Hi-Affi™ Rabbit Anti-APP Recombinant Antibody (clone M204) (CAT#: PABX-014)

Figure 1 ELISA showing immunoreactivity of monoclonal M204 antibody with chromatographed S75 fractions containing tau-K18 oligomer (fractions 44-49, colored red), but not tau monomer (fractions 58-61, colored black).

A volume of 20 µl of the indicated SEC fraction was diluted into 100 µl of coating buffer and applied to ELISA plate. In fractions 46 and 61 (the highest peaks), 20 µl = 100 ng of protein/well.

Abskharon, R., Seidler, P. M., Sawaya, M. R., Cascio, D., Yang, T. P., Philipp, S., ... & Eisenberg, D. S. (2020). Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease. Journal of Biological Chemistry, 295(31), 10662-10676.

Figure 2 ELISA showing immunoreactivity of monoclonal M204 with chromatographed S200 fractions containing tau40 oligomer (fractions 10-15, colored red) but not tau monomer (fractions 17-19, colored black).

A volume of 50 µl of the indicated SEC fraction containing tau40 oligomer (fractions 8-15) were diluted into 100 µl of coating buffer. For tau40 monomer fractions 17 and 19, 10 µl was used to coat ELISA plates rather than 50 µl because the peak monomer fraction was about 5 times the intensity of the oligomer peak. For reference, the amount of protein used to coat the ELISA plate in fractions 13 and 19 (the highest peaks) was 100 ng of protein.

Abskharon, R., Seidler, P. M., Sawaya, M. R., Cascio, D., Yang, T. P., Philipp, S., ... & Eisenberg, D. S. (2020). Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease. Journal of Biological Chemistry, 295(31), 10662-10676.

Figure 3 Epitope mapping using a peptide array with overlapping synthetic tau peptides shows M204 binding most strongly to peptides containing the sequences KVQIINK and SVQIVY (in red).

Abskharon, R., Seidler, P. M., Sawaya, M. R., Cascio, D., Yang, T. P., Philipp, S., ... & Eisenberg, D. S. (2020). Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease. Journal of Biological Chemistry, 295(31), 10662-10676.

Figure 4 Seeding by IL15-mediated tau-K18 purified oligomers in HEK293 biosensor cells expressing YFP-tagged tau-K18. C, as in B, except following treatment of tau-K18 oligomer with 10 μm M204 antibody.

Seeding inhibition by M204 is interpreted based on the appearance of fewer puncta compared with B. Representative cells containing aggregated tau are marked by red arrows, and cells without by white arrows.

Abskharon, R., Seidler, P. M., Sawaya, M. R., Cascio, D., Yang, T. P., Philipp, S., ... & Eisenberg, D. S. (2020). Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease. Journal of Biological Chemistry, 295(31), 10662-10676.

Figure 5 Quantification of seeding from representative images shown in B and C.

Tau oligomer seeding and inhibition by M204 antibody were determined by calculating the number of normalized puncta per well of a 96-well-plate. Statistical analysis was performed using one-way ANOVA (****, p < 0.0001; ***, p < 0.0006; **, p < 0.003) and Tukey's multiple comparison in GraphPad Prism. Error bars show the S.D. of three replicates.

Abskharon, R., Seidler, P. M., Sawaya, M. R., Cascio, D., Yang, T. P., Philipp, S., ... & Eisenberg, D. S. (2020). Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease. Journal of Biological Chemistry, 295(31), 10662-10676.

Figure 6 Western blotting of tissue from the brain of a donor with AD, fractionated into: crude brain homogenate, Sarkosyl-insoluble, and Sarkosyl-soluble fractions, each probed with M204 or A11 anti-oligomer antibodies.

Abskharon, R., Seidler, P. M., Sawaya, M. R., Cascio, D., Yang, T. P., Philipp, S., ... & Eisenberg, D. S. (2020). Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease. Journal of Biological Chemistry, 295(31), 10662-10676.

Figure 7 M204-scFv antibody delays in vitro aggregation.

B-D, ELISA showing immunoreactivity of the corresponding tau-K18 oligomer fractions from A with M204-scFv (B) monomer, (C) dimer, and (D) trimer. E-G, M204-scFv mediated inhibition of tau-K18 aggregation. Aggregation was measured by ThT fluorescence with M204-scFv added at molar ratios of 1:1 and 1:0.5 (tau-K18: M204-scFv), as indicated. Inhibition of IL15-induced tau aggregation by M204-scFv is shown for the M204-scFv monomer (E), dimer (F), and trimer (G). Tau-k18 is a positive control that was run in parallel but without the addition of M204-scFv, and is the same in aggregation experiments shown in E-G, colored black.

Abskharon, R., Seidler, P. M., Sawaya, M. R., Cascio, D., Yang, T. P., Philipp, S., ... & Eisenberg, D. S. (2020). Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease. Journal of Biological Chemistry, 295(31), 10662-10676.

Figure 8 B-E, quantification of the inhibitory effect of M204-scFv antibodies on inhibition of seeding by Sarkosyl-insoluble fractions from AD brain tissue, measured in HEK293 biosensor cells expressing YFP-tagged tau-K18.

Statistical analysis was performed using one-way ANOVA (****, p < 0.0001; ***, p < 0.0002; **, p < 0.001; *, p < 0.01, n.s., no significance) followed by a Tukey's multiple comparison test in GraphPad Prism. Error bars show the S.D. of three replicates.

Abskharon, R., Seidler, P. M., Sawaya, M. R., Cascio, D., Yang, T. P., Philipp, S., ... & Eisenberg, D. S. (2020). Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease. Journal of Biological Chemistry, 295(31), 10662-10676.

Figure 9 Representative images of seeding and inhibition in HEK293 biosensor cells expressing YFP-tagged tau-K18.

Cells seeded with the Sarkosyl-insoluble fraction from AD Donor 1 without pre-treatment with M204-scFv (left panel) or no inhibitor (No I), and following overnight incubation with M204-scFv trimer, dimer, or monomer (as indicated). The seeding can vary dramatically from different regions of a given brain tissue section, and this reflects a naturally occurring nonuniform distribution of tau pathology in the brain. Representative cells that contain aggregates are marked by red arrows, and cells without by white arrows.

Abskharon, R., Seidler, P. M., Sawaya, M. R., Cascio, D., Yang, T. P., Philipp, S., ... & Eisenberg, D. S. (2020). Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease. Journal of Biological Chemistry, 295(31), 10662-10676.

Figure 10 A-D, tests of the effectiveness of M204-scFv for inhibition of tau fibril seeding by diseased brain extracts in our HEK293 biosensor cell-based assay.

M204-scFv dimer and trimer inhibit seeding by extract from (A) CTE Donor 1, but not (B) CTE Donor 2, (C) CBD donor, or (D) a donor with a P301L familial mutation. Statistical analysis was performed using one-way ANOVA (****, p < 0.0001; ***, p < 0.0008; **, p < 0.005; *, p < 0.01, n.s., no significance) followed by a Tukey's multiple comparison test in GraphPad Prism. Error bars show the S.D. of three replicates.

Abskharon, R., Seidler, P. M., Sawaya, M. R., Cascio, D., Yang, T. P., Philipp, S., ... & Eisenberg, D. S. (2020). Crystal structure of a conformational antibody that binds tau oligomers and inhibits pathological seeding by extracts from donors with Alzheimer's disease. Journal of Biological Chemistry, 295(31), 10662-10676.