P-tau immunoassay

Abstract

The present embodiments relate to an immunoassay kit capable of measuring p-tau205 in a sample and to methods involving the use of such an immunoassay kit. The present embodiment also relates to a monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to p-tau205 and that can be used in such an immunoassay kit.

Description

[0001] P-TAU IMMUNOASSAY TECHNICAL FIELD The invention generally relates to an immunoassay specifically measuring p-tau205 and to the use thereof in monitoring AD and tau pathology across other neurological disorders. BACKGROUND Neuropathological examination confirming the presence of Aβ plaques and tau NFTs remains the gold standard for definitive diagnosis of AD. Aβ plaques are the result of the aberrant accumulation of Aβ peptides in the extracellular space, while NFTs are constituted by intraneuronal fibrils of abnormally phosphorylated tau protein. The spatiotemporal spreading pattern of Aβ plaques and NFTs in brain are referred to as Thal and Braak stages, respectively. Interestingly, unlike Thal amyloid stages, Braak stages have been shown to correlate with disease progression and cognitive decline. Post-mortem, Braak stages are commonly determined by immunostaining using AT8, an antibody obtained by immunizing mice with PHF or PHF-tau, which targets phosphorylation at amino acids serine 202, threonine 205 and serine 208. In recent years, the development of ultra-sensitive immunoassays and MS methods have greatly expanded the knowledge regarding p-tau species in fluid, and most importantly, highlighted their potential value of as biomarkers for AD. Several studies using immunoassays have shown that most p-tau species, i.e., p-tau217, p-tau231 or p-tau235, share common features: all are highly specific for AD, they increase during preclinical AD, and associate well with both amyloid and tau pathology as measured with PET. However, they do present some dissimilarities. For example, p-tau217 has been shown to display the largest fold-changes and various reports indicate it may provide the best performance for AD diagnosis and disease monitoring. p-Tau231 has been demonstrated to be the earliest p-tau biomarker to abnormally emerge during preclinical AD stages, even prior to amyloid pathology becoming abnormal using PET. p-Tau235 has been proposed as a biomarker capable of staging preclinical AD, due to its involvement in a sequential phosphorylation event observed in neuropathology confirmed brain tissue. WO 2022 / 212756 discloses methods to quantify tau phosphorylation at specific amino acid residues and Aβ species to diagnose a subject, guide treatment decisions, and select subjects for clinical trials. US 11,085,935, US 11,402,392 and US 11,635,440 disclose methods to quantify tau phosphorylation at specific amino acid residues to predict time to onset of mild cognitive impairment due to Alzheimer's disease, stage Alzheimer's disease, guide treatment decisions, select subjects for clinical trials, and evaluate the clinical efficacy of certain therapeutic interventions. WO 2022 / 159766 discloses methods to quantify and analyze various CSF tau species and the use thereof to measure pathological features and / or clinical symptoms of tauopathies, including determining the amount of time to dementia due to Alzheimer's disease, determining the time from dementia onset, staging Alzheimer's disease, guiding treatment decisions, and evaluate the clinical efficacy of certain therapeutic interventions. SUMMARY It is a general objective to provide an immunoassay kit that can be used in AD monitoring. It is a particular objective to provide such an immunoassay kit capable of quantifying tau phosphorylation. These and other objectives are met by embodiments as disclosed herein. The present invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims. The present embodiments relate an immunoassay kit capable of measuring p-tau205 in a sample and to methods involving the use of such an immunoassay kit. The present embodiments also relate to a monoclonal antibody that can be used in such an immunoassay kit. The immunoassay kit of the embodiments was used to show that CSF p-tau205 increased progressively across the AD continuum in human subjects, CSF p-tau205 followed a step-wise increase across AT groups, CSF p-tau205 was more tightly associated with tau-PET than Aβ-PET, and CSF p-tau205 was associated with cognition. Hence, p-tau205 as measured using the immunoassay kit is a specific AD biomarker with superior performance with large dynamic ranges and strong association with amyloid PET, tau PET, neurodegeneration and cognition. The monoclonal antibody, or antigen-binding fragment thereof, of the invention can be used to measure p-tau205 not only in CSF sampled but also in blood samples. BRIEF DESCRIPTION OF THE DRAWINGS The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which: Figure 1. CSF p-tau205 and p-tau202 levels across diagnostic groups. In the Discovery cohort, (A) CSF p-tau205 and (B) CSF p-tau202 were increased in AD compared with control cases. In the Paris cohort, (C) CSF p-tau205 was increased in MCI+ and AD+ compared with CSF amyloid-negative groups, (D) whereas CSF p-tau202 was only significantly increased in AD+ compared with CSF amyloid-negative groups. In the TRIAD cohort, (E) CSF p-tau205 was increased across CSF amyloid-positive groups compared with CSF amyloid-negative groups, (F) while high levels of CSF p-tau202 were mostly circumscribed to the AD+ group. Data information: Boxplots show the median, IQR and all participants. Participants are grouped based on the presence or absence of CSF amyloidosis measured with Lumipulse® CSF Aβ42 / 40. P-values were determined using Mann-Whitney U test and one-way ANOVA adjusted by age and sex, followed by Bonferroni-corrected post hoc comparison (*P <0.05, **P <0.01, ***P <0.001, ****P <0.0001). Figure 2. CSF p-tau205 and p-tau202 levels across AT groups. In the Paris cohort, (A) CSF p-tau205 increased progressively across the AT groups, (B) whereas CSF p-tau202 was only increased in A+T+ group. In the TRIAD cohort, (C) CSF p-tau205 increased in a stepwise manner across AT groups (D) while high levels of CSF p-tau202 were only present in A+T+. Data information: Boxplots show the median, IQR and all participants. Participants are grouped based on the presence or absence of CSF amyloidosis measured with Lumipulse® CSF Aβ42 / 40. P-values were determined using one-way ANOVA adjusted by age and sex, followed by Bonferroni-corrected post hoc comparison (*P <0.05, **P <0.01, ***P <0.001, ****P <0.0001). Figure 3. CSF p-tau205 and p-tau202 associations with global and regional Aβ-PET uptake (TRIAD cohort). (A) CSF p-tau205 correlated with global Aβ-PET SUVRs across all cases and CSF amyloid- positive and negative participants. (B) CSF p-tau202 only correlated with global Aβ-PET SUVRs across all participants. At the voxel level, both (C) CSF p-tau205 and (D) CSF p-tau202 displayed regional associations with Aβ-PET. Data information: Participants are grouped based on the presence or absence of CSF amyloidosis measured with Lumipulse® CSF Aβ42 / 40. Spearman’s rank correlation is displayed for all participants, CSF amyloid-positive and negative groups. Simple linear regression with 95% confidence intervals of CSF amyloid-positive and negative groups is also presented. Voxel maps display the adjusted R-squared and t values of the linear associations between CSF biomarkers and [18F]AZD4694, adjusted by age and sex. Figure 4. CSF p-tau205 and p-tau202 association with global and regional tau-PET uptake (TRIAD cohort). (A) CSF p-tau205 and (B) CSF p-tau202 correlated with global tau-PET SUVRs across all cases and CSF amyloid-positive participants. At the voxel level, both (C) CSF p-tau205 and (D) CSF p-tau202 displayed regional associations with tau-PET. Data information: Participants are grouped based on the presence or absence of CSF amyloidosis measured with Lumipulse® CSF Aβ42 / 40. Spearman’s rank correlation is displayed for all participants, CSF amyloid-positive and negative groups. Simple linear regression with 95% confidence intervals of CSF amyloid-positive and negative groups is also presented. Voxel maps display the adjusted R-squared and t values of the linear associations between CSF biomarkers and [18F]MK6240, adjusted by age and sex. Figure 5. Proportion of variation in CSF p-tau205 and p-tau202 levels explained by Aβ and tau pathology measured by PET (TRIAD cohort). The performance of three regression models (Aβ-PET: A, tau-PET: T, Aβ and tau-PET: A+T) predicting CSF p-tau205 and p-tau202 concentrations was evaluated. The best model predicting the variation in the concentration of CSF p-tau205 was A+T, whereas for CSF p-tau202 it was T. Data information: Each barplot represents one model. Independent variables included Aβ-PET (A) and tau-PET (T). All models include age and sex as covariates (represented in light grey). AIC of each model is displayed on top of each bar plot, within a dashed square. R-squared values for each model are displayed on top of the respective bar plot, whereas the partial R-squared of each variable within the model is presented inside the bar plot. Figure 6. CSF p-tau205 and p-tau202 association neurodegeneration. (A) CSF p-tau205 correlated with global measures of grey matter atrophy assessed by VBM across all cases and CSF amyloid-positive and negative participants. (B) CSF p-tau202 correlated with global measures of grey matter atrophy across all cases and CSF amyloid-negative participants. Both (C) CSF p-tau205 and (D) CSF p-tau202 displayed regional associations with neurodegeneration. Data information: Participants are grouped based on the presence or absence of CSF amyloidosis measured with Lumipulse® CSF Aβ42 / 40. Spearman’s rank correlation is displayed for all participants, CSF amyloid-positive and negative groups. Simple linear regression with 95% confidence intervals of CSF amyloid-positive and negative groups is also presented. Voxel maps display the adjusted R-squared and t values of the linear associations between CSF biomarkers and VBM, adjusted by age and sex. Figure 7. CSF p-tau205 and p-tau202 diagnostic performance in the Discovery cohort. ROC analysis showing the higher diagnostic performance of CSF p-tau205 and p-tau202 when discriminating AD from control cases in the Discovery cohort. Data information: AUC values are presented followed by 95% confidence intervals. DeLong test (dashed square) was used to determine the statistical differences between biomarker performances (P˂0.05 is indicated in bold). Figure 8. Spearman’s rank correlation between CSF p-tau205 and p-tau202 assays with antibody-free mass spectrometry in the TRIAD cohort. (A) CSF p-tau205 and (B) CSF p-tau202 correlated with antibody-free mass spectrometry measurements of tryptic CSF p-tau205 and p-tau202 peptides in the TRIAD cohort. Data information: Participants are grouped based on the presence or absence of CSF amyloidosis determined with Lumipulse® CSF Aβ42 / 40. Spearman’s rank correlation and simple linear regression (with 95% confidence intervals) for all participants are displayed. Figure 9. CSF p-tau205 and p-tau202 concentrations and diagnostic performance in in CI participants in Paris and TRIAD cohorts. In the Paris cohort, both (A) CSF p-tau205 and (B) p-tau202 were increased in CI+ compared with CI- cases. (C) ROC analysis showing the higher diagnostic performance (in AUC values) of CSF p-tau205 and p-tau202 when discriminating CI+ from CI- cases in the Paris cohort. In the TRIAD cohort, both (D) CSF p-tau205 and (E) p-tau202 were increased in CI+ compared with CI- cases. (F) ROC analysis showing the higher diagnostic performance of CSF p-tau205 and p-tau202 when discriminating CI+ from CI- cases in the TRIAD cohort. Data information: Boxplots show the median, IQR and all participants. Participants are grouped based on the presence or absence of CSF amyloidosis measured with Lumipulse® CSF Aβ42 / 40. Group differences were determined using Mann-Whitney U test (****P <0.0001). AUC values are presented followed by 95% confidence intervals. DeLong test (dashed square) was used to determine the statistical differences between biomarker performances (P˂0.05 is indicated in bold). Figure 10. CSF p-tau205 and p-tau202 diagnostic performance identifying CSF amyloid-positive groups in the Paris cohort. ROC analysis showing and comparing the performance of CSF p-tau205 and p-tau202 discriminating AD+ (A, B), MCI+ (C, D) and nonAD+ (E, F) from CSF amyloid-negative diagnostic groups. Data information: AUC values are presented followed by 95% confidence intervals. DeLong test (dashed square) was used to determine the statistical differences between biomarker performances (P˂0.05 is indicated in bold). Figure 11. CSF p-tau205 and p-tau202 diagnostic performance discriminating diagnostic groups in the TRIAD cohort. ROC analysis showing and comparing the performance of CSF p-tau205 and p-tau202 discriminating AD+ (A, B), MCI+ (C, D), nonAD+ (E, F) and CU+ (G, H) from CSF amyloid-negative diagnostic groups. Data information: AUC values are presented followed by 95% confidence intervals. DeLong test (dashed square) was used to determine the statistical differences between biomarker performances (P˂0.05 is indicated in bold). Figure 12. CSF p-tau205 and p-tau202 concentrations and diagnostic performance in A- and A+ participants in the Paris and TRIAD cohorts. In the Paris cohort, both (A) CSF p-tau205 and (B) p-tau202 were increased in A+ compared with A- cases. (C) ROC analysis showing the higher diagnostic performance (in AUC values) of CSF p-tau205 and p-tau202 when discriminating A+ from A- cases in the Paris cohort. In the TRIAD cohort, both (D) CSF p-tau205 and (E) p-tau202 were increased in A+ compared with A- cases. (F) ROC analysis showing the higher diagnostic performance of CSF p-tau205 and p-tau202 when discriminating A+ from A- cases in the TRIAD cohort. Data information: Boxplots show the median, IQR and all participants. Participants are grouped based on the presence or absence of CSF amyloidosis measured with Lumipulse® CSF Aβ42 / 40. Group differences were determined using Mann- Whitney U test (****P <0.0001). AUC values are presented followed by 95% confidence intervals. DeLong test (dashed square) was used to determine the statistical differences between biomarker performances (P˂0.05 is indicated in bold). Figure 13. CSF p-tau205 and p-tau202 diagnostic performance discriminating AT groups in the Paris and TRIAD cohorts. ROC analysis showing and comparing the performance of (A) CSF p-tau205 and (B) p- tau202 discriminating AT groups in the Paris cohort. ROC analysis showing and comparing the performance of (C) CSF p-tau205 and (D) p-tau202 discriminating AT groups in the TRIAD cohort. Data information: AUC values are presented followed by 95% confidence intervals. DeLong test (dashed square) was used to determine the statistical differences between biomarker performances (P˂0.05 is indicated in bold). Figure 14. CSF p-tau205 and p-tau202 concentrations and diagnostic performance in in Aβ-PET negative and positive participants in the TRIAD cohorts. In the TRIAD cohort, both (A) CSF p-tau205 and (B) p- tau202 were increased in Aβ-PET positive compared with Aβ-PET negative cases. (C) ROC