Characterization of Tau ELISAs for Evaluating Tau Accumulation in the Brains With Alzheimer’s Disease


 Background: One main pathological hallmark of Alzheimer’s disease (AD) is tau accumulation as neurofibrillary tangles (NFTs) in the brain. Although sandwich enzyme-linked immunosorbent assays (ELISAs) are useful for quantifying tau levels, including those in CSF, plasma and brain, it has not yet been determined which antibody combination is the most appropriate for assessing the neuropathological accumulation of tau in the brain. Methods: We developed several sandwich tau ELISAs by introducing antibodies against several tau epitopes, including from its N-terminal and C-terminal regions, and evaluated tau levels depending on disease stage, brain areas, and other AD-related changes. Results: We observed that tau levels in insoluble brain fraction determined by each ELISAs differ depending on the epitopes of the antibodies: there is a trend that non-AD control samples yield relatively high signals when an antibody against the N-terminal region of tau is used. On the other hand, ELISAs combining two antibodies against the later-middle to C-terminal regions of tau produced substantially increased signals from AD samples, compared to those from non-AD controls. Such ELISAs better distinguish AD and non-AD controls, and the results are more closely associated with Braak NFT stage, Aβ accumulation, and neuroinflammatory markers. In addition, these ELISAs can reflect the pattern of tau spread across brain regions. Conclusions: Tau ELISAs that combine two antibodies against the later-middle to C-terminal regions of tau can better reflect neuropathological tau accumulation, which would enable to evaluate tau accumulation in the brain at a biochemical level.

accumulation in the brain as well as the subtle changes in bio uids (24)(25)(26)(27)(28)(29). On the other hand, although tau ELISAs are used to measure change in tau levels in CSF or blood (29)(30)(31)(32), they are rarely used to evaluate the amount of tau accumulation in the brain. To begin to use tau ELISAs to evaluate tau accumulation in the brain, it should be considered that the reactivity of these ELISAs could be very different depending on the epitopes recognized by antibodies, similar to the results obtained in evaluation of tau in CSF (32). Thus, by using several antibodies against N-terminal to C-terminal regions of tau, this study aims to identify the appropriate set of antibodies for use in tau ELISAs to evaluate neuropathological tau accumulation in the brain.

