Figure 1.
Detection of tau fragments in human CSF.
Human control and AD CSF subjected to RP-HPLC, fractions collected and run on SDS-PAGE gels followed by western-blotting with different tau antibodies. A) HT7 (mid domain antibody). B) Tau 12 (N-terminal antibody). C) K9JA (C-terminal microtubule repeat domain antibody). D) IgG1 isotype control. On each blot, human recombinant tau441 (tau) is included in lane 1 and molecular weight markers (mw) in lane 2 followed by the HPLC fractions from 1 to 6 or HPLC fractions 7 to 11. Fractions 1 and 2 were pooled and run as a single sample, while fractions 3-10 were run as individual samples. Control CSF (C) and AD CSF (D) samples for each fraction were run side by side for comparison.
Figure 2.
Schematic of tau 441 protein with the approximate location of various linear epitope antibodies Tau12, HT7, BT2, Tau5, AT120 and 77G7 and phospho-site specific antibodies AT270 (p181) and PHF6 (p231) indicated; additional antibody epitope and clone information included in Table 1. Antibody combinations used for the different tau and ptau ELISAs are shown. For each assay, capture antibodies are highlighted in red, detection antibodies in black and the minimal tau region required (aa numbering based on tau 441) is indicated. Antibodies used in the INNOTEST/INNO-BIA AlzBio3 total tau and p181 tau assays are also shown for comparison.
Figure 3.
Characterization of tau ELISAs.
Representative tau 441 standard curves (left panels) and CSF dilution linearity results (right panels) shown for A) HT7-BT2, B) HT7-Tau5, C) Tau12-BT2 and D) Tau12-HT7 tau ELISAs. On each standard curve graph, tau 441 calibrators (Standards) and results for CSF sample dilutions (Samples) are shown. The assay lower limit of quantitation (LLQ, vertical dashed line) is also indicated. On each dilution linearity graph, dilution-corrected tau levels for a pooled control CSF sample and a pooled AD CSF sample relative to sample dilution are shown. The vertical dashed lines indicate the dilution determined to be optimal for CSF analysis.
Figure 4.
Characterization of HT7+77G7 tau ELISA.
Representative tau 441 standard curve (left panel) and CSF dilution linearity results (right panel) shown. On the standard curve graph, tau 441 calibrators (Standards) and results for CSF sample dilutions (Samples) are shown. The assay lower limit of quantitation (LLQ, vertical dashed line) is also indicated. On the dilution linearity graph, tau levels for a pooled control and pooled AD CSF samples tested with or without a 100 pg/ml tau 441 spike are shown.
Figure 5.
Characterization of ptau assays.
Representative ptau standard curves (left panels) and CSF dilution linearity results (right panels) shown for A) HT7-AT270, B) HT7-PHF6, and C) Tau12-AT270 ptau ELISAs. On each standard curve graph, ptau calibrators (Standards) and results for CSF sample dilutions (Samples) are shown. The assay lower limit of quantitation (LLQ, vertical dashed line) is also indicated. On each dilution linearity graph, dilution-corrected ptau levels for a pooled control and a pooled AD CSF samples relative to sample dilution are shown. The vertical dashed lines indicate the dilution determined to be optimal for CSF analysis.
Figure 6.
Tau and ptau levels in 20 AD and 20 control CSF samples.
A set of 20 AD and 20 age-matched normal control CSF samples were analyzed using the tau ELISAs (HT7-BT2, HT7-Tau5, Tau12-BT2, Tau12-HT7 and HT7-77G7) and pTau ELISAs (HT7-AT270, HT7-PHF6 and Tau12-AT270). Dashed lines indicate the assay LLQ corrected for CSF dilution. Statistics based on 2-tailed Student’s t test comparison of log-transformed data, * p < 0.05; ** p< 0.01; *** p < 0.001.