analysis showing the higher diagnostic performance (in AUC values) of CSF p-tau205 and p-tau202 when discriminating Aβ-PET positive from Aβ-PET negative cases. Data information: Boxplots show the median, IQR and all participants. Group differences were determined using Mann-Whitney U test (****P <0.0001). AUC values are presented followed by 95% confidence intervals. DeLong test (dashed square) was used to determine the statistical differences between biomarker performances (P˂0.05 is indicated in bold). Figure 15. Spearman’s rank correlation between CSF p-tau205 and p-tau202 concentrations with tau- PET SUVRs across diagnostic groups in the TRIAD cohort. (A) CSF p-tau205 levels correlated with tau- PET SUVRs across all CSF amyloid-positive diagnostic groups (CU+, MCI+ and AD+). (B) CSF p-tau202 levels only correlated with tau-PET SUVRs across in AD+ cases. Data information: Participants are grouped based on the presence or absence of CSF amyloidosis determined with Lumipulse® CSF Aβ42 / 40. Spearman’s rank correlations of all diagnostic groups are displayed. Simple linear regressions (with 95% confidence intervals) of diagnostic groups which showed significant correlations with tau-PET SUVRs are presented. Figure 16. CSF p-tau205 and p-tau202 concentrations and diagnostic performance in tau-PET negative and positive participants in the TRIAD cohort. In the TRIAD cohort, both (A) CSF p-tau205 and (B) p- tau202 were increased in tau-PET positive compared with tau-PET negative cases. (C) ROC analysis showing the higher diagnostic performance (in AUC values) of CSF p-tau205 and p-tau202 when discriminating tau-PET positive from tau-PET negative cases. Data information: Boxplots show the median, IQR and all participants. Group differences were determined using Mann-Whitney U test (****P <0.0001). AUC values are presented followed by 95% confidence intervals. DeLong test (dashed square) was used to determine the statistical differences between biomarker performances (P˂0.05 is indicated in bold). Figure 17. CSF p-tau205 and p-tau202 concentrations across tau-PET Braak stages and regional association with tau-PET Braak V-VI. (A) CSF p-tau205 increased progressively across tau-PET Braak stages, whereas (B) CSF p-tau202 was only increased in tau-PET Braak V-VI individuals. Regional association between (C) CSF p-tau205 and (D) CSF p-tau202 with tau-PET Braak V-VI participants. Data information: Boxplots show the median, IQR and all participants. P-values were determined using one- way ANOVA adjusted by age and sex, followed by Bonferroni-corrected post hoc comparison (*P <0.05, **P <0.01, ***P <0.001, ****P <0.0001). Voxel maps display the adjusted R-squared and t values of the linear associations between CSF biomarkers and [18F]MK6240 at Braak V-VI, adjusted by age and sex. Figure 18. CSF p-tau205 diagnostic performance discriminating controls and AD patients was evaluated with (A) a CSF p-tau205 immunoassay using Tau12 as detector antibody and (B) CSF p-tau205 immunoassay using Tau13 as detector antibody. Data information: Boxplots show the median, IQR and all participants. Participants are grouped in Controls or AD based on the presence or absence of CSF amyloidosis measured with Lumipulse® CSF Aβ42 / 40. P-values were determined using one-way ANOVA adjusted by age and sex, followed by Bonferroni-corrected post hoc comparison (*P <0.05, **P <0.01, ***P <0.001, ****P <0.0001). AUC values are presented followed by 95% confidence intervals. Figure 19. Comparison of the signal to noise ratio of CSF p-tau205 immunoassays using Tau12 as detector antibody (top) or Tau13 as detector antibody (bottom). Figure 20. Comparison of the binding kinetics of the p-tau205 antibody clones 38C8, 37A2 and 2F10 by BIAcore measurements. The three clones (37A2, 38C8, and 2F10) were evaluated using a combination of GSK3b and DYRK1A phospho-Tau441. Figure 21. Simoa® p-tau2052F10 test with helper beads (SIMOA) using human CSF and plasma. Three conditions were tested: HB1 (immunoassay without helper beads), HB2 (immunoassay with 50% helper beads), and HB3 (immunoassay with 70% helper beads). Figure 22. P-tau205 measurement in human CSF (A) and plasma samples (B) using immunoassay kit. The in-house developed immunoassay targeting p-tau205 using 2F10 clone successfully discriminated AD from controls cases in both CSF and plasma. Mann-Whitney non-parametric test (****p<0.0001; **p<0.01). Figure 23. P-tau205 measurement in human CSF using the 2F10 immunoassay kit (A) and the ThermoFisher immunoassay (B). The in-house developed immunoassay targeting p-tau205 using 2F10 clone outperformed the immunoassay based on antibody from ThermoFisher (C) with a higher fold change. Mann-Whitney non-parametric test (****p<0.0001; ***p<0.001). Figure 24. P-tau205 measurement in human blood plasma using the 2F10 immunoassay kit (A) and the ThermoFisher immunoassay (B). The in-house developed immunoassay targeting p-tau205 using 2F10 clone was able to significantly discriminate AD from control samples in blood plasma but the immunoassay based on antibody from ThermoFisher could not. Mann-Whitney non-parametric test (**p<0.01; ns non- significant). ABBREVATIONS A - Aβ Aβ - amyloid-β Aβ42 / 40 - ratio β-amyloid 42 and 40 ABTS - 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid AD - Alzheimer’s disease ADNI - Alzheimer's disease neuroimaging initiative AIC – Akaike information criterion APOE - apolipoprotein E AT – Aβ and tau AT(N) – Aβ, tau and neurodegeneration AUC - area under the receiver operating characteristics curve CDR - clinical dementia rating CSF - cerebrospinal fluid CU – cognitively unimpaired DAB - 3,3’-diaminobenzidine DLB - dementia with Lewy bodies ELISA - enzyme-linked immunosorbent assay FTB - frontotemporal dementia GSK-3β - glycogen synthase kinase-3 beta HRP - horseradish peroxidase iQC - internal quality control IQR - interquartile range MCI – mild cognitive impairment MCP - microtiter plate MMSE – mini-mental state examination MRI - magnetic resonance imaging MS - mass spectrometry N1 / N2 - N-terminal domain 1 / 2 NFT - neurofibrillary tangle nonAD - non-Alzheimer’s disease P1 / P2 – proline rich domain 1 / 2 PET - positron emission tomography PHF - paired helical filament PPA - primary progressive aphasia PSP - progressive supranuclear palsy p-tau - phosphorylated tau p-tau181 / 202 / 205 / 217 / 231 / 235 - tau phosphorylated at threonine 181 / serine 202 / threonine 205 / threonine 217 / threonine 231 / serine 235 R1 / R2 / R3 / R4 – repeat domain 1 / 2 / 3 / 4 SD – standard deviation SNAP - suspected non-AD pathology SUVR – standardized uptake value ratio T – tau TMB - 3,3’,5,5’-tetramethylbenzidine TRIAD - Translational Biomarkers of Aging and Dementia t-tau – total tau VBM - voxel-based morphometry VCID - vascular cognitive impairment and dementia DETAILED DESCRIPTION The invention generally relates to an immunoassay specifically measuring p-tau205 and to the use thereof in monitoring AD and tau pathology across other neurological disorders. The recent advances in the development of anti-Aβ therapies for the treatment of AD has expanded the use of biomarkers not only for diagnostic purposes, but as tools to screen potential candidates for a treatment and monitor drug effects. Due to the long preclinical asymptomatic phase in AD, the time gap for the administration of these therapies has been shown paramount to optimize their efficacy. Recently, the TRAILBLAZER-2 donanemab trial had a great success based on recruiting participants with intermediate tau PET burden. However, tau PET presents significant cost, and requires highly specialized centers and personnel with expertise in this technique. Thus, a fluid biomarker reflecting tau burden and deposition as well as atrophy and cognitive decline would be highly valuable in clinical trials, as a cost- effective tool for recruitment, participant stratification and staging, and for evaluating if disease progression has been tackled. Moreover, such a biomarker would represent a clinically relevant tool in clinical settings for AD diagnosis and patient management and monitoring. The results present herein show that p-tau205, such as CSF p-tau205, as measured by an immunoassay kit is a highly AD specific biomarker, which follows a continuous and steep increase along with AD progression, reflecting tau burden, and correlating with brain atrophy and cognitive performance, indicating that this phosphorylation could be a potential biomarker to stage tau pathology. Since this would be of great use in the clinics and clinical trial recruitment, a high throughput method, such as an immunoassay kit disclosed herein, would be very helpful. The first immunoassay measuring p-tau205 levels in CSF is disclosed herein and its biomarker potential for the diagnosis of AD was investigated using three independent cohorts. The findings indicated that (i) CSF p-tau205 increased progressively across the AD continuum (abnormally emerging during preclinical AD stages); (ii) CSF p-tau205 followed a step-wise increase across AT groups; (iii) CSF p-tau205 was more tightly associated with tau-PET than Aβ-PET, both in terms of global PET measures and at the voxel level; (iv) CSF p-tau205 concentration was mostly explained by in vivo measurements of tau pathology using tau PET; (v) CSF p-tau205 was associated with grey matter atrophy globally and at the voxel level; and (vi) CSF p-tau205 was associated with cognition. Overall, CSF p-tau205 is shown herein to be a specific AD biomarker with superior performance with large dynamic ranges, small overlap between groups and strong association with amyloid PET, tau PET, neurodegeneration and cognition. The p-tau205 immunoassay was validated in a discovery cohort showing high accuracies to discriminate AD and controls. Importantly, while the immunoassay quantifies phosphorylated tau fragments elongating from the N-terminus to tau mid-region (requiring the presence of the N-terminal and phosphorylated epitopes), quantifications with the p-tau205 immunoassay correlated with the measurements of tryptic phosphorylated p-tau205 peptide in CSF using a MS antibody-free method (Gobom, J., et al., Antibody- free measurement of cerebrospinal fluid tau phosphorylation across the Alzheimer's disease continuum. Mol Neurodegener, 2022.17(1): 81). This indicates that the p-tau205 immunoassay is specific in detecting the levels of phosphorylation. The novel p-tau205 immunoassay was used to assess the levels of CSF p-tau205 across clinical groups in Paris and TRIAD cohorts. In both cohorts, CSF p-tau205 concentrations were higher in all CSF Aβ42 / 40 positive groups compared with CSF Aβ42 / 40 negative individuals, and showed a continuous increase along the AD continuum. Interestingly, in the TRIAD cohort, CSF p-tau205 was significantly increased in CU+ compared with CU-. Thus, the results demonstrate that CSF p-tau205 starts increasing during preclinical AD stages. Further, CSF p-tau205 had high dynamic ranges in CSF Aβ42 / 40 positive groups. This resulted in CSF p-tau205 displaying high diagnostic accuracies when discriminating CSF Aβ42 / 40 positive from negative groups in both cohorts, especially for cognitively impaired CSF Aβ42 / 40 positive groups (MCI+, nonAD+ and AD+). The levels of CSF p-tau205 were also assessed across AT groups in both cohorts. While the AT stratification was applied cross-sectional samples, abnormalities in CSF Aβ42 / 40 and p-tau181 are considered consecutive in AD. Therefore, AT classification can be interpreted as a proxy of disease progression. CSF p-tau205 showed a stepwise increase across AT groups, from A-T- to A+T-, and from A+T- to A+T+. Moreover, CSF p-tau205 was pronouncedly increased in A+T+ group, suggesting that the bulk of the increase in this biomarker occurs during late AD stages. Phosphorylated tau species arguably represent the most promising AD biomarkers, as they are highly specific for AD, they emerge early during asymptomatic AD stages and are tightly associated with both amyloid and tau accumulation. However, while p-tau is classified as a tau pathology or “T” biomarker according to the AT(N) framework, accumulating evidence in recent years suggests that existing p-tau biomarkers are not exclusively reflective of NFTs deposition in AD brain. P-tau species in CSF are tightly associated with early CSF amyloidosis, emerging early during asymptomatic stages, when subtle abnormalities in CSF Aβ42 / 40 are detectable. In addition, existing p-tau biomarkers are more associated with amyloid PET than tau PET, and post-mortem confirmed samples, blood p-tau species are more strongly associated with Aβ plaques than tau tangles. Therefore, due to the strong association between fluid p-tau measurements and Aβ pathology across the AD continuum, it cannot be determined whether the increase in existing p-tau biomarkers, such as p-tau181, p-tau217 or p-tau231, is reflective of aggregated Aβ or tau in the brain. This is especially evident in symptomatic AD cases, where p-tau biomarkers strongly associate with both pathologies. Thus, fluid biomarkers capable of specifically reflecting aggregated tau in brain are highly needed. In this context, CSF p-tau205 represents a useful biomarker alternative to current p-tau species for tracking tau pathology in AD brain. Across all participants in the TRIAD cohort, CSF p-tau205 displayed strong correlations with global measures of Aβ and tau accumulation measured with PET. Additionally, the strength of the correlation between CSF p-tau205 with Aβ and tau PET was similar. However, when participants were stratified according to CSF Aβ42 / 40 status, the p-tau205 biomarker was more strongly associated with tau than Aβ PET SUVRs. Moreover, the correlation of the p-tau205 biomarker with tau PET SUVRs was investigated across clinical groups. For CSF p-tau205, significant correlations with tau PET were only found in CSF Aβ42 / 40 positive groups, and these increased in strength along the AD continuum (CU+ < MCI+ ˂ AD+). These results indicate that CSF p-tau205 is strongly associated with tau-PET across the AD continuum and strengthens with disease progression. Furthermore, the p-tau205 biomarker displayed higher accuracies discriminating tau PET positivity compared with Aβ PET, suggesting the increase in CSF p-tau205 is closer to tau PET positivity threshold rather than Aβ PET becoming abnormal. Further supporting its close link with tau pathology, the p-tau205 biomarker was more strongly associated with tau pathology than Aβ pathology at the voxel level. In particular, CSF p- tau205 displayed large adjusted-R2values with tau PET. The association of CSF p-tau205 with tau PET was investigated within groups of participants at different Braak stages. Interestingly, voxel-wise analysis demonstrated that CSF p-tau205 was significantly associated with tau PET only in Braak V-VI participants, which indicates high concentrations of this p-tau205 biomarker may be reflective of advance stages of tau deposition. This was also observed in terms of global uptake, with CSF p-tau205 showing pronounced concentrations in tau PET Braak V-VI participants. The association of CSF p-tau205 with tau deposition was demonstrated by determining the proportion of variance of the CSF p-tau205 biomarker explained by Aβ-PET and tau-PET. The model that better