Methods
Human brain tissues-Postmortem brain tissues were obtained from the Brain Bank for Aging Research (BBAR) at the Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology under the approval of the institutional ethics committees. The BBAR routinely performs the standardized neuropathological evaluations as previously described (33,34). In the rst cohort, we analyzed the gray matter of the frontal cortex (Brodmann area (BA) = 8 or 9) of 24 AD patients and 36 non-AD controls whose demographics are shown in Supplementary Table 1. In the second cohort, we analyzed ve neocortical areas (dorsolateral prefrontal (BA = 9), orbitofrontal (BA = 12), inferior temporal (BA = 20), inferior parietal (BA = 39/40), and primary visual (BA = 17)), four limbic areas (posterior cingulate (BA = 31), entorhinal (BA = 28), amygdala, and insular), and ve subcortical areas (striatum (caudate), thalamus, cerebellum, diagonal band of Broca, and nucleus accumbens) of 18 individuals; these individuals included 5 normal elderly controls, 4 cases with plaque dominant senile change (PSC), 4 cases with NFT-predominant change (NFTC), and 5 AD cases. AD was de ned according to the BBAR de nition (Braak NFT stage ≥ IV and Braak senile plaque stage = C (or 3)) and the presence of clinical dementia (35). Among the cases who did not meet the criteria of AD, cases who had severe senile plaques (Braak senile plaque stage ≥ B (or 2)) were diagnosed with PSC (plaque dominant senile change), and cases who had signi cant NFT pathology (Braak NFT stage ≥ III) without severe senile plaques (Braak SP stage ≤ A (0 or 1)) were diagnosed with NFTC (NFT-predominant change) (36).
Sample preparation-We extracted proteins according to a previously-described method with some modi cations (37)(38)(39)(40). In brief, after the removal of meninges and blood vessels, brain specimens were pulverized in a prechilled (on dry ice) BioPulverizer (BioSpec) and homogenized with a polytron homogenizer (Kinematica) at a ratio of 10 ml/g of wetweight brain tissue in ice-cold RIPA lysis buffer (Millipore) containing 0.1% SDS, a complete protease inhibitor cocktail, and PhosSTOP phosphatase inhibitor cocktail (Roche) on ice. In a small pilot study, we also extracted brain tissue in icecold TBS with 1% Sarkosyl (Merck) containing a complete protease inhibitor cocktail, and PhosSTOP phosphatase inhibitor cocktail (Roche), instead of RIPA lysis buffer. After centrifugation at 100,000 g for 1 hour at 4 °C, the supernatant was aliquoted and stored at -80 °C (referred to as the RIPA-soluble fraction if RIPA was used, or the Sarkosylsoluble fraction if Sarkosyl was used). The residual pellet was rehomogenized in TBS plus 5 M guanidine hydrochloride (GuHCl), pH 7.6, and incubated with mild agitation for 12-16 hour at room temperature. After centrifugation at 15,000 g for 30 minutes, the resultant supernatant (referred to as the GuHCl fraction, or RIPA-insoluble fraction if RIPA was used or the Sarkosyl-insoluble fraction if Sarkosyl was used) was diluted with 9 volumes of TBS, aliquoted and stored at -80 °C until the analysis by ELISA.
Tau ELISAs-Tau (or total-tau) ELISAs were developed by combining several commercially available antibodies as follows: Tau13 (epitope: 20 One of these antibodies was used for capture, and the other antibody conjugated to biotin was used for detection. Recombinant human Tau-441 protein (Wako) was used as a standard. We also developed an ELISA against phosphorylated forms of tau (phospho-tau) by using Tau5 as a capture antibody and the biotin-conjugated mouse monoclonal AT270 antibody (epitope: phospho-tau 181, Thermo Scienti c) as a detection antibody. Synthetic peptides were generated: "TPPAPKT(p)PPSSGEPPPSLPTPPTREPKKVA" consisted of two partial fragments from 175 a.a. to 189 a.a. of Tau-441 including phosphorylated-threonine at 181 a.a. (corresponding to the residue of Tau-441), and from 213 a.a. to 227 a.a. of Tau-441. This peptide was used as standards. Colorimetric quanti cation was performed using an iMark plate reader (Bio-Rad) after incubations with horseradish peroxidase (HRP)-linked Avidin-D (Vector) or streptavidin-PolyHRP40 (Stereospeci c Detection Technologies), followed by incubation with 3,3',5,5'-tetramethylbenzidine substrate (Nacalai Tesque, Kyoto, Japan).
Quanti cation of other proteins-The levels of full-length Aβ40 (Aβ 1−40 ) and Aβ42 (Aβ 1−42 ), glial brillary acidic protein (GFAP), CD11b, and apoE were determined by ELISA as previously described (37,38,40). The RIPA-insoluble (GuHCl) fraction was used to measure Aβ40, Aβ42, and apoE levels, and the RIPA-soluble fraction was used to measure the GFAP, and CD11b levels. The levels of the total proteins in each fraction were determined by a Protein Assay BCA kit according to the manufacturer's instructions (Fuji lm).
Western blotting-GuHCl fraction samples were rst dialyzed with 8 M urea before electrophoresis. The samples were mixed with 4x Laemmli Sample Buffer (BIO-RAD) and were run on the Tris-Glycine electrophoresis system (NIHON EIDO Co., Ltd.). The immunoreactive bands by each tau antibody and the appropriate HRP-conjugated secondary antibodies were detected and quanti ed using a chemiluminescent imaging system, ImageQuant LAS 3000 (Fuji lm).
Statistical analysis -All the values measured by ELISAs were rst normalized to the total protein levels in the sample. Comparisons of these normalized values between AD cases and non-AD controls were performed by the Wilcoxon signed-rank test. The nonparametric Spearman rank correlation coe cient was used to summarize the degree of correlation between tau levels and neuropathological or biochemical measurements. ROC (receiver operating characteristic) curve analyses were performed considering AD as an event. All the statistical analyses were performed by JMP Pro (version 13.0.0; SAS, Cary, NC). P-values of less than 0.05 were considered signi cant.

