Fig. 4

DesAb-O detects Aβ₄₂ oligomers in the CSFs of AD patients in vitro. A Representative sandwich dot-blot analysis of Aβ₄₂ species and CSFs. The capture Abs, 6E10 and DesAb-O were spotted onto nitrocellulose membranes (2 µl corresponding to 0.01 mg/ml and 10 µM). The membranes were incubated with solutions containing different Aβ₄₂ species (monomeric Aβ₄₂ (M), A + oligomers, fibrils (F1) at 0.01 mg/ml, the CSF from a representative AD patient (n = 9) and that from a representative control subject (n = 4) at 0.1 mg/ml. Finally, the membranes were probed with the detection 6E10 Ab. B Indirect sandwich ELISA. 0.25 mg/ml of CSFs from AD patients and control subjects were adsorbed and quantified by using DesAb-O at 1 µM. Standard curve was obtained with decreasing concentration of Aβ₄₂ species formed in vitro. αSyn monomeric protein was used as a negative control. Data were normalised for the corresponding average value at concentration 0 pg/ml. Experimental errors are S.E.M (n = 4 for synthetic samples and control CSFs and n = 9 for AD CSFs). Samples were analysed by Student t test relative to 0 pg/ml (*P < 0.05, **P < 0.01, ***P < 0.001) or to A + (§P < 0.05, §§P < 0.01, §§§P < 0.001) or to control CSF (°P < 0.05). C Representative STED images showing Aβ₄₂ species (M, A + , F1, a mixture containing A + and F1 at 1:1 molar ratio) and CSFs collected from AD patients and controls spotted in a glass coverslip at 25 µM and 0.5 mg/ml, respectively (n = 4 for synthetic samples and control CSFs and n = 9 for AD CSFs). The green fluorescent signals arise from the staining with 6E10 and DesAb-O Abs. Higher magnifications of the Aβ₄₂ species are shown in the boxed areas