explained the variation of CSF p-tau205 was the A+T model, and within this model, tau contributed with a 70% to the proportion of variation. These results confirm that p-tau205 levels are reflecting tau brain pathology. The association of CSF p-tau205 with neurodegeneration and cognition was investigated. The p-tau205 biomarker showed significant correlations with global measures of grey matter quantified with voxel-based morphometry and cognitive assessments using MMSE. This link with brain atrophy might also translate into a better association with cognitive performance. Interestingly, Braak stages have been shown to better correlate with disease progression and cognitive decline than amyloid staging. The fact that CSF p-tau205 reflects tau pathology, indicates it is a better indicator of cognitive deterioration. CSF p-tau205 had a significant correlation with MMSE scores in both Paris and TRIAD cohort. Phosphorylated forms of tau have traditionally been measured using MS methods. Such MS methods, however, require access to special equipment (mass spectrometer) and experience in operating such an equipment. Mass spectrometers are generally not available in clinics. There is, therefore, a need for a clinically acceptable technique for measuring phosphorylated forms of tau, and in particular p-tau205, i.e., tau phosphorylated at threonine residue 205. Reference to amino acid residues in tau herein is to the amino acid residues in human tau as defined in SEQ ID NO: 1 and further presented here below, in which threonine residue 205 is marked in bold. MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L The present invention relates to an immunoassay specifically measuring p-tau205 in a biological sample, and in particular a biological fluid sample. An aspect of the embodiments therefore relates to an immunoassay kit for determining an amount of p- tau205 in a sample. The immunoassay kit comprises a first antibody, or an antigen-binding fragment thereof, immobilized to a support or intended to be immobilized to the support and a second antibody, or an antigen-binding fragment thereof. According to the embodiments, one of the first antibody, or the antigen-binding fragment thereof, and the second antibody, or the antigen-binding fragment thereof, has specificity for p-tau205, i.e., tau phosphorylated at threonine residue 205. The other of the first antibody, or the antigen-binding fragment thereof, and second antibody, or the antigen-binding fragment thereof, has specificity for tau. The antibody, or the antigen-binding fragment thereof, having specificity for p-tau205 preferably binds specifically to an epitope in p-tau205 that comprises threonine residue 205 in phosphorylated form. In particular, the antibody, or the antigen-binding fragment thereof, preferably binds specifically to a peptide of consecutive amino acid residues in tau and where the peptide comprises threonine residue 205. Illustrative, but non-limiting, examples of such peptides include SGYSSPGSPGTPGSR (SEQ ID NO: 2), GYSSPGSPGTPGSRS (SEQ ID NO: 3), YSSPGSPGTPGSRSR (SEQ ID NO: 4), SSPGSPGTPGSRSRT (SEQ ID NO: 5), SPGSPGTPGSRSRTP (SEQ ID NO: 6), PGSPGTPGSRSRT (SEQ ID NO: 7), GSPGTPGSRSR (SEQ ID NO: 8), SPGTPGSRS (SEQ ID NO: 9), SPGTPGSR (SEQ ID NO: 10) or SPGTPGS (SEQ ID NO: 11). In an embodiment, the peptide comprises any of SEQ ID NO: 2 to 11. In another embodiment, the peptide consists of any of SEQ ID NO: 2 to 11. The antibody, or the antigen-binding fragment thereof, having specificity for tau preferably binds specifically to an epitope in tau, such as in p-tau205, not encompassing threonine residue 205. For instance, the antibody, or the antigen-binding fragment thereof, could bind specifically to an epitope in the N-terminal part (amino acid residues 1-44), in N1 (amino acid residues 45-74), in N2 (amino acid residues 75-103), in the amino acid sequence between N2 and P1 (amino acid residues 104-150), in P1 (amino acid residues 151-198), in R1 (amino acid residues 244-274), in R2 (amino acid residues 281-305), in R3 (amino acid residues 312-337), in R4 (amino acid residues 338-370) or in the C-terminal part (amino acid residues 371-441). It is also possible, but generally less preferred, to use an antibody, or an antigen- binding fragment thereof, binding specifically to an epitope in P2 (amino acid residues 199-243) as long as the binding of the antibody, or the antigen-binding fragment thereof, does not interfere with the binding of the other antibody, or the antigen-binding fragment thereof, having specificity for an epitope encompassing phosphorylated threonine 205. In a preferred embodiment, the antibody, or the antigen-binding fragment thereof, having specificity for tau binds specifically the N-terminal part of tau and p-tau205. Illustrative, but non-limiting, examples of epitopes in the N-terminal part of tau, to which one of the antibody, or the antigen-binding fragment thereof, could bind specifically include amino acids 6-18 and amino acids 15-25 in SEQ ID NO: 1. Hence, in a particular embodiment, one of the first antibody, or the antigen-binding fragment thereof, and the second antibody, or the antigen-binding fragment thereof, has specificity for an epitope encompassing phosphorylated threonine 205 in p-tau205. In this particular embodiment, the other of the first antibody, or the antigen-binding fragment thereof, and second antibody, or the antigen-binding fragment thereof, has specificity for the N-terminal part of tau and p-tau205. The antibodies of the immunoassay kits could be polyclonal antibodies, monoclonal antibodies or a combination of a polyclonal antibody and a monoclonal antibody. One or both of the antibodies may be an antigen-binding fragment having specificity for the relevant epitope or peptide. In such a case, the antigen-binding fragment can be selected from a group consisting of a single chain antibody, a Fv fragment, a scFv fragment, a Fab fragment, a F(ab’)2 fragment, a Fab’ fragment, a Fd fragment, a single-domain antibody (sdAb), a scFv-Fc fragment, and a di-scFv fragment. An antibody, or an antigen-binding fragment thereof, having specificity for an epitope or peptide means that the antibody, or the antigen-binding fragment thereof, binds specifically to the epitope or peptide. The specificity of an antibody, or an antigen-binding thereof, can be determined based on affinity and / or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with the antibody, or the antigen-binding fragment thereof, (KD), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antibody, or the antigen-binding fragment thereof. The lesser the value of KD, the stronger the binding strength between the antigenic determinant and the antibody, or the antigen-binding fragment thereof. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1 / KD. As will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest. Avidity is the measure of the strength of binding between the antibody, or the antigen-binding fragment thereof, and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antibody, or the antigen-binding fragment thereof, and the number of pertinent binding sites present on the antibody, or the antigen-binding fragment thereof. Typically, antibodies, or antigen-binding fragments thereof, will bind to their antigen with a dissociation constant (KD) of 10-7to 10-12moles / liter (M) or less, and preferably 10-8to 10-12M or less and more preferably 10-9to 10-12M, i.e., with an association constant (KA) of 107to 1012M-1or more, and preferably 108to 1012M-1or more and more preferably 109to 1012M-1. Generally, any KD value greater than 10-4M (or any KA value lower than 104M-1) is generally considered to indicate non-specific binding. Preferably, an antibody, or an antigen-binding fragment thereof, of the embodiments will bind to p-tau205 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 5 nM, preferably equal to or less than 2.5 nM, such as equal to or less than 1 nM. In a particular embodiment, the immunoassay kit is a sandwich immunoassay kit. This means that the kit uses antibodies, or antigen-binding fragments thereof, binding to different epitopes of p-tau205 so that both the first and second antibodies, or the antigen-binding fragments thereof, can simultaneously bind to the same p-tau205 molecule. In a particular embodiment, the immunoassay kit is an ELISA kit and preferably a sandwich ELISA. A sandwich ELISA can be used to detect p-tau205 in a sample by preparing a surface of a support, such as a solid support, to which the first antibody, or the antigen-binding fragment thereof, is bound as so- called capture antibody. In a preferred embodiment, a known quantity of the first antibody, or the antigen- binding fragment thereof, is bound to the surface of the support. Any non-specific binding sites on the surface are optionally but preferably blocked. The sample is then applied to the surface so that any p- tau205 present therein will be captured by the immobilized first antibodies, or the antigen-binding fragments thereof. Unbound material is optionally but preferably removed by one or multiple washing steps. The second antibody, or the antigen-binding fragment thereof, typically denoted detection antibody, is then added and is allowed to bind to any p-tau205 captured by the first antibody, or the antigen-binding fragment thereof. The amount of bound detection antibody, or an antigen-binding fragment thereof, such as the second antibody, or the antigen-binding fragment thereof, is then determined by direct or indirect detection methods. For instance, a label or enzyme can be attached directly to the second antibody, or the antigen- binding fragment thereof, or indirectly via a link, such as a biotin-streptavidin or a biotin-avidin link. It is, alternatively, possible to use a secondary or auxiliary antibody that is labeled or connected to an enzyme and binds specifically to the second antibody, or the antigen-binding fragment thereof. Hence, in an embodiment the second antibody, or the antigen-binding fragment thereof, has a covalently attached biotin. Alternatively, the second antibody, or the antigen-binding fragment thereof, has a covalently attached streptavidin or avidin. The immunoassay kit preferably also comprises an HRP-labeled streptavidin or an HRP-labeled avidin. Alternatively, the immunoassay kit also comprises an HRP-labeled biotin. The immunoassay kit also comprises a HRP substrate, such as a TMB substrate, a DAB substrate or an ABTS substrate. In such a case, the amount of p-tau205 in the sample can be determined by spectrophotometric methods that detect the conversion of the chromogenic substrate by HRP into a colored product that is detectable. In an embodiment, the immunoassay kit also comprises a MCP as the support to which capture antibody, or an antigen-binding fragment thereof, such as the first antibody, or the antigen-binding fragment thereof, is immobilized or is intended to be immobilized. The support could alternatively be magnetic beads, such as Dynabeads® magnetic beads. In a preferred embodiment, the capture antibody of the sandwich ELISA is the antibody, or the antigen- binding fragment thereof, binding specifically to an epitope encompassing phosphorylated threonine 205 in p-tau205. In such an embodiment, the antibody, or the antigen-binding fragment thereof, binding specifically to tau, including p-tau205, such as to the N-terminal part of tau, including p-tau205, is then used as detection antibody. In another embodiment, the capture antibody of the sandwich ELISA is the antibody, or the antigen- binding fragment thereof, binding specifically to tau, including p-tau205, such as to the N-terminal part of tau, including p-tau205, and the detection antibody is the antibody, or the antigen-binding fragment thereof, binding specifically to an epitope encompassing phosphorylated threonine 205 in p-tau205. The immunoassay kit does not necessarily have to be an ELISA kit. In another embodiment, the immunoassay kit uses affinity chromatography where the first antibody, or the antigen-binding fragment thereof, is bound to a stationary phase, such as to a gel matrix or beads in a column. For instance, the gel matrix or beads could be made of agarose, such as Sepharose®. In such a case, p-tau205 present in a sample will be entrapped in the column through binding to the immobilized first antibodies, or the antigen-binding fragments thereof. Following washing, the bound p- tau205 can be eluted and detected using the second antibody, or the antigen-binding fragment thereof. For instance, the amount of eluted p-tau205 can be determined using Western blotting and with the second antibody, or the antigen-binding fragment thereof, for p-tau205 detection using direct or indirect detection methods. In a further embodiment, the immunoassay kit is based on the Simoa® HD-X platform. In such a case, the capture antibody is immobilized onto beads, such as paramagnetic beads. The paramagnetic beads coupled with antibodies are then added to the sample. The detection antibody, which is enzymatically labeled to be able to generate a fluorescent product in the presence of a fluorescent substrate, is added to the sample. An illustrative example of a fluorescent substrate and enzymatic label is resorufin ^-D- galactopyranoside, which is a fluorogenic substrate for ^-galactosidase yielding the hydrolysis product resorufin with absorption / emission maxima at 571 / 585 nm. Immunocomplexes comprising the paramagnetic beads and detection antibodies will form if the sample comprises p-tau205. The sample is then loaded into an array, such as a Simoa® disc, comprising a plurality of microwells sized to encompass preferably one paramagnetic bead (immunocomplex) each. The fluorescent substrate is added to the plurality of microwells to enable enzymatic signal amplification and fluorescence imaging. The immunoassay kit could be based on other immunoassay platforms than ELISA and single-molecule immunosorbent assay (Simoa®). Illustrative, but non-limiting, examples of such other immunoassay platforms include NUcleic acid Linked Immuno-Sandwich Assay (NULISA™) by Alamar Biosciences (Feng et al., NULISA: a proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing, Nature Communications, 2023, 14: 7238), Olink® platform based on proximity extension assay (PEA), Meso Scale Discovery (MSDTM) high performance electrochemiluminescence (ECL) assay, Lumipulse® assay, Elecsys® assay, Ella® Immunoassay (ProteinSimple, Bio-Techne), ARCHITECT® immunoassay (Abbott), Atellica® Immunoassay (Siemens Healthineers), O-link and ARGOTMHT System Immunoassay analysis (Alamar Biosciences). The monoclonal antibody, or an antigen-binding fragment thereof, according to the embodiments, described further herein can also be used other types of analysis methods including, but not limited, to Western blot analysis, dot blot analysis, flow cytometry-based immunoassay, lateral flow immunoassay, radioimmunoassay (RIA), competition immunoassay, dual antibody sandwich assay, chemiluminescent assay, bioluminescent assay, fluorescent assay, agglutination assay, immunoprecipitation-mass spectrometry (IP-MS). Examples 3 to 16 as presented herein disclose a monoclonal antibody binding specifically to p-tau205 with binding characteristics, including dissociation profile, that make it