAV1–hGFP, a total of 4 × 1012 genomes. AAV expression in a mouse brain 3 weeks and 10 months after injection. Magnification: top, 40×; bottom, 200×. B, Newborn CRND8 mice were injected intracerebroventricularly with AAV1 scFv. After 3 weeks, brain paraffin sections were analyzed for scFv expression using anti-His primary antibody and anti-rabbit secondary antibody. Magnification, 200×. C, Representative Western blot of a rabbit anti-His immunoprecipitation from brains of scFv-treated CRND8 mice, detected with chicken anti-c-myc primary antibody and anti-chicken HRP secondary antibody, showing the anti-Aβ scFvs expressed in the brain.
- Figure 3.
Anti-Aβ scFvs attenuate Aβ deposition in 5-month-old CRND8 mice. Newborn CRND8 mice were injected intracerebroventricularly with AAV1 scFv9 and scFv42.2. Control mice received AAV1–hGFP. Five months later, mice were killed after treatment; one hemibrain was processed for immunohistochemistry, and the other was processed for biochemical analysis. A, Representative immunostained sections for amyloid plaques from brains of scFv-treated CRND8 mice. Magnification, 40×. B, Aβ levels in the SDS-soluble and SDS-insoluble FA-soluble fractions analyzed by Aβ sandwich ELISA. n = 5; *p < 0.05 versus control.
- Figure 4.
Anti-Aβ scFvs attenuate Aβ deposition in 3-month-old CRND8 mice. Newborn CRND8 mice were injected intracerebroventricularly with AAV1 expressing scFv9, scFv40.1, and scFv42.2. Control mice received AAV1–scFv ns or PBS. Three months later, mice were killed after treatment. One hemibrain was used for immunohistochemistry, and the other was used for biochemical analysis. A, Representative immunostained sections for amyloid plaques from brains of scFv-treated CRND8 mice. Magnification, 40×. B, Quantitative image analysis of amyloid plaque burden in the neocortex of scFv-treated CRND8 mice. *p < 0.05 versus control. C, Aβ levels in SDS-soluble extracts. D, An Aβ–scFv complex in plasma was detected by ELISA with a capture antibody specific to the free end of Aβ (for scFv9, mAb40.1; for scFv40.1 and scFv42.2, mAb9) and anti-myc–HRP as detection. n = 7; *p < 0.05 versus nonspecific scFv; **p < 0.01 versus nonspecific scFv; ***p < 0.005 versus nonspecific scFv. Error bars indicate SEM.