Results
Pilot tests of tau ELISAs to analyze the brains of AD patients and non-AD controls.
We developed several tau ELISAs by combining several antibodies against epitopes raging from the N-terminal to Cterminal regions of tau (Fig. 1A). All these ELISAs showed good dose-dependent standard curves using recombinant human tau-441 protein ( Supplementary Fig. 1). We then tested samples pooled from the RIPA-insoluble fraction (i.e., GuHCl fraction) of the frontal cortex of 8 individuals with AD or without AD (control) by using these ELISAs. The results are summarized in Fig. 1B. When antibodies against the N-terminal to middle regions of tau were used in ELISA, the control sample showed relatively high levels of tau. In particular, including an antibody against the N-terminal region (i.e., Tau13) showed relatively high tau levels (25-400 ng/mg). In the ELISAs using Tau13 antibody, a mild difference between the control and AD samples was observed; AD sample showed approximately 1.2-to 5-fold higher levels of tau than those in the control sample. On the other hand, when antibodies against the middle to C-terminal regions of tau were used, the control sample showed relatively low levels of tau (below 100 ng/mg). More importantly, when antibodies against the later-middle (i.e., 218-225 a.a. of Tau-441: epitope of Tau5 antibody) to C-terminal regions of tau were combined, substantial differences between the control and AD samples were generally observed; AD sample showed more than 10-to 100-fold higher levels of tau than the control sample. These results suggest that tau ELISAs have different reactivities depending on the epitopes targeted by tau antibodies, which might affect the evaluation of tau pathology in brains of AD patients.
Detailed comparison of tau ELISAs to distinguish the brains of AD patients and non-AD controls.
To con rm our pilot results, we next analyzed individual RIPA-insoluble fraction of frontal cortex of AD and non-AD control brains (n = 60, Supplementary Table. 1) by using representative tau ELISAs that used Tau13 (epitope: N-terminal 20-35 a.a. of Tau-441) or OST (epitope: MTBR 323-363 a.a. of Tau-441) as the capture antibody. The results are shown in Fig. 2A. When Tau13 was used as a capture antibody, the brains of control cases showed relatively higher levels of tau (100-600 ng/mg, also shown in Supplementary Table 2), and the brains of AD patients showed 1.5-to 4-fold increases in tau levels, compared to those in control brains; moreover, this difference was not always signi cant. When OST was ued as a capture antibody, the brains of control cases showed relatively lower levels of tau, especially when antibodies against the middle to C-terminal regions of tau were used as detection antibodies (less than 100 ng/mg, also shown in Supplementary Table 2). Moreover, consistent with our initial results, ELISAs using antibodies against the later-middle to C-terminal regions showed robust differences between control and AD; the brains of AD patients had over 500-fold higher levels of tau accumulation than the brains of controls (also shown in Supplementary Table 2). To con rm that ELISAs using antibodies against the later-middle to C-terminal regions of tau would be better to distinguish AD patients and non-AD controls, we performed ROC curve analyses. Indeed, when OST antibody was used as a capture antibody and antibodies against the middle to C-terminal regions of tau were used as detection antibodies, the AUC was more than 0.96 (Fig. 2B). In particular, OST-77G7 ELISA showed the best result (AUC = 0.97). On the other hand, when Tau 13 was used as a capture antibody, the AUC was relatively lower, ranging from 0.65 to 0.79, and Tau13-Tau5 ELISA yielded the lowest AUC value (AUC = 0.65). The signi cant difference in AUC between each ELISA is described in detail in Supplementary Table 3. These results con rm that tau ELISAs using antibodies against the later-middle to C-terminal regions of tau can better distinguish AD patients and non-AD controls.
We also analyzed the RIPA-soluble fraction to determine how far this easily-extractable fraction can distinguish AD and non-AD controls by these ELISAs. We tested OST-77G7 and Tau13-Tau5 ELISAs, which showed the best and worst AUC, respectively, for the RIPA-insoluble fraction. When tested by Tau13-Tau5 ELISA, the control cases and AD patients showed almost similar values without a signi cant difference (p = 0.1283, Supplementary Fig. 2A). On the other hand, when tested by OST-77G7 ELISA, AD patients showed a signi cant approximately 1.5-fold increase (median value) in tau levels (p = 0.0053, Supplementary Fig. 2A). Notably, in OST-77G7 ELISA, a strong correlation was observed (r = 0.85) between RIPA-soluble tau and RIPA-insoluble tau, while the RIPA-soluble tau levels in some AD patients overlapped with those of controls ( Supplementary Fig. 2B). Indeed, in the ROC curve analysis to distinguish AD patients and non-AD controls, the AUC was 0.72 ( Supplementary Fig. 2C), which was weaker than that of the GuHCl fraction. These results indicate that the RIPA-insoluble (i.e., GuHCl) fraction is more suitable than the RIPA-soluble fraction for distinguishing AD patients and non-AD controls by this tau ELISA.
To assess whether other popular extraction methods, such as using Sarkosyl instead of RIPA, give a similar result, we also tested the Sarkosyl-insoluble fraction from a small number of subjects. In OST-77G7 ELISA, AD samples showed signi cantly higher Sarkosyl-insoluble tau levels than non-AD control samples (p = 0.0003), while in Tau13-Tau5 ELISA, there was no signi cant difference between these samples (p = 0.1046) ( Supplementary Fig. 3A). Notably, tau levels in the Sarkosyl-insoluble fraction measured by OST-77G7 ELISA correlated well with tau levels in the RIPA-insoluble fraction measured by the same OST-77G7 ELISA ( Supplementary Fig. 3C); in addition, these results showed good AUC values (AUC = 0.97) for distinguishing AD patients and non-AD controls, similar to those of the RIPA-insoluble fraction ( Supplementary Fig. 3D). These results indicate that our ndings can be applied to other popular extraction methods, including a method using Sarkosyl.
Correlation of tau ELISA results with NFT neuropathological stage and other AD-related neurodegenerative markers.
To determine whether these tau ELISAs indeed re ect tau accumulation in the brain, we analyzed the correlation between Braak NFT stage and tau levels in the RIPA-insoluble fraction determined by each ELISA. The results are summarized in Table 1. When Tau13 was used as the capture antibody, we observed mild-to-moderate correlations between tau levels by ELISA and Braak NFT stage (r = 0.35-0.67). In particular, tau levels measured by Tau13-Tau5 ELISA showed the lowest correlation (r = 0.35, p = 0.056, Fig. 3A). On the other hand, with ELISAs using OST as a capture antibody, we obtained better correlations with Braak NFT stage, especially when detection antibodies against the later-middle to Cterminal regions of tau were used (r > 0.80), including OST-77G7 ELISA (r = 0.81, p < 0.001, Fig. 3D). These ndings indicate that tau ELISAs using antibodies against the later-middle region to C-terminal regions of tau can better re ect the pathological accumulation of tau in the brain. We also analyzed the correlation with the levels of Aβ, in ammatory cell markers, GFAP and CD11b, and apoE protein.
These results are also summarized in Table 1, and representative results of the correlations of Tau13-Tau5 ELISA, and OST-77G7 ELISA are also shown as graphs in Fig. 3B, C, E, and F. In brief, while both Aβ40 and Aβ42 in the GuHCl fraction were increased in the brains of AD patients (Supplementary Table 2), Aβ40 in particular tended to have a better correlation with tau levels measured by ELISAs that combined antibodies against the middle region to C-terminal regions of tau (r > 0.60), likely because Aβ40 increases during AD progression while Aβ42 reaches a plateau at an early stage (39). Regarding in ammatory markers, we con rmed that the levels of in ammatory cell markers tended to be increased in AD patients compared to controls (GFAP levels were signi cant, but CD11b levels only exhibited a trend, Supplementary Table 2). The results of the ELISAs using Tau13 as a capture antibody were generally not well correlated with the levels of glial markers (GFAP: r = 0.04-0.19; CD11b: r = -0.01-0.12). On the other hand, the results of ELISAs using OST as a capture antibody tended to be better correlated with the levels of these markers (GFAP: r = 0.21-0.45; CD11b: r = 0.09-0.42). In particular, their correlations with tau levels measured by OST-77G7 ELISA were signi cant (GFAP: r = 0.45, p = 0.003; CD11b: r = 0.42, p = 0.008). It is known that apoE accumulates on NFTs, especially extracellular NFTs (41,42), in addition to amyloid plaques. Since accumulated apoE could be evaluated in the GuHCl fraction (38), we analyzed the correlation with apoE levels in the GuHCl fraction, and we observed that the apoE levels were better correlated with tau levels measured by ELISAs using OST as a capture antibody (r = 0.43-0.60), rather than those using Tau13 (r = 0.21-0.37). These results indicate that Tau ELISAs with antibodies against the later-middle to C-terminal regions of tau (especially OST-77G7 ELISA) can better re ect AD-associated pathological changes, including Aβ, in ammatory cells, and apoE accumulation in addition to tau accumulation.