suitable for use in the immunoassay kit. An aspect of the embodiments therefore relates to a monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to human p-tau205. The monoclonal antibody, or the antigen-binding fragment thereof, has a heavy chain variable region (VH) complementarity determining region 1 (CDR1) consisting of SNVMS as defined in SEQ ID NO: 12, a VH CDR2 consisting of TINTRGITYYASWAKG as defined in SEQ ID NO: 13 and a VH CDR3 consisting of AGSGSISYFNL as defined in SEQ ID NO: 14. The monoclonal antibody, or the antigen-binding fragment thereof, also has a light chain variable region (VL) CDR1 consisting of QSSQSVYDNNRLS as defined in SEQ ID NO: 15, a VL CDR2 consisting of RASTLES as defined in SEQ ID NO: 16 and a VL CDR3 consisting of QSYSYSDSGGAGFA as defined in SEQ ID NO: 17. The VH and VL of the monoclonal antibodies, or the antigen-binding fragments thereof, consist of alternative framework regions (FRs) and CDRs in the form of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In an embodiment, VH FR1 comprises, preferably consists of, QSVEESGGNLVTPGGSLTLTCTVSGFSLS as defined in SEQ ID NO: 22. In an embodiment, VH FR2 comprises, preferably consists of, SWVRQAPGEGLEWIG as defined in SEQ ID NO: 23. In an embodiment, VH FR3 comprises, preferably consists of, RLTISKTSTTVVLKMTSLTTEDTATYFCAT as defined in SEQ ID NO: 24. In an embodiment, VH FR4 comprises, preferably consists of, WGPGTLVTVSS as defined in SEQ ID NO: 25. In an embodiment, the VH comprises the amino acid sequence as defined in SEQ ID NO: 19 or 33. In a particular embodiment, the VH consists of the amino acid sequence as defined in SEQ ID NO: 19 or 33. The amino acid sequence as defined in SEQ ID NO: 33 corresponds to the amino acid sequence as defined in SEQ ID NO: 19 without the N-terminal signal peptide (SEQ ID NO: 30). In an embodiment, VL FR1 comprises, preferably consists of, ADIVMTQTPASVEAAVGGTVTINC as defined in SEQ ID NO: 26. In an embodiment, VL FR2 comprises, preferably consists of, WYQQKPGQPPKLLIY as defined in SEQ ID NO: 27. In an embodiment, VL FR3 comprises, preferably consists of, GVPSRFKGSGSGTEFTLTISDLECGDAATYYC as defined in SEQ ID NO: 28. In an embodiment, VL FR4 comprises, preferably consists of, FGGGTEVVVK as defined in SEQ ID NO: 29. In an embodiment, the VL comprises the amino acid sequence as defined in SEQ ID NO: 21 or 34. In a particular embodiment, the VL consists of the amino acid sequence as defined in SEQ ID NO: 21 or 34. The amino acid sequence as defined in SEQ ID NO: 34 corresponds to the amino acid sequence as defined in SEQ ID NO: 21 without the N-terminal signal peptide (SEQ ID NO: 31). In an embodiment, the heavy chain of the monoclonal antibodies, or the antigen-binding fragment thereof, comprises the heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain of the monoclonal antibodies, or the antigen-binding fragments thereof, comprises the light chain variable region (LH) and a light chain constant region (CL). In an embodiment, the heavy chain of the monoclonal antibodies, or the antigen-binding fragments thereof, comprises an optional N-terminal signal peptide. The optional signal peptide destinates the heavy chain, when synthesized in a host cell, toward the secretory pathway. Any such signal peptide that promotes secretion of the heavy chain when produced in a host cell can be used according to the embodiments. An illustrative, but non-limiting, example of such a signal peptide is defined in SEQ ID NO: 30. In an embodiment, the light chain of the monoclonal antibodies, or the antigen-binding fragments thereof, comprises an optional N-terminal signal peptide. The optional signal peptide destinates the light chain, when synthesized in a host cell, toward the secretory pathway. Any such signal peptide that promotes secretion of the light chain when produced in a host cell can be used according to the embodiments. An illustrative, but non-limiting, example of such a signal peptide is defined in SEQ ID NO: 31. The antigen-binding fragment of the monoclonal antibody can be any fragment of a monoclonal antibody capable of binding specifically to p-tau205 and can be selected from a group consisting of a single chain antibody, a Fv fragment, a scFv fragment, a Fab fragment, a F(ab’)2 fragment, a Fab’ fragment, a Fd fragment, a single-domain antibody (sdAb), a scFv-Fc fragment, and a di-scFv fragment. The monoclonal antibody, or antigen-binding fragment thereof, is preferably an isolated monoclonal antibody, or an antigen-binding fragment thereof, such as isolated from the supernatant of a hybridoma or host cell used for antibody production. The monoclonal antibody, or the antigen-binding fragment thereof, can be a humanized monoclonal antibody, or a humanized antigen-binding fragment thereof, or a chimeric monoclonal antibody, or a chimeric antigen-binding fragment thereof, capable of binding specifically to p-tau205. A further aspect of the embodiments includes a nucleic acid molecule encoding a monoclonal antibody, or an antigen-binding fragment thereof, according to the embodiments. Nucleic acid molecule as used herein includes polynucleotide, oligonucleotide, and nucleic acid sequence, and generally means a polymer of DNA or RNA, which may be single-stranded or double-stranded, which may contain natural, non-natural or altered nucleotides, and which may contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. Another aspect of the embodiments relates to an expression vector comprising a promoter and a nucleic acid molecule encoding a monoclonal antibody, or an antigen-binding fragment thereof, according to the embodiments operatively controlled by the promoter. The expression vector comprises at least one nucleic acid molecule comprising coding sequences that can be expressed, such as transcribed and translated, in a host cell comprising the expression vector. The expression vector is in an embodiment selected among DNA molecules, RNA molecules, plasmids, episomal plasmids and virus vectors. The nucleic acid molecule encoding the monoclonal antibody, or an antigen-binding fragment thereof, is operatively controlled by the promoter in the expression vector, i.e., is under transcriptional control of the promoter. In an embodiment, the promoter is selected from the group consisting of the human EF1 ^ promoter, the CMV promoter, the CAG promoter, the PGK promoter, the TRE promoter, the U6 promoter and the UAS promoter if the host cell is an eukaryotic cell, such as a human cell. Illustrative, but non- limiting, examples of a promoter that could be used if the host cell is a bacterial cell include the T5 promoter, the T7 promoter, the rham promoter, the phoA promoter, the Sp6 promoter, the lac promoter, the AraBad promoter, the trp promoter and the Ptac promoter. If the host cell is a yeast cell the promoter could be selected from the group consisting of the CYC1 promoter, the ADH1 promoter, the TEF2 promoter, the pCYC promoter, a PGAL promoter and the GFD promoter as illustrative, but non-limiting, examples. A further aspect of the embodiments relates to a host cell comprising an expression vector according to the embodiments. In such an aspect, the promoter is constitutively active in the host cell or inducible active in the host cell. This means that the nucleic acid molecule encoding that the monoclonal antibody, or an antigen-binding fragment thereof, can then be transcribed in the host cell to produce the monoclonal antibody, or an antigen-binding fragment thereof, in the host cell. In an embodiment, the host cell is selected from the group consisting of a bacterial cell, a yeast cell, and an eukaryotic cell, such as a human cell. SEQ ID NO: 18 is an example a nucleotide sequence encoding the heavy chain of the monoclonal antibody with a N-terminal signal peptide, whereas SEQ ID NO: 20 is an example of a nucleotide sequence encoding the light chain of the monoclonal antibody with a N-terminal signal peptide. In a currently preferred embodiment, the immunoassay kit is based on the Simoa® HD-X platform. In such a case, the capture antibody is the monoclonal antibody, or an antigen-binding fragment thereof, disclosed above and referred to as 2F10 herein. This capture antibody is immobilized onto paramagnetic beads pre-activated by EDC. The detection antibody used in such an immunoassay kit is an antibody, or an antigen-binding fragment thereof, having specificity for an epitope in the N-terminal part of tau, preferably an epitope consisting of amino acid residues 6-18 in SEQ ID NO: 1. This detection antibody is preferably biotinylated with an excess of biotin to get a biotinylated detection antibody. For instance, 1 µg / ml detection antibody is biotinylated with a 80x biotin excess. The biotinylated detection antibody is preferably used at a concentration of 2 µg / ml in the immunoassay kit. The immunoassay kit further streptavidin covalently coupled to ^–D–galactosidase (SBG) to generate a detectable signal. In a preferred embodiment, the immunoassay kit uses a concentration of 400 pM SBG. In an embodiment, the immunoassay kit further comprises paramagnetic beads without any bound capture antibody. In a preferred embodiment, the concentration of such so called helper paramagnetic beads is preferably selected within an interval of from 25 up to 95 %, preferably from 40 up to 80 %, and more preferably from 45 up to 75%, such as from 50 up to 70% of the concentration of paramagnetic beads with bound capture antibody. In a further embodiment, the immunoassay kit is based on the Simoa® HD-X platform. In such a case, the capture antibody is immobilized onto beads, such as paramagnetic beads. The paramagnetic beads coupled with antibodies are then added to the sample. The detection antibody, which is enzymatically labeled to be able to generate a fluorescent product in the presence of a fluorescent substrate, is added to the sample. An illustrative example of a fluorescent substrate and enzymatic label is resorufin ^-D- galactopyranoside, which is a fluorogenic substrate for ^-galactosidase yielding the hydrolysis product resorufin with absorption / emission maxima at 571 / 585 nm. Immunocomplexes comprising the paramagnetic beads and detection antibodies will form if the sample comprises p-tau205. The sample is then loaded into an array, such as a Simoa® disc, comprising a plurality of microwells sized to encompass preferably one paramagnetic bead (immunocomplex) each. The fluorescent substrate is added to the The sample is preferably a biological sample and preferably a body fluid sample. In an embodiment, the body fluid sample is selected from the group consisting of a CSF sample, a brain interstitial fluid, a blood sample, a plasma sample, and a serum sample. In a preferred embodiment, the body fluid sample is selected from the group consisting of a CSF sample, a blood sample a plasma sample, and a serum sample. In a particular embodiment, the body fluid sample is a CSF sample. In another particular embodiment, the body fluid sample is a blood sample, a plasma sample, or a serum sample. Tau is normally expressed within neurons and is also secreted into the brain interstitial fluid that communicates freely with CSF and, in a more restricted manner, blood via the glymphatic clearance system of the brain. The immunoassay kit of the present embodiments can be used in a method for determining the amount of p-tau205 in a sample. Another aspect of the embodiments relates to a method for determining an amount of p-tau205 in a sample. The method comprises contacting the sample with a first antibody, or an antigen-binding fragment thereof, and a second antibody, or an antigen-binding fragment thereof, of an immunoassay kit according to the embodiments. The method also comprises detecting an amount of bound second antibody, or the antigen-binding fragment thereof. The amount of p-tau205 in the sample is then determined based on the detected amount of bound second antibody, or the antigen-binding fragment thereof. Any prior art techniques to detect the amount of bound antibody, or the antigen-binding fragment thereof, can be used in the present method. For instance, the detection can be direct or indicted, and may generate a fluorescent or chromogenic signal. Direct detection typically involves the use of an antibody, or an antigen-binding fragment thereof, that is conjugated to a label. Indirect detection utilizes a labeled secondary or auxiliary antibody raised against the host species of the antibody, or the antigen-binding fragment thereof. Commonly used labels for visualization of binding of an antibody, or an antigen-binding fragment thereof, to epitope includes fluorophores and enzymes that convert soluble substrates into chromogenic end products. In an embodiment, the method also comprises correlating the detected or measured amount of second antibody, or the antigen-binding fragment thereof, bound to p-tau205 to an amount of p-tau205. This may be performed using a pre-defined correlation between detected or measured amount of second antibody, or the antigen-binding fragment thereof, bound to a reference p-tau205 protein and concentration of the reference p-tau205 protein. A typical reference p-tau205 protein that can be used when generating such a pre-defined correlation is recombinant full-length tau (SEQ ID NO: 1), which has been in vitro phosphorylated, such as by GSK-3 ^. The pre-defined correlation may, thus, be generated by contacting different samples comprising different concentrations of the reference p-tau205 protein with the first antibody, or the antigen-binding fragment thereof, and the second antibody, or the antigen-binding fragment thereof, of the immunoassay kit according to the embodiments. The amount of second antibody, or the antigen-binding fragment thereof, bound to the reference p-tau205 protein is then measured in the different samples to thereby get a standard curve, function or relationship between concentration of reference p-tau205 protein and the measured amount of second antibody, or the antigen-binding fragment thereof, bound to the reference p- tau205 protein. This pre-defined correlation, such as standard curve, function or relationship, can then be used to map or convert the detected or measured amount of second antibody, or the antigen-binding fragment thereof, bound to p-tau205 in the sample to a concentration of p-tau205 in the sample. Hence, in an embodiment, an amount of p-tau205 in a sample as referred to herein is a concentration of p-tau205 in the sample. Monitoring AD as used herein include detection, screening or diagnosis of AD unless otherwise discussed herein. As disclosed herein, p-tau205 measured in a body fluid sample is a highly specific AD biomarker. The immunoassay kit and method of the embodiments could, thus, be used in diagnosing AD in a human subject. The method comprises determining an amount of p-tau205 in a body fluid sample obtained from the human subject with the immunoassay kit or method of the embodiments and diagnosing the human subject with AD based on the determined amount of p-tau205 in the body fluid sample. p-tau205 as measured in a body fluid sample follows a continuous and steep increase along with AD progression. Accordingly, the immunoassay kit and method of the embodiments could be used in monitoring AD progression in a human