Fig. 12

4E6 and 6B2 differ in their binding to human derived PHF material. a Different tau species were spotted onto nitrocellulose and incubated with either 4E6 or 6B2 as the primary antibody. 4E6 bound better to solubilized PHF but 6B2 reacted more strongly with the sarkosyl insoluble tau fraction. Both antibodies had limited reactivity with sarkosyl soluble tau protein. b The same three tau fractions were prepared from control brain, and spotted onto nitrocellulose. Neither antibody showed binding to the sarkosyl soluble fraction, and only limited binding to the solubilized PHF and sarkosyl insoluble tau. (Images for all three samples for the 4E6 and 6B2 treated control brains were taken from the same strip, the order has been changed for clarity.) c Plates were coated with solubilized PHF from AD and control brains. 6B2 showed significantly higher binding to AD than control, and than 4E6 to either AD or control at dilutions, 1/200-1/125 (p < 0.0001–0.05). 4E6 did not show significantly higher binding to AD versus control. d The assay plate was coated with sarkosyl soluble tau from AD and control brains, and serial dilutions of 4E6 and 6B2 were added. At the 1/200 dilution 6B2 showed significantly higher binding to AD than control, and higher binding than 4E6 to either fraction (p < 0.01, 0.05 and 0.05 respectively). e Assay plates were coated with sarkosyl insoluble tau. 6B2 showed significantly higher binding to AD relative to control, and than 4E6 to either AD or control, from dilutions 1/200-1/125 k (p < 0.0001–0.05). As before, no significant differences between AD and control sample were seen with 4E6. f Competitive ELISA assays were performed by pre-incubating the antibodies with increasing concentrations of solubilized PHF material (0.01 -1 μg/ml). 6B2 binding was not inhibited at any PHF concentration. However, 4E6 binding was inhibited in a dose-dependent manner with an IC50 of 71 nM. All of the results show that 4E6 preferentially binds solubilized tau species, while 6B2 primarily binds to insoluble highly aggregated tau. All columns or points on each graph have SEM error bars, however some of those are too small to be visible