Distinct reactivity of tau antibodies by western blotting analysis
To address the reason why ELISA results are different depending on the epitopes targeted by tau antibodies, we performed western blotting analysis. In the GuHCl fraction, tau was generally detected as the monomer form (50-64 kDa) and aggregated form (> 64 kDa) by western blotting (Fig. 4A). When an antibody against the N-terminal region of tau was used (i.e., Tau13), there was a trend that the monomer form of tau was more clearly visible than its aggregated form. On the other hand, when antibodies against the middle to C-terminal regions of tau were used, the aggregated form was clearly observed in AD patients, especially with antibodies against MTBR to the C-terminal region of tau (i.e., 77G7 and Tau46); these results were con rmed by densitometric analysis (Fig. 4B). These difference in the reactivity of tau depending on antibody recognition of epitopes from the N-terminal to C-terminal regions of tau might explain the observed difference in the ELISA results.
Tau accumulation across brain regions during disease development To address whether these tau ELISAs can evaluate the pattern of tau spread during AD development, we analyzed multiple brain regions of 18 individuals with different stages of AD (demographic information of each case is shown in Supplementary Table 4). We used OST-77G7 ELISA as one of the best tau ELISAs. Results are shown in each individual case (Fig. 5). Indeed, AD patients showed increased insoluble tau levels in several brain regions, including the limbic areas and neocortical areas, but less remarkable levels in several subcortical areas, including the striatum and thalamus, and cerebellum. On the other hand, individuals with early AD pathology (PSC or NFTC), showed increased insoluble tau levels only in the entorhinal cortex, but not apparent in other brain regions. Interestingly, some of these individuals showed somewhat increased insoluble tau levels in the amygdala and temporal cortex, although the extent of tau levels was much fewer compared to that in the entorhinal cortex. The control groups (Braak NFT stage I) generally showed low insoluble tau levels. Notably, one individual showed apparent insoluble tau levels in the entorhinal cortex (Cont#5). When Tau13-Tau5 ELISA was used, there were no such trends recapitulating the progression of Braak NFT stage ( Supplementary Fig. 4). These results indicate that OST-77G7 ELISA can address the pattern of tau spread pattern across brain regions during AD development.