subject. The method comprises determining an amount of p- tau205 in a body fluid sample obtained from the human subject with the immunoassay kit or method of the embodiments and monitoring AD progression in the human subject with AD based on the determined amount of p-tau205 in the body fluid sample. p-tau205 as measured in a body fluid sample correlated with brain atrophy and cognitive performance. The immunoassay kit and method of the embodiments could be used in monitoring brain atrophy and / or cognitive performance in a human subject. The method comprises determining an amount of p-tau205 in a body fluid sample obtained from the human subject with the immunoassay kit or method of the embodiments and monitoring brain atrophy and / or cognitive performance in the human subject with AD based on the determined amount of p-tau205 in the body fluid sample. This embodiment is in particular advantageous in detecting any deterioration in cognitive performance and / or any progression in brain atrophy. As disclosed herein, p-tau205 measured in a body fluid sample can be used to detect and monitor tau brain pathology, also referred to as tau proteinopathy or AD tau proteinopathy. The immunoassay kit and method of the embodiments could, thus, be used in diagnosing and / or monitoring tau brain pathology in a human subject. The method comprises determining an amount of p-tau205 in a body fluid sample obtained from the human subject with the immunoassay kit or method of the embodiments and diagnosing and / or monitoring tau brain pathology in the human subject based on the determined amount of p-tau205 in the body fluid sample. Tau brain pathology as used herein involves formation of neuropathological lesions in the form of NFTs. Such NFTs are constituted of tau proteins in hyperphosphorylated and aggregated forms and are present in the brain of AD and other neurodegenerative diseases. The immunoassay kit and method of the embodiments can be used to evaluate or determine efficiency of a treatment of AD in human subjects. Thus, by comparing determined amounts of p-tau205 during or after the treatment with corresponding amounts of p-tau205 previously determined before the start of the treatment or in connection with the start of the treatment it is possible to determine whether the selected treatment has any medical effect in terms of reducing the amount of p-tau205 in the human subject or at least slow any increase in p-tau205 in the human subject. Hence, a reduction in amount of p-tau205 or a reduction in the increase in p-tau205 over time in a treated human subject is determined to correlate with a treatment that has effect with regard to AD, i.e., an efficient treatment. However, if no significant reduction in the amount of p-tau205 or in the increase in p-tau205 over time is detected in a treated human subject, then the particular treatment is not efficient and does not have the desired effect with regard to AD. EXAMPLE EXAMPLE 1 Post-mortem staging of AD neurofibrillary pathology is commonly performed by immunoreaction (AT8) for hyperphosphorylated tau (p-tau) at positions 202 / 205. Given the tight link between these phosphorylated residues and NFTs, we hypothesized that CSF measurements of p-tau205 and p-tau202 may be more reflective of NFTs in AD than currently available immunoassays for p-tau181, p-tau217 and p-tau231. Therefore, two ultrasensitive Simoa® immunoassays were developed for the quantification of CSF p- tau205 and p-tau202 and measured these phosphorylations in three independent cohorts comprising the AD continuum, non-AD cases and healthy participants: a discovery cohort (n=48), an unselected clinical cohort (BioCogBank Paris Lariboisière cohort [n=217]) and a research cohort well-characterized by clinically validated fluid and imaging biomarkers (TRIAD cohort [n=222]). In TRIAD, the association of the biomarkers with Aβ and tau pathologies was tested, indexed by PET. CSF p-tau205 levels were increased in all CSF Aβ positive (Aβ+) clinical groups compared to Aβ-, while CSF p-tau202 was significantly higher only at late AD stages. CSF p-tau205 showed high diagnostic accuracy discriminating Aβ+ from Aβ- participants across the AD continuum (AUCs = 94.2-99.3). In contrast, CSF p-tau202 showed modest accuracies. In all cases, the performance of CSF p-tau205 was superior to that of CSF p-tau202. In TRIAD, CSF p-tau205 showed a significant correlation with Aβ PET SUVRs (rS=0.68, P<0.0001) and tau PET SUVRs (rS= 0.61, P<0.0001). This association was stronger in CSF Aβ+ cases (rS=0.67, P<0.0001), but not significant in CSF Aβ- participants. In this case, CSF p-tau202 also presented weaker correlations with amyloid and tau PET (rS=0.37 and rS=0.36, respectively, P<0.0001 for both). Independent multivariable regression analyses indicated that CSF p-tau205 concentrations were explained by the combination of both amyloid and tau pathologies, but the tau variable contributed with a 70% to the model. The best-fitting model for CSF p-tau202 was the one including only tau PET. The results indicate that CSF p-tau205 is a superior biomarker than p-tau202 and could be used as a surrogate marker of the intensity of brain pathology in AD. Materials and methods Sample cohorts The diagnostic performance of the in-house developed Simoa® CSF p-tau202 and p-tau205 assays was assessed in three different cohorts: discovery cohort, Partis cohort and TRIAD cohort. Discovery cohort The discovery cohort was contained CSF samples from biologically defined AD (n=21) and neurological controls (n=26) (Table 3). AD cases were accepted for clinical evaluation for suspected AD, underwent lumbar puncture, and core AD CSF biomarkers were measured. AD participants displayed the typical AD CSF biomarker profile (CSF Aβ1–42 < 530 ng / L, p-tau181 > 60 ng / L, t-tau > 350 ng / L, all measured using Innotest® ELISA). Neurological controls included patients with subjective cognitive complains but no CSF biomarkers abnormalities. Individuals with other neurological disorders or with concomitant inflammatory diseases were excluded. Ethical approval for research use of these samples has been granted by the Ethics Committee at the University of Gothenburg (EPN 140811). Paris cohort Paris cohort is a memory clinic based cohort consisting of a total of 212 subjects, which underwent clinical assessment and core CSF AD biomarker analysis at the Centre of Cognitive Neurology at Lariboisière Fernand-Widal Hospital, Université de Paris. All patients underwent a detailed clinical evaluation including both personal and family histories, extensive neurological and neuropsychological assessment, APOE genotyping, brain MRI and CSF collection. Clinical diagnosis was made by specialists in multidisciplinary consensus meetings, taking into account CSF biomarkers results and the validated criteria for the clinical diagnostic of for AD dementia, AD-MCI, DLB and FTD. AD patients exhibited abnormalities for core CSF biomarkers on the AD continuum. MCI of other causes (non-AD MCI) included subjects with psychiatric disorder, systemic disease or sleep apnea. Non-AD MCI showed normal core CSF biomarkers profile or suspected non-Alzheimer pathophysiology (normal Aβ1-42 / 40, high p-tau and / or high t-tau). Non-AD dementia patients comprised patients with dementia with DLB, FTD (n=15), VCID, and Creutzfeldt Jakob disease. Subjective cognitive decline patients included individuals with several years of clinical follow-up for clinical complains but presenting with normal cognitive testing and no abnormalities at CSF and imaging examinations. Participants were classified according to the clinical syndrome (CU, MCI or dementia) and the CSF amyloid status (Aβ- / Aβ+, as defined by Lumipulse® CSF Aβ42 / 40) into: CU- (n=21), MCI- (n=39), MCI+ (n=48), AD+ (n=73), nonAD- (non-AD dementia Aβ-, n=25) and nonAD+ (non- AD dementia Aβ+, n=6). Participants were also stratified based on the Aβ (A) and tau (T) status defined using Lumipulse® CSF Aβ42 / 40 and p-tau181, respectively, into A-T- (n=82), A+T- (n=27) and A+T+ (n=99) (the A-T+ [n=4] group is considered SNAP, and was therefore not included in the statistical analysis of the AT groups, but is depicted in the AT boxplots) (Table 4). TRIAD cohort The cross-sectional samples presented here belong to the TRIAD cohort (McGill University, Montreal, Canada). Participants from this research cohort were stratified according to the clinical syndrome (CU, MCI or dementia) and the CSF amyloid status (Aβ- / Aβ+, as defined by Lumipulse® CSF Aβ42 / 40) into: young (n=27), CU- (n=74), CU+ (n=33), MCI- (n=17), MCI+ (n=35), AD+ (n=43), non-AD+ (n=5) and non- AD- (n=28). Young and CU participants scored 0 on the CDR scale with no objective cognitive abnormalities. MCI cases had CDR score of 0.5 and presented objective and subjective memory impairment but preserved the ability to perform daily life activities. AD cases were diagnosed according the National Institute of Aging and the Alzheimer’s Association criteria for probable AD. Non-AD participants included cases suspected of non-Alzheimer pathophysiology with diagnosis including with FTP (behavioral or semantic variant), PSP or PPA, all with CDR>0.5 and negative Aβ-PET scan. Core CSF biomarkers were measured in all participants using a Lumipulse® platform (G1200, Fujirebio) at the Clinical Neuochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. Similarly as in the Paris cohort, participants were further stratified based on the Aβ (A) and tau (T) status defined using Lumipulse® CSF Aβ42 / 40 and p-tau181, respectively, into A-T- (n=135), A+T- (n=28) and A+T+ (n=88) (as in Paris cohort, the A-T+ [n=11] group is considered SNAP, and was therefore not included in the statistical analysis of the AT groups, but is depicted in the AT boxplots) (Table 5). Simoa® CSF p-tau202 and p-tau205 measurements CSF levels of p-tau202 and p-tau205 were quantified in all previously described cohorts (Discovery, Paris, TRIAD) with two in-house immunoassays developed using a Simoa® HD-X platform (Quanterix) at the Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. CSF p-tau202 immunoassay comprises a rabbit polyclonal antibody selective against phosphorylated tau at serine 202 (Immunogen: synthesized human tau peptide around the phosphorylation site of serine202, ThermoFisher Scientific, Phospho-Tau (Ser202) polyclonal antibody #PA5-104664, ThermoFisher Scientific) used as captured antibody, whereas a biotinylated mouse monoclonal antibody targeting N-terminal tau (Tau12; Purified anti-Tau, 6-18 Antibody #806501, Biolegend) was used for detection. Similarly, CSF p-tau205 immunoassay comprised a rabbit polyclonal antibody selective against phosphorylated tau at threonine 205 (Immunogen: synthesized human tau peptide around the phosphorylation site of threonine205, Phospho-Tau (Thr205, Thr522) Polyclonal Antibody #PA-114657, ThermoFisher Scientific) used as captured antibody and biotinylated Tau12 for detection. Eight-point calibration curves in both assays were generated using commercially available recombinant full-length tau411 in vitro phosphorylated by GSK- 3β (SignalChem) and run in duplicates. Prior to the analysis, samples were allowed to thaw at room temperature for 45 minutes. Thawed CSF samples were vortexed for 30 seconds at 2000 rpm and diluted using commercially available Tau2.0 assay diluent (Quanterix). All samples were randomized and analyzed blinded. Two iQCs, one low and one high, were run in duplicates at the beginning and the end of each plate. Intra- and inter- assay precision was ˂15% for both p-tau202 and p-tau205 immunoassays. Imaging analysis (TRIAD cohort) A subset of participants who had amyloid and tau pathologies indexed by PET imaging was included in this Example. The structural MRI data was acquired on a 3T Siemens Magnetom® where high-resolution T1-weighted images were acquired. The SPM12 tool was used for segmentation of T1-weighted images, which were then non-linearly registered to the ADNI template using DARTEL, as previously reported (Therriault, J., et al., Association of Apolipoprotein E ε4 With Medial Temporal Tau Independent of Amyloid-β. JAMA Neurol, 2020.77(4): 470-479). MRI images were also processed with an optimized VBM protocol. The grey matter VBM images provided an estimation of global brain neurodegeneration, which was generated with an AD-signature mask that is a composite of the entorhinal, inferior temporal, middle temporal, and fusiform regions. Amyloid and tau PET used [18F]AZD4694 and [18F]MK6240 as radiotracers, which were acquired with a Siemens High Resolution Research Tomograph (Siemens Medical Solutions, Knoxville, TN) respectively 40-70 and 90-110 minutes post-tracer injection. Images were co-registered to individual’s MRI T1- weighted scans and processed following published protocols (Pascoal, T.A., et al., In vivo quantification of neurofibrillary tangles with [(18)F]MK-6240. Alzheimers Res Ther, 2018.10(1): 74; Therriault, J., et al., Determining Amyloid-β Positivity Using (18)F-AZD4694 PET Imaging. J Nucl Med, 2021.62(2): 247-252). The global amyloid load was inferred by the average SUVR of the precuneus, cingulate, inferior parietal, medial prefrontal, lateral temporal, and orbitofrontal cortices and had the cerebellar grey matter as reference region. Amyloid PET positivity was established as equals or greater than 1.55 SUVR. For tau PET, the average SUVR in the meta-region of interest (meta-ROI), having the inferior cerebellar grey matter as reference region, was used to estimate a global tau load and the cutoff for tau positivity was 1.24 SUVR. In addition, in vivo classification of Braak stages was performed as previously described (Pascoal, T.A., et al., 18F-MK-6240 PET for early and late detection of neurofibrillary tangles. Brain, 2020. 