Discussion
Compared to biomarker studies that analyze tau levels in CSF or plasma, the number of studies utilizing ELISAs to detect tau accumulation in brains is much smaller. However, previous studies exist that used ELISAs to detect tau levels in the brain (43)(44)(45)(46)(47)(48)(49). Direct ELISAs, where brain homogenate or puri ed paired helical lament (PHF) is coated on microwells and then detected by anti-tau antibodies, were rst developed by several groups (43)(44)(45)(46). One major disadvantage of direct ELISAs is high background signals due to the nonspeci c binding of samples to the plate, which could overestimate or underestimate the amount of target proteins even if standard proteins are used. Indeed, most of these studies neither used standard proteins nor calculated tau levels (44)(45)(46). Sandwich ELISAs have also been developed by some researchers to detect phosphorylated tau levels in the brain (47)(48)(49). However, they used a buffer -soluble (i.e., no detergent) fraction of brain tissue, which might signi cantly underestimate the amount of tau accumulation that is highly insoluble (50). Additionally, some phospho-speci c tau antibodies could lose their epitope in the advanced stage of NFT formation (14, 16, 51, 52). On the other hand, there are few studies using sandwich ELISAs to detect accumulation of total tau (i.e., using buffer-or detergent-insoluble fractions) in the brain, except for our previous study that used OST-HT7 ELISA (38). However, our previous study also did not elucidate whether OST-HT7 ELISA is the best option, or whether other ELISAs exist that can better evaluate neuropathological tau accumulation.
Thus, the current study comprehensively compared combinations of tau antibodies, and observed that ELISAs using antibodies, especially against MTBR to C-terminal regions of tau can assess neuropathological tau accumulation that well distinguish AD patients and non-AD controls, and correlate with Braak NFT stage, Aβ accumulation, and neuroin ammation. Our results appear to be consistent with the previous studies showing that MTBR to C-terminal regions form a core and aggregate as PHF, while the N-terminal region is easily truncated (20,53). Interestingly, secreted tau in the CSF or cellular medium mostly loses its C-terminal regions, likely from the region corresponding to the epitope of Tau5 (54,55). This truncation physiologically occurs irrespective of the disease, while Aβ accumulation would promote the secretion (55,56). Although previous studies have observed that tau ELISAs using a C-terminal antibody are not appropriate for detecting secreted tau in CSF (32), it would be interesting to examine whether the currently optimal ELISAs to detect tau in the brain, such as OST-77G7 ELISA, indeed cannot detect tau levels in CSF.
Western blotting analyses showed a clear difference in the reactivity of each tau antibody with aggregated tau and monomer tau in the insoluble fraction of brains: the reactivity of aggregated tau to monomer tau is increased from antibodies targeting the N-terminal epitope to antibodies targeting the C-terminal epitope (Fig. 4), which likely explains the difference in reactivity of each ELISA. One plausible explanation for this result is that antibodies targeting the Nterminal epitope of tau cannot recognize aggregated tau due to the absence of the N-terminal region (20,53). Additionally, monomer tau in the insoluble fraction appears to be less recognized by antibodies against the middle to Cterminal regions of tau, compared to an antibody against the N-terminal region, suggesting that the middle to C-terminal regions of monomer tau in the insoluble fraction would be masked for unknown reasons. Further studies are necessary to address the mechanism.
One advantage of sandwich ELISAs is their quantitative feature. The Braak NFT staging system assigns seven stages based on the topological distribution of tau, with the limitation that such a semiquantitative method is insu cient to precisely estimate the degree of tau accumulation in each area (16). Indeed, we observed a wide range of tau levels with OST-77G7 ELISA among AD patients with the same Braak stage VI (in the frontal cortex of the rst cohort: 1,630 to 245,100 ng/mg, Supplementary Table 2). Notably, a few control individuals pathologically diagnosed by Braak NFT stage I indeed had a signi cant amount of tau accumulation in the entorhinal cortex (Fig. 5), suggesting that our ELISA method could be more sensitive for detecting neuropathological tau accumulation in the brain than conventional pathological assessment. In future studies, it would be interesting to address its relationship with cognitive decline, disease progression, and other tauopathies. Nonetheless, this method would be useful to determine the degree of pathological tau accumulation, in addition to the diagnosis of neuropathological Braak stage.
By analyzing multiple brain regions, we observed that the pattern of tau spread is consistent with the scheme of Braak NFT stage: tau rst accumulate in the entorhinal cortex (Braak stage I-II), then involves the limbic area or its-surrounding areas (Braak stage III-VI), and nally reaches neocortical areas (Braak stage V-VI) (8). Such analysis of region-speci c patterns of AD pathologies would provide important insights into the pathological mechanism of the disease, as shown in our recent studies focusing on Aβ accumulation (37)(38)(39)57). Thus, when combined with additional data, such as region-speci c Aβ data or comprehensive gene expression data, the current data would be useful to address the pathological mechanism of region-speci c tau accumulation as well as the crosstalk between Aβ and tau accumulation.

Limitations
One potential limitation in the current study is the lack of evaluation of phospho-tau accumulation. In general, it is di cult to evaluate phosphorylated proteins in postmortem brains due to their instability through dephosphorylation during autopsy, sample storage or extraction processes (58)(59)(60). Notably, some phospho-tau epitopes might also be lost in the advanced stage of NFT formation (14,16,51,52). Despite such disadvantages, we tested one phospho-tau ELISA using AT270 antibody, which is widely used to detect phospho-tau in CSF and plasma (32,61,62). This phospho-tau ELISA indeed showed weaker performance (AUC = 0.80) in distinguishing AD patients and non-AD controls, compared to total tau levels by OST-77G7 ELISA ( Supplementary Fig. 5). Future studies are necessary to address whether other phospho-tau ELISAs also show different performances depending on the epitope targeted by the antibodies and to determine which total tau ELISA or phospho-tau ELISA is better for evaluating neuropathological tau accumulation.

Conclusions And Future Directions
In conclusion, we evaluated several total tau ELISA, and observed that ELISA combining antibodies against the latermiddle to C-terminal regions of tau can better address neuropathological tau accumulation that associates with other neurodegenerative changes during AD development. The current nding proposes useful total tau ELISA methods for evaluating neuropathological tau accumulation in the brain, which will facilitate understanding the pathological mechanism as well as evaluating therapeutic e cacy in future studies focusing on tau accumulation.

Consent for publication
All participants or study partners/caregivers gave written informed consent to participate in this study for scienti c purposes, including publications. The informed consents are available from the corresponding author upon reasonable request. All reported data are anonymized, and no individual participant information can be identi ed from the presented datasets.

Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information les.

Competing interests
The authors declare that they have no competing interests.     describes each brain region analyzed in the current study, strati ed by neocortical areas, limbic areas, and subcortical areas. Blue notation indicates neocortical areas; purple notation indicates limbic areas; and orange notation indicates subcortical areas. The demographic information of each subject is described in supplementary Table 4.

Supplementary Files
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