143(9): 2818-2830). Statistical analysis Analyses were performed on GraphPad Prism® (v79, San Diego, California, USA), unless otherwise specified. Parametric and non-parametric tests were used when appropriate, based on the data distribution. Thus, comparisons between groups were performed with Mann–Whitney U test (two categories), and one-way ANCOVA adjusted by age and sex, followed by Bonferroni-corrected post-hoc analysis. Spearman’s rank (rS) tested the correlation between biomarkers. The accuracy of CSF p-tau biomarkers to distinguish binary outcomes was determined using the AUC, which were compared between each other using DeLong test (MedCalc, Ostend, Belgium). Linear regression models tested the association between biomarkers adjusting for age and sex (R Studio v4.0) and the AIC and adjusted R2values are reported for model comparisons. In addition, mediation analysis was performed using the mediate function (psych) in R. Statistical analysis on imaging data was performed using Rminc, where linear regression models were applied voxel-wise to evaluate the association between CSF and imaging biomarkers adjusting by age and sex in all participants or within groups, as described in the results. Adjusted R2and t-parametric maps are presented. Random-field theory (Worsley, K.J., et al., Unified univariate and multivariate random field theory. Neuroimage, 2004.23 Suppl 1: S189-95) was applied on the t-parametric maps to correct for multiple comparisons. Ethics approval and consent All participants or their legal relatives in case of severe dementia gave written informed consent to their participation in this study. Collection and analysis of samples were approved by Ethics Committee at the University of Gothenburg (EPN 140811), for the Discovery cohort, the ethic committee of Bichat University, Paris, France (CEERB GHU Nord n°10-037) for Paris cohort, and by the Research Ethics Board of the Montreal Neurological Institute as well as the Faculty of Medicine Research Ethics Office, McGill University, for the TRIAD cohort. Results Participant characteristics 47 individuals were studied in the discovery cohort, 212 from a clinical cohort (Paris cohort) and 262 from a research cohort (TRIAD cohort). There were no significant differences in age across groups in the discovery cohort. Significant differences in age across groups existed in both Paris and TRIAD cohort (p<0.0001). No significant differences in sex between groups were observed for none of the three cohorts. In both Paris and TRIAD cohort, significant differences in APOE-ε4 carriers and MMSE score existed between groups. Full demographic information, clinical features and biomarker concentrations are shown in Table 1 (Paris cohort), Table 2 (TRIAD cohort) and Table 3 (Discovery cohort). CSF p-tau205 and CSF p-tau202 immunoassays validation We first investigated CSF p-tau205 and p-tau202 levels in the Discovery cohort using the novel in-house Simoa® immunoassays. This cohort was comprised by biologically defined AD cases with a typical AD profile and neurological controls with minor psychiatric symptoms and normal CSF biomarker profile. Both CSF p-tau205 and p-tau202 were significantly increased in AD compared to controls (P <0.0001) (Figure 1A and 1B). Both phosphorylations showed high diagnostic performance identifying AD cases (AUCp- tau205=0.99, AUCp-tau202=0.86), but CSF p-tau205 was significantly superior to CSF p-tau202 (DeLong´s test, p=0.0089) (Figure 7). To further explore the accuracy of the methods to quantify p-tau205 and p- tau202 in CSF, the measurements from the in-house Simoa® immunoassays were correlated with previous analysis using an antibody-free MS method (Gobom, J., et al., Antibody-free measurement of cerebrospinal fluid tau phosphorylation across the Alzheimer's disease continuum. Mol Neurodegener, 2022.17(1) p.81) in the TRIAD cohort (Figure 8A and 8B). CSF p-tau205 showed strong and significant correlation with the MS measurements (rS =0.81 p<0.0001), whereas CSF p-tau202 displayed a moderate yet significant correlation with the MS counterpart (rS=0.40 p<0.0001). CSF p-tau205 and CSF p-tau202 across clinical diagnosis groups In the Paris cohort, CSF p-tau205 was significantly increased in AD+ when compared with all Aβ- groups (CU-, MCI-, nonAD-; P ˂0.0001 for all), with MCI+ (P ˂0.001) and nonAD+ (P ˂0.01). CSF p-tau205 concentrations were also higher in MCI+ compared with all Aβ- groups (CU-, MCI-, nonAD-; P ˂0.0001 for all) (Figure 1C). CSF p-tau202 was only increased in AD+ compared with Aβ- groups (CU-, MCI-, nonAD-; P ˂0.05 for all) (Figure 1D). In the TRIAD cohort, CSF p-tau205 was increased in AD+ and MCI+ compared with Aβ- groups (P ˂0.0001 for all). Additionally, CSF p-tau205 levels were higher in CU+ and nonAD+ compared with all Aβ- groups excepting MCI- (P ˂0.01 for all). Between Aβ+ groups, CSF p- tau205 was increased in AD+ compared with CU+ (P ˂0.0001) and MCI+ (P ˂0.01), and in MCI+ compared with CU+ (P ˂0.01) (Figure 1E). Increased CSF p-tau202 levels were mostly circumscribed to the AD+ group. CSF p-tau202 was significantly higher in AD+ compared with all Aβ- groups (P ˂0.05, for all), and also in MCI+ compared with young and MCI- cases (P ˂0.05, for all). CSF p-tau202 levels in both CU+ and nonAD+ were only increased when compared with young subjects (P 0.01, for all) (Figure 1F). When participants were grouped according to both cognitive and Aβ status, CSF p-tau205 and p- tau202 levels were significantly increased in CI+ (MCI+, nonAD+ and AD+) compared with CI- cases (MCI-, nonAD-) in both Paris and TRIAD cohorts (P ˂0.0001, for all) (Figure 9A, 9B, 9D and 9E). In the Paris cohort, CSF p-tau205 showed high diagnostic accuracy discriminating AD+, nonAD+ and MCI+ from CU-, MCI- and nonAD- (AD+: AUCs = 94.2-99.3, nonAD+: AUCs = 81.6-96.0, MCI+: AUCs = 87.1-94.1) (Figure 10A, 10C and 10E). In contrast, CSF p-tau202 showed modest accuracies in all three scenarios (AD+: AUCs = 72.7-81.6, nonAD+: AUCs = 50.7-61.5, MCI+: AUCs = 62.8-72.8) (Figure 10B, 10D and 10F). In all cases, the performance of CSF p-tau205 was superior to that of CSF p-tau202 (DeLong: P ˂0.05 for all; excepting nonAD+ vs MCI-, which was not significant). In the TRIAD cohort, CSF p-tau205 displayed high accuracies discriminating AD+, nonAD+ and MCI+ from Aβ- groups (AD+: AUCs = 97.4-99.9, nonAD+: AUCs = 96.2-100, MCI+: AUCs =91.9-98.5) (Figure 11A, 11C and 11E). When discriminating CU+ from Aβ- groups, CSF p-tau205 showed moderate to high accuracies (CU+: AUCs = 75.6-92.5) (Figure 11G). CSF p-tau202 AUC values were for the most part lower than those of CSF p-tau205: (AD+: AUCs = 73.3-92.3, nonAD+: AUCs = 72.9-98.5, MCI+: AUCs = 62.6-86.5, CU+: AUCs =53.2-80.0) (Figure 11B, 11D, 11F and 11H). In both the Paris and the TRIAD cohort, the discriminatory accuracy of CSF p-tau205 discriminating CI+ from CI- (AUC=96.0-92.6) was significantly higher than that of CSF p-tau202 (AUC=75.0-69.8, DeLong: P ˂0.0001 for both) (Figure 9C and 9F). CSF p-tau205 and CSF p-tau202 across AT groups In the Paris and the TRIAD cohort, CSF p-tau205 increased progressively across the AT groups (Figure 2A and 2c). In both cohorts, a slight yet significant increase was observed from A-T- to A+T- (P ˂0.01, for both). This was followed by a pronounced increase between A+T- and A+T+ (P ˂0.0001, for both). Contrarily, CSF p-tau202 levels in Paris and TRIAD cohorts were only significantly increased in A+T+ cases compared to A-T- and A+T- (P 0.05, for all) (Figure 2B and 2D). In both cohorts, the concentrations of CSF p-tau205 and p-tau202 were significantly higher in A+ compared with A- (Figure 12A, 12B, 12D and 12E). In terms of discriminatory accuracies, in both cohorts CSF p-tau205 showed high performance discriminating A+T+ from A+T- (AUCs = 92.7-93.7) and A-T- (AUCs = 97.4-99.3), and moderate-to-high when discriminating A-T- from A+T- (AUCs = 74.7-82.7) (Figure 13A and 13C). CSF p-tau202 performed better discriminating A+T+ from A+T- (AUCs = 70.8-81.6) and A-T- (AUCs = 78.1-80.3) than distinguishing A-T- from A+T- (AUCs = 54.9-59.9) (Figure 13B and 13D). The performance of CSF p-tau205 was superior to that of CSF p-tau202 when discriminating AT groups in both cohorts (P ˂0.05, for all). In both cohorts, CSF p-tau205 and p-tau202 concentrations were significantly higher in A+ compared with A- cases (P ˂0.0001, for all), however the discriminatory accuracy of CSF p-tau205 (AUC=92.3-93.5) was significantly higher than that of CSF p-tau202 (AUC=70.7-74.0, DeLong: P ˂0.0001) (Figure 12C and 12F). Associations of CSF p-tau205 and p-tau202 with Aβ pathology indexed by Aβ PET In the subset of TRIAD samples with available imaging data (Table 6), CSF p-tau205 showed a significant correlation with Aβ PET SUVRs (rS= 0.68, P <0.0001). The same was true for CSF p-tau202, but the strength of the correlation was weaker than that of CSF p-tau205 (rS= 0.37, P <0.0001). When stratified by CSF Aβ42 / 40, CSF p-tau205 was correlated with Aβ PET SUVRs in CSF Aβ42 / 40 positive cases (rS= 0.40, P <0.0001) and showed a weak correlation in CSF Aβ42 / 40 negative participants (rS= 0.21, P =0.0174) (Figure 3A). CSF p-tau202 did not correlated with Aβ PET SUVRs in participants stratified by CSF Aβ42 / 40 (Figure 3B). When participants were stratified into Aβ PET negative and Aβ PET positive cases, both CSF p-tau205 and p-tau202 were significantly increased in latter group (P ˂0.0001), but CSF p-tau205 displayed higher accuracy than CSF p-tau202 discriminating the two groups (AUC205=90.3, AUC202=71.1; DeLong: P ˂0.0001) (Figure 14). In the voxel-wise analysis, the association between Aβ PET and the two CSF biomarker was localized in AD-related regions, being them the posterior cingulate, praecuneus, temporal and frontal cortices (Figure 3C and 3D). However, results suggest a stronger association between CSF p-tau205 and Aβ PET as compared with CSF p-tau202, which is evidenced by the higher adjusted-R2values and higher significant T-values that also encompassed wider regions for CSF p-tau205. CSF p-tau205 and CSF p-tau202 associations with tau pathology indexed by tau PET In the subset of TRIAD samples with available tau-PET imaging (Table 6), CSF p-tau205 and p-tau202 significantly correlated with tau PET SUVRs, but the strength of the correlation was stronger for CSF p- tau205 (rS= 0.61, P <0.0001) than for p-tau202 (rS= 0.36, P <0.0001) (Figure 4A and 4B). CSF p-tau205 strongly and significantly associated with tau PET SUVRs in CSF Aβ42 / 40 positive cases (rS= 0.67, P <0.0001), but not in CSF Aβ42 / 40 negative participants. The same was observed for CSF p-tau202, but the correlation with CSF Aβ42 / 40 positive was more moderate (rS= 0.45, P <0.0001). The association of CSF p-tau205 and p-tau202 with tau PET SUVRs across clinical groups was investigated. CSF p-tau205 displayed moderate-to-strong correlations with tau PET SUVRs across clinical groups within the AD continuum, and these progressively increased in strength from CU+ (rS= 0.43, P <0.0147) to MCI+ (rS= 0.56, P <0.001), and from MCI+ to AD+ (rS= 0.64, P <0.0001) (Figure 15A). CSF p-tau202 associated with tau PET SUVRs only in the AD+ group, showing a moderate correlation (rS= 0.41, P=0.0208) (Figure 15B). Both CSF p-tau205 and p-tau202 were significantly increased in tau PET positive individuals compared with tau PET negative (P ˂0.0001), but CSF p-tau205 showed higher accuracy than CSF p-tau202 discriminating the two groups (AUC205=94.5, AUC202=81.4; DeLong: P ˂0.0001) (Figure 16). Both CSF p-tau biomarkers displayed higher accuracies when discriminating tau PET than Aβ PET status. When stratifying participants into tau PET Braak stages, CSF p-tau205 showed a stepwise increase across tau PET Braak stages, displaying significant increases from Braak 0 to I-II (P ˂0.01), from Braak I-II to III-IV (P ˂0.0001), and from Braak III-IV to V-VI (P ˂0.0001) (Figure 17A). The increase in CSF p-tau205 concentration was especially pronounced at Braak III-IV and V-VI. CSF p-tau202 also displayed a seemingly stepwise increase across tau PET Braak stages, but was only significantly increased in Braak stage V-VI compared with Braak stages 0 and I-II (P ˂0.0001) (Figure 17B). At the voxel level, the association between tau PET and CSF p-tau205 and p-tau202 was more prominent in temporal regions, with highest adjusted-R2values localized on the medial temporal regions (Figures 4C and 4D). Once again, the model including CSF p-tau205 better explained tau PET than the one with CSF p-tau202, given the higher adjusted-R2values and the wider significant brain regions observed for CSF p-tau205. In addition, for CSF p-tau205, the adjusted-R2values found with tau PET were also larger as compared to Aβ PET, which may suggest a closer association between CSF p-tau205 and tau PET than with Aβ PET. The same linear models were performed within groups of participants at different Braak stages (within Braak I-II, Braak III-IV and Braak V-VI independently of each other). The association between CSF p-tau205 and p-tau202 and tau PET was found to be significant only within the group of Braak V-VI stages, and only for p-tau205, suggesting a potential association between this biomarker and advanced tau pathology (Figures 17C and 17D). Proportion of variation of CSF p-tau205 and p-tau202 concentrations explained by Aβ and tau PET The proportion of variation in CSF p-tau205 and p-tau202 explained by Aβ and tau PET was investigated using regression models. First, independent multivariable regression analyses were performed to determine which variables optimally described the variation of CSF p-tau205 and p-tau202 concentrations across all groups. Three models were generated using Aβ and tau PET as independent variables, and CSF p-tau205 or p-tau202 as dependent variables (using age and sex as covariates). The regression models included (i) Aβ PET (A), (ii) tau PET (T), (iii) Aβ and tau PET (A&T). The assessment of the models was determined based on the R-squared (R2) and the Akaike criterion (ΔAIC>2 was considered significant). According to this, the model that better explained CSF p-tau205 concentrations was A+T, (R2=0.625, AIC=736, ΔAIC=15). The proportion of variation explained by each of the independent variables was investigated in the A+T model (partial R2): T accounted for a partial R2= 0.436 (69.7%), followed by A with a partial R2= 0.162 (25.9%). For CSF p-tau202 both the T and the A+T model showed the same R2and AIC (R2=0.315, AIC=685), therefore the simplest model, that is the T only model, was considered best-fitting model (Figure 5). As a complementary assessment, mediation analysis was performed to verify the direct and indirect contribution of PET biomarkers to the CSF biomarker levels. When considering tau PET as mediator in the association between Aβ PET and CSF p-tau205 (adjusting for age and sex), Aβ PET was found to have significant direct and indirect (through tau PET) effects on CSF p-tau205 (βdirect=0.32, Pdirect<0.001; βindirect=0.29, Pindirect<0.001), whilst the direct effect of tau PET alone was 0.49 (P<0.001). For CSF p- tau202, however, the effect of Aβ PET was totally mediated by tau PET (βdirect=0.07, Pdirect=0.22; βindirect=0.18, Pindirect<0.001) and again tau PET alone had a larger effect on this biomarker (β=0.32, P<0.001). CSF p-tau205 and p-tau202 association with neurodegeneration and cognition A subset of 213 individuals in TRIAD cohort had available structural MRI measurements (Table 7). Both CSF p-tau205 and p-tau202 showed significant and negative correlations with global measures of grey matter quantified with VBM (CSF p-tau205: rS = -0.36, CSF p-tau202: rS = -0.33; P ˂ 0.0001 for both) (Figure 6A and 6B). When stratified by CSF Aβ42 / 40, CSF p-tau205 significantly correlated with both amyloid-positive (rS = -0.29, P = 0.0047) and negative cases (rS = -0.25, P = 0.0064), whereas CSF p- tau202 was significantly correlated in amyloid-negative cases (rS = -0.27, P = 0.0023) and approached significance in amyloid-positive (rS = -0.20, P = 0.0626). At the voxel level, significantly associated regions were restricted to the posterior cingulate and medial temporal cortices and mostly detected for CSF p- tau205 (Figure 6C and 6D). In both Paris and TRIAD cohort, cognitive assessments using MMSE were available in a subset of 207 and 218 participants, respectively. CSF p-tau205 showed a significant and negative correlation with MMSE in Paris (rS = -0.38; P ˂ 0.0001) and TRIAD cohort (rS = -0.40; P ˂ 0.0001). CSF p-tau202 also correlated with MMSE scores in Paris (rS = -0.20; P = 0.0056) and TRIAD cohort (rS = -0.29; P ˂ 0.0001), but these were weaker than those of CSF p-tau205. When stratified by CSF Aβ42 / 40, CSF p-tau205 only correlated with MMSE in amyloid-positive participants (Paris cohort: rS = -0.23, P = 0.0115; TRIAD cohort: rS = -0.32, P = 0.0015), but not CSF p-tau202.

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p a s d b Dten a a T 5 e ul1 a 0 1 .0s 0 1 .00 1 .00 1 .00 1 .00 1 . 0 10 10 v- 0 n 0 0 0 000.00.00.00 P ) ) ) ) ) 8 1 9 7 ) 0.4) ) ).3).1 5.5.91.36 1 1+ 07 7.63 6 4.05 9.5.9D 3 A =(n(1 .9 4( (7(9 82(00(84(64(3 ) 030(0 7 30(0 4 4.0 / 2.56 1 51 91 12.0 80.60.2 3.22.42 1 68 ) ) ) ) ) )1 4 . ) ) ) 7 .5) 9 .6 7.2 1.4 44 0 6I+035(3.32(0 6( .072( .008 1 32() 0.500.5C = 84(3 8 2 0( (7 3(30(Mn( .33.5 / 2.05 62.7 7 17 1 51 71 82.00.548 7.32 2.31 5 )6 ) )2 ) 7 )0 ) )1 2 .1) ) ). .1.03 7 4+ 08 0. .56.9.04 9.4.2U 3 C =6(n(4 0 .5 4(3(33(0 920(00(3(1() 030(920(0 2 7 1.34 / 31 1.15 6 7 2 5 1 92.00.584.5 2.8.92 1 0 4 ) ) ) ) ) ) 7-D ) 2 ) 17.7 1 7.59 )3 )1A 81.48(8. .33(6.046( .0.98(4 1(7.28.1no = n 5 7 .7 2(11(.18 1 30() .99 3 . 8 10(7 10(2 N(3 5 6 41 / 13 52.0 80 77.2 9.2.87 1 0 2 )6 ) ) ) 0I- )4.20 ) 7 ) .5 3.3 )2 3 4.5C 11. .0.4 8.) 6 6 ) .19 .0M =1n(73(1(3 ( 1 11(0(2 1(9(7 ) 410(410(.3 5(6 0 8.84 0 3 / 1.09 .06 .6.07 1 6.3.87 1 2 82 0 14 23 1 0 ) ) ) 4 3 ) 7 ) 0.)6 )1 7.52 )3 )9- )4U 6.59(1.9.83(5 2( .920( .0.30 0 2.1.0C = n(2 .2 3(60(1(1() 60(460(9 5 0 .74 6 9 3 1 0 3 6 2 5 / 6.21 61 92.00.538. .3.83 48 1 0 2 )1 ) 6g ).9) 3 2 ) ) ..5 1.1 ) ) .51 8 .20.2.) 8 7 ) .08n 621(21(30(7(4( .0uo =2(620(2 ) 620(50(Yn(7 .0 4(3 1 9 .76 7 2 1 61 / 2.79 4 6 92.0.4.41 2 4 0 22 69.2.81 1 0 )L , )le% bam / no( liigpl leatsra ) a 3 ciev( eb F ba al1 urra(eeSli ia av 2=srn a ) d a % efcror4 c oCe va a ( eyscsl0 1( (T M,(s olε-E E )ssE u pi4 / 8 2 1 TE )s R E P )s R B eleesra v a O S e S u u M s M m 4 a V g A M e L e y P A M a c M u L β At- patP- es V -u es V -tβ A a c U S a T a c U S spah1 uit0 1.00 1 .00or.0 g wn d 0 0 0 eeera twp e m b o) ec7 )5 )9 ger.0.4.0 aee0(1(3( raw9.31 0.42 3.6pls5 m e o v c elot srd e) e6 ) skra.07 )0( .29 .5 us m aio31(1(wb.46 . 4ts dn062.04 et asillE a S M ) )WM5 )l ,.01 .13 ak n0(61( .4s oti31(ura.401.4 27.2Kc t.u n d a e ipfic o)7 ) )trsra a.06( .3peY0 511( .50en..45(.39 s .5 od pu 0 2 1 edorlucgne) )ni e4.06 ) 6.0l.y4nwto e0(0(0(pb4.0 u s42 0.51.91or iegc + ne D u A q)5 ) ) noerf.02 .84 N 40(60( .4a 60(7.εteE.40.71.71 a O irP pAord p n) ) ) pax.x6 2ae e.0( .78 . ss s070(3 a,20( )erd a n.50.09 % 1.11(pan e r m g o o )c ao y DSt b( erd strn aea u s ekre qsjamius - d hacA m ia’V o nsO b w n 2 5 o o N F ) S L 02 0 h 2s sraAe yamCm Um / u g a usi Gp(t- a pt- patP w- a d e D n a n o EXAMPLE 2 This Example compared the performance of two p-tau205 immunoassays. In this example, the same paramagnetic beads conjugated with the p-tau205 antibody were tested against two different detector antibodies – Tau12 and Tau13 – in the Simoa® platform. The same samples with typical AD CSF biomarker profile as assessed by Lumipulse® CSF Aβ42 / 40 (n=54) and neurological controls (n=56) were measured. Materials and Methods Simoa® CSF p-tau205 measurements CSF levels of p-tau205 were quantified in a cohort using two in-house immunoassays developed using a Simoa® HD-X platform (Quanterix) at the Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. The first CSF p-tau205 immunoassay in accordance with Example 1, i.e., comprised a rabbit polyclonal antibody selective against phosphorylated tau at threonine 205 (Immunogen: synthesized human tau peptide around the phosphorylation site of threonine205, Phospho- Tau (Thr205, Thr522) Polyclonal Antibody #PA-114657, ThermoFisher Scientific) used as captured antibody and a biotinylated mouse monoclonal antibody targeting N-terminal tau (Tau 12; Purified anti- Tau, 6-18 Antibody #806501, Biolegend) was used as detection antibody. The second CSF p-tau205 immunoassay comprised the rabbit polyclonal antibody selective against phosphorylated tau at threonine 205 (Immunogen: synthesized human tau peptide around the phosphorylation site of threonine205, Phospho-Tau (Thr205, Thr522) Polyclonal Antibody #PA-114657, ThermoFisher Scientific) as capture antibody and a biotinylated mouse monoclonal antibody targeting N-terminal tau (Tau13; Purified anti- Tau, 15-25 Antibody #835204, Biolegend) as detection antibody. Calibration curves and sample preparation was done in accordance with Example 1. Cohort The cohort consisted of biologically defined AD cases (n=54) with typical AD CSF biomarker profile as assessed by Lumipulse® CSF Aβ42 / 40 and neurological controls (n=56) with normal core CSF biomarker profile. Results Figure 18A (Tau12 as detector antibody) and Figure 18B (Tau13 as detector antibody) show the performance of the two p-tau205 immunoassays in discriminating AD and control cases. The two p-tau205 immunoassays showed statistically (no significant difference in AUC values, DeLong Test) the same performance in discriminating AD and control cases. The signal to noise ratio of the two p-tau205 immunoassays is shown in Figure 19 indicating a signal to noise ratio of 1 to 10 for the p-tau205 immunoassay using Tau12 as detector antibody and 10 to 100 for the p-tau205 immunoassay Tau13 as detector antibody. Hence, a 10-fold improvement in signal to noise ratio was obtained when using Tau13 instead of Tau12 as detector antibody in the p-tau205 immunoassay. Such an improvement in signal to noise ratio means that less CSF sample volume is needed for quantification of p-tau205. EXAMPLE 3 This Example tested different anti-p-tau205 monoclonal antibody clones as capture antibody and the Tau12 antibody as detector antibody in an ELISA set-up. Reagents and Antibodies Capture Antibodies: Different clones of p-tau205-specific generated antibodies (2D1, 2F10, 31A6, 37A2, 38C2) and commercial p-tau205 (Immunogen: synthesized human tau peptide around the phosphorylation site of threonine205, Phospho-Tau (Thr205, Thr522) Polyclonal Antibody #PA-114657, ThermoFisher Scientific). Detector Antibody: Tau12 (Purified anti-Tau, 6-18 Antibody #806501, Biolegend), which specifically recognizes amino acid residues 6-18 in the N-terminal of the tau protein. Samples ^ Recombinant phosphorylated tau: In vitro phosphorylated using GSK3β kinase ^ Non-Phosphorylated Tau (Tau441): Full-length tau protein without phosphorylation ^ Brain Sample: Homogenized brain tissue extract containing tau protein ^ Cerebrospinal Fluid (CSF): Biological fluid sample containing tau protein Procedures 1. Coating the Plate: 96-well ELISA plates were coated with 100 µL of capture antibody solution (p-tau205 antibody clones) at a concentration of 1 µg / mL in a carbonate-bicarbonate buffer (pH 9.6). Plates were incubated overnight at 4°C to allow proper adsorption of the capture antibodies to the plate surface. After incubation, the ELISA plates were washed three times with phosphate-buffered saline (PBS) containing 0.05 % Tween-20 (PBST). The wells were blocked with 300 µL of blocking buffer (1% BSA in PBST) for 1 hour at room temperature to prevent non-specific binding. After blocking, the wells were washed three times with PBST. 100 µL of samples (recombinant phosphorylated tau, non-phosphorylated tau, brain sample, or CSF) were added to the wells. The samples were diluted in sample buffer (PBS with 1% BSA). The ELISA plates were incubated for 2 hours at room temperature or overnight at 4°C. Following incubation, the plates were washed five times with PBST. 100 µL of detection antibody, i.e., tau12 antibody conjugated to the enzyme horseradish peroxidase (HRP) was added to each well at an appropriate dilution in PBST with 1% BSA. The ELISA plates were incubated for 2 hours at room temperature. The ELISA plates were then washed five times with PBST to remove unbound detector antibody. 100 µL of TMB substrate solution (3,3',5,5'-tetramethylbenzidine) was added to each well. The ELISA plates were incubated in the dark for 15-30 minutes at room temperature to allow color development. The enzyme-substrate reaction was stopped by adding 100 µL of stopping solution to each well. The optical density (OD) was measured at 450 nm using a microplate reader. The OD values correlated with the amount of phosphorylated tau present in the samples. A standard curve was generated using known concentrations of recombinant phosphorylated tau. Sample concentrations were determined by comparing their OD values to the standard curve. Comparison of signal intensity between different p-tau205 antibody clones provided information about their binding affinity and specificity. Table 8. ELISA plate layout. GSK3b (µg / ml) Tau441 (µg / ml) Brain CSF / Plasma / Brain A 2 1 1:1000 CSF pool neat B 1 0.5 1:2000 CSF pool 1:2 C 0.5 0.25 1:4000 Plasma 1:2 D 0.25 0.125 1:8000 Plasma 1:4 E 0.125 0.0625 1:16000 Brain 1:1000 F 0.0625 0.03125 1:32000 Brain 1:4000 G 0.03125 0.015625 1:64000 Brain 1:1000 H Blank Blank Blank Brain 1:4000 The results are presented in Tables 9-14 for the tested capture antibodies. Table 9. Antibody clone 2D1 (1 µg / ml) GSK3b (µg / ml) Tau441 (µg / ml) Brain CSF / Plasma / Brain A 3.49 2.93 1.25 0.04 B 3.40 2.62 0.69 0.02 C 3.42 1.81 0.40 0.17 D 3.43 1.27 0.22 0.10 E 3.35 0.84 0.14 0.14 F 3.27 0.45 0.07 0.05 G 3.05 0.24 0.04 0.12 H 0.03 0.01 0.03 0.06 Table 10. Antibody clone 2F10 (1 µg / ml) GSK3b Tau441 Brain CSF / Plasma / Brain (µg / ml) (µg / ml) A 3.22 2.21 0.36 0.03 B 3.11 1.66 0.20 0.05 C 3.06 1.15 0.12 0.09 D 2.87 0.79 0.08 0.06 E 2.77 0.49 0.06 0.14 F 1.86 0.25 0.05 0.05 G 0.98 0.14 0.05 0.12 H 0.04 0.02 0.03 0.06 Table 11. Antibody clone 31A6 (1 µg / ml) GSK3b (µg / ml) Tau441 (µg / ml) Brain CSF / Plasma / Brain A 3.27 1.86 0.23 0.05 B 3.36 1.27 0.14 0.04 C 3.38 0.97 0.10 0.18 D 3.28 0.78 0.06 0.11 E 3.21 0.45 0.05 0.12 F 3.01 0.20 0.04 0.04 G 2.68 0.11 0.04 0.13 H 0.03 0.02 0.04 0.06 Table 12. Antibody clone 37A2 (1 µg / ml) GSK3b (µg / ml) Tau441 (µg / ml) Brain CSF / Plasma / Brain A 3.86 2.64 1.13 0.03 B 3.48 2.30 0.67 0.03 C 3.43 1.63 0.38 0.14 D 3.36 1.24 0.20 0.09 E 3.34 0.75 0.13 0.13 F 3.23 0.32 0.08 0.04 G 3.02 0.18 0.05 0.12 H 0.03 0.02 0.03 0.05 Table 13. Antibody clone 38C8 (1 µg / ml) GSK3b (µg / ml) Tau441 (µg / ml) Brain CSF / Plasma / Brain A 3.41 2.36 1.21 0.03 B 3.34 1.60 0.72 0.02 C 3.29 1.23 0.40 0.12 D 3.34 0.88 0.23 0.08 E 3.28 0.54 0.13 0.13 F 3.20 0.26 0.08 0.05 G 2.97 0.16 0.06 0.13 H 0.05 0.02 0.04 0.05 Table 14. Reference antibody Invitrogen (1 µg / ml) GSK3b (µg / ml) Tau441 (µg / ml) Brain CSF / Plasma / Brain A 3.23 2.22 0.06 0.03 B 3.19 1.22 0.05 0.05 C 3.06 0.67 0.03 0.05 D 2.87 0.37 0.03 0.04 E 2.45 0.20 0.02 0.06 F 1.76 0.12 0.02 0.04 G 1.25 0.08 0.03 0.06 H 0.02 0.02 0.03 0.04 The antibody clones generated higher signal to noise as compared to the commercial anti-p-tau205 antibody when using conventional ELISA set-up. EXAMPLE 4 The primary aim of this Example was to evaluate and compare different anti-p-tau205 antibody clones to compare performance. Additionally, the cross-reactivity of these antibody clones with other tau species was assessed. Reagents and Antibodies Capture Antibodies: Different clones of p-tau205-specific generated antibodies (2D1, 2F10, 31A6, 37A2, 38C2) and commercial p-tau205 (Immunogen: synthesized human tau peptide around the phosphorylation site of threonine205, Phospho-Tau (Thr205, Thr522) Polyclonal Antibody #PA-114657, ThermoFisher Scientific). Detector Antibody: Tau12 (Purified anti-Tau, 6-18 Antibody #806501, Biolegend), which specifically recognizes amino acid residues 6-18 in the N-terminal of the tau protein. Samples ^ Recombinant phosphorylated tau: In vitro phosphorylated using GSK3β kinase (H: 1500 pg / mL, G: 500 pg / mL, F: 167 pg / mL, E: 56 pg / mL, D: 19 pg / mL, C: 6 pg / mL, B: 2 p / mL, A: 0.7 pg / ml, Blank: 0 pg / mL) ^ Non-Phosphorylated Tau (Tau441): Full-length tau protein without phosphorylation (H: 1500 pg / mL, G: 500 pg / mL, F: 167 pg / mL, E: 56 pg / mL, D: 19 pg / mL, C: 6 pg / mL, B: 2 p / mL, A: 0.7 pg / ml) ^ Plasma samples: QC C, QC E, QC H, QC L ^ Cerebrospinal Fluid samples (CSF): CSF 1, CSF 2, CSF, 3, CSF 4 Procedures Beads were coated with capture antibody by incubating paramagnetic beads with the p-tau205 antibody clones in a suitable MES buffer at room temperature with gentle shaking. After antibody attachment, the beads were blocked with 1% BSA in PBS for 1 hour at room temperature to reduce non-specific binding. Then, the beads were washed multiple times with PBST using a magnetic separation rack to remove unbound antibodies and blocking buffer. Biotinylated mouse monoclonal antibody targeting N-terminal tau (Tau12, BioLegend) was used for detection. Biotin was used in an excess of 40x. Prior to the analysis, the samples were allowed to thaw at room temperature for 45 min. Thawed CSF samples were vortexed for 30 s at 2000 rpm and diluted using commercially available Tau2.0 assay diluent (Quanterix). Plasma samples were vortexed for 30 s at 2000 rpm, and centrifuged for 10 min at 4000 rpm, and diluted using commercially available Tau2.0 assay diluent (Quanterix). Co-incubation of sample and detector antibody or 2-steps (47 min co-incubation of beads, sample and detector followed by a 7 min incubation with SBG), no helper beads, 25 µL of beads, 20 µL of detector antibody and 100 µL of SBG. The SIMOA® analyzer was used to detect and quantify the signal. The SIMOA® analyzer detects fluorescence signals generated by the enzyme-substrate reaction, correlating with the concentration of the target protein in the samples. The results are presented in Tables 15-20. Table 15. Antibody clone 2F10 Sample AEB1 AEB2 Mean S / N H 17.639 17.192 17.416 6319.004 G 4.390 4.855 4.622 1677.137 GSK3 ^ F 1.191 0.846 1.019 369.626 E 0.355 0.432 0.393 142.735 D 0.130 0.101 0.115 41.811 C 0.045 0.038 0.042 15.087 B 0.016 0.018 0.017 6.220 A 0.006 0.006 0.006 2.190 Blank 0.003 0.003 0.003 H 2.041 1.661 1.851 671.642 G 0.504 0.639 0.571 207.245 F 0.219 0.226 0.223 80.786 E 0.085 0.077 0.081 29.305 Tau441 D 0.027 0.025 0.026 9.514 C 0.012 0.012 0.012 4.326 B 0.012 0.006 0.009 3.284 A 0.002 0.002 0.002 0.858 QC C 0.003 0.003 0.003 0.966 QC E 0.004 0.005 0.004 1.544 Plasma QC H 0.002 0.002 0.002 0.839 QC L 0.002 0.002 0.002 0.662 CSF 1 0.072 0.069 0.070 25.494 CSF 2 0.039 0.039 0.039 14.031 CSF CSF 3 0.021 0.026 0.023 8.516 CSF 4 0.031 0.035 0.033 12.005 Table 16. Antibody clone 37A2 Sample AEB1 AEB2 Mean S / N H * * G * * F 11.970 10.036 11.003 2698.173 E 2.668 3.404 3.036 744.520 GSK D 0.764 0.768 0.766 187.805 C 0.268 0.233 0.250 61.395 B 0.058 0.056 0.057 13.975 A ** 0.022 0.022 5.311 Blank 0.003 0.005 0.004 Tau441 H 2.271 2.333 2.302 564.504 G 1.246 0.631 0.938 230.134 F 0.243 0.270 0.257 63.004 E 0.083 0.080 0.082 20.055 D 0.034 0.028 0.031 7.599 C 0.013 0.014 0.013 3.269 B 0.008 0.008 0.008 1.940 A 0.004 0.003 0.003 0.804 QC C 0.004 0.005 0.005 1.205 QC E 0.005 0.004 0.004 1.080 Plasma QC H 0.008 0.009 0.009 2.125 QC L 0.001 0.002 0.002 0.398 CSF 1 0.026 0.025 0.025 6.230 CSF 2 0.019 0.025 0.022 5.476 CSF CSF 3 0.022 0.019 0.021 5.027 CSF 4 0.018 0.019 0.019 4.591 Table 17. Antibody clone 38C8 Sample AEB1 AEB2 Mean S / N H ‘ * G * 20.996 20.996 5511.328 F 8.164 8.245 8.204 2153.544 E 1.961 1.909 1.935 507.998 GSK D 0.729 0.534 0.631 165.722 C 0.145 0.107 0.126 33.010 B 0.041 0.035 0.038 9.852 A 0.010 0.015 0.012 3.225 Blank 0.004 0.004 0.004 H 2.219 1.658 1.939 508.903 G 0.713 0.614 0.664 174.230 F 0.256 0.216 0.236 61.966 Tau441 E 0.098 0.076 0.087 22.866 D 0.037 0.032 0.035 9.072 C 0.013 0.014 0.014 3.549 B 0.006 0.006 0.006 1.486 A 0.002 0.004 0.003 0.770 QC C 0.003 0.003 0.003 0.868 QC E 0.004 0.003 0.003 0.906 Plasma QC H 0.003 0.003 0.003 0.865 QC L 0.001 0.001 0.001 0.298 CSF 1 0.020 0.026 0.023 6.044 CSF 2 0.024 0.027 0.025 6.653 CSF CSF 3 0.019 0.017 0.018 4.762 CSF 4 0.015 0.018 0.016 4.260 Table 18. Antibody clone 31A6 Sample AEB1 AEB2 Mean S / N H * * G 15.838 16.341 16.090 1355.558 F 5.121 4.318 4.719 397.607 E 0.989 0.883 0.936 78.862 GSK D 0.324 0.257 0.290 24.445 C 0.100 0.105 0.102 8.633 B 0.038 0.065 0.052 4.343 A 0.022 0.024 0.023 1.926 Blank 0.022 0.001 0.012 H 2.151 1.491 1.821 153.427 G 0.694 0.767 0.731 61.580 F 0.257 0.240 0.248 20.908 E 0.087 0.111 0.099 8.339 Tau441 D 0.056 0.052 0.054 4.562 C 0.035 0.032 0.033 2.785 B 0.024 0.022 0.023 1.924 A 0.024 0.022 0.023 1.918 QC C 0.002 0.002 0.002 0.194 Plasma QC E 0.002 0.002 0.002 0.162 QC H 0.004 0.004 0.004 0.315 QC L 0.004 0.005 0.005 0.397 CSF 1 0.016 0.018 0.017 1.454 CSF 2 0.018 0.019 0.019 1.567 CSF CSF 3 0.014 0.012 0.013 1.095 CSF 4 0.012 0.014 0.013 1.116 Table 19. Antibody clone 2D1 Sample AEB1 AEB2 Mean S / N H * * G 21.700 20.141 20.921 1027.677 F 7.012 7.284 7.148 351.134 E 1.768 1.718 1.743 85.626 GSK D 0.496 0.394 0.445 21.864 C 0.131 0.188 0.159 7.832 B 0.052 0.059 0.056 2.736 A 0.030 0.029 0.029 1.448 Blank 0.018 0.023 0.020 H 1.516 1.953 1.734 85.194 G 0.584 0.612 0.598 29.362 F 0.228 0.219 0.223 10.975 E 0.090 0.102 0.096 4.716 Tau441 D 0.046 0.047 0.047 2.288 C 0.031 0.029 0.030 1.477 B 0.025 0.023 0.024 1.178 A 0.022 0.020 0.021 1.029 QC C 0.003 0.003 0.003 0.156 QC E 0.002 0.002 0.002 0.078 Plasma QC H 0.005 0.005 0.005 0.243 QC L 0.002 0.003 0.003 0.136 CSF 1 0.018 0.015 0.017 0.818 CSF 2 0.020 0.020 0.020 0.994 CSF CSF 3 0.013 0.014 0.013 0.654 CSF 4 0.014 0.013 0.013 0.658 Table 20. Reference antibody Invitrogen Sample AEB1 AEB2 Mean S / N H * * G * * F 23.579 22.876 23.227 606.695 E 11.373 12.266 11.820 308.727 GSK D 4.936 4.606 4.771 124.619 C 1.857 2.010 1.934 50.508 B 0.702 0.751 0.726 18.969 A 0.260 0.258 0.259 6.759 Blank 0.040 0.037 0.038 H 5.500 5.221 5.361 140.018 G 1.673 1.497 1.585 41.408 F 0.456 0.473 0.464 12.128 E 0.225 0.257 0.241 6.302 Tau441 D 0.107 0.106 0.107 2.789 C 0.062 0.038 0.050 1.304 B 0.030 0.044 0.037 0.972 A 0.036 0.039 0.037 0.976 QC C 0.164 0.170 0.167 4.361 QC E 0.006 0.008 0.007 0.177 Plasma QC H 0.204 0.212 0.208 5.428 QC L 0.005 0.004 0.005 0.127 CSF 1 0.055 0.055 0.055 1.427 CSF 2 0.065 0.064 0.065 1.686 CSF CSF 3 0.051 0.044 0.048 1.244 CSF 4 0.058 0.061 0.060 1.556 Bold has % CV > 15%; * Too high signal, ** Invalid result. The antibody clone 2F10 showed the best performance and therefore the highest potential for a blood assay. In particular, the antibody clone 2F10 had high signal-to-noise ratio (S / N) with GSK3b and low S / N with non-phosphorylated tau441, indicating high specificity for the phosphorylated form of the tau protein. Further, the antibody clone 2F10 had the highest S / N in human CSF samples as well as in some plasma samples. Two other antibody clones (37A2 and 38C8) had similar performance but smaller S / N in CSF samples. These three clones were selected for continuing immunoassay development in blood. EXAMPLE 5 This Example investigated the affinity of the different antibody clones for tau phosphorylated at position T205. Biacore is a label-free technology used to analyze biomolecular interactions in real-time. It employs surface plasmon resonance (SPR) to measure binding events between molecules, providing quantitative data on kinetics, affinity, concentration, and specificity. The three antibody clones (37A2, 38C8 and 2F10) selected from Example 4 were tested with a mixture of GSK3b and DYRK1A phospho-Tau441. From all the antibody clones, there was a better response with a mixture of GSK3b and DYRK1A phospho-Tau441. The highest on-rate was for 37A2, followed by 38C8 and 2F10. Interestingly, 2F10 had the lowest off-rate of all the tested antibody clones, see Table 21 and Figure 20. This antibody clone 2F10 therefore binds less protein but binds stronger than the other tested antibody clones. Based on the dissociation profiles, 2F10 was selected as the most promising anti-p-tau205 monoclonal antibody, which was in agreement with the results from Example 4. Table 21. Biacore binding results. Antibody clone Max on-rate response Min off-rate response % dissociation (at 1485 s) (at 1990 s) 2F10 63 44 30 % 37A2 170 47 72 % 38C8 108 17 84 % EXAMPLE 6 The Example tested the best protocol for bead conjugation to optimize the immunoassay. SIMOA® is a paramagnetic bead-based immunoassay technology. Thus, bead conjugation, that is the linkage of antibodies to beads, represents one critical element in assay development. Naked beads, i.e., beads without capture antibody, were carboxylated on their surface. To crosslink the naked beads with the capture antibody, beads were activated by incubating the naked bead in a solution containing 1-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride (EDC), as well as Sulfo-NHS, which makes them reactive to the capture antibody. Reagents and Antibodies p-tau205 Antibody Clones: 2F10, 37A2, 38C2 Beads: 2F10, 37A2, 38C2; conjugated with EDC and Sulfo-NHS Detection antibody: Biotinylated secondary antibody specific for tau or phosphorylated tau (Tau12) 40x biotin excess Samples: in order; 2 blanks, 1 calibrator point (GSK3β p-tau 441), 3 plasma QC samples, 3 high tau CSF samples, 3 low tau CSF samples. Table 22. EDC vs. S-NHS conjugation Old EDC conjugation S / N beads 2F10 S / N 37A2 S / N 38C8 S / N Blank1 0.00229 0.00444 0.00297 0.00376 0.00264 0.00277 0.00382 0.00406 Blank2 0.00659 0.00455 0.00289 0.00430 Cal E 9.19202 2072.09 0.04892 13.01 1.75178 632.95 0.86625 213.13 iQC H 0.24096 54.32 0.00489 1.30 0.01025 3.70 0.00871 2.14 iQC L 0.00808 1.82 0.00359 0.95 0.00264 0.95 0.00318 0.78 PDGFRb 0.00967 2.18 0.01683 4.47 0.00407 1.47 0.00608 1.50 1 High tau 0.10723 24.17 0.15800 42.01 0.00784 2.83 0.00952 2.34 2 High tau 0.08064 18.18 0.08812 23.43 0.00703 2.54 0.00625 1.54 3 High tau 0.13641 30.75 0.18094 48.11 0.00682 2.47 0.00641 1.58 4 Low tau 0.02307 5.20 0.01156 3.07 0.00396 1.43 0.00924 2.27 5 Low tau 0.01677 3.78 0.01155 3.07 0.00573 2.07 NaN 6 Low tau NaN NaN NaN NaN S-NHS conjugation Blank1 2F10 S / N 37A2 S / N 38C8 S / N Blank2 0.00357 0.00361 0.01128 0.01206 0.00774 0.00633 Cal E 0.00366 0.01283 0.00493 iQC H 0.12177 33.72 0.44665 37.04 0.77585 122.50 iQC L 0.00443 1.23 0.01012 0.84 0.00917 1.45 PDGFRb 0.00343 0.95 0.00803 0.67 0.00407 0.64 1 High tau 0.01226 3.39 0.00974 0.81 0.00334 0.53 2 High tau 0.11466 31.76 0.01145 0.95 0.00892 1.41 3 High tau 0.06303 17.46 0.00991 0.82 0.00563 0.89 4 Low tau 0.12428 34.42 0.00949 0.79 0.00570 0.90 5 Low tau 0.01030 2.85 0.00948 0.79 0.00788 1.24 6 Low tau NaN NaN NaN EDC conjugated 2F10 gave high signal in CSF samples with high tau compared to those with low tau, thereby indicating good diagnostic performance. EDC conjugated 2F10 also generated signals in plasma samples. The antibody clone 2F10 clone was selected among the three tested antibody clones for continuing immunoassay development. EXAMPLE 7 Biotinylation refers to the covalent addition of a biotin to another molecule, in our case to detection antibodies. Commercial sources of biotin are generally linked to amine-reactive groups, such as N- hydroxysuccinimide ester (NHS), which specifically react with N-terminal and lysine amino groups of proteins forming amide bonds. In our case, the final purpose of linking biotin to detection antibodies is to bind streptavidinhorseradish peroxidase (HRP) complexes. Biotin displays an extremely high affinity and specificity for streptavidin, and this interaction can sustain high temperatures and extreme pH, making it very suitable for large variety of molecular biology techniques. Additionally, due to the small size of biotin, biotinylation does not interfere with the binding abilities of the detection antibody, which are preserved intact. Similarly to bead conjugation, biotinylation of the detection antibody can be optimized in order to improve the immunoassay performance. Procedures A starting point for antibody biotinylation was an antibody concentration of 1 µg / mL and a biotin excess of 40x. We also included Tau13 (Tau13; Purified anti-Tau, 15-25 Antibody #835204, Biolegend), a mouse monoclonal antibody that binds to amino acid residues 15-25 at the N-terminal of human tau. Reagents and Antibodies ^ p-tau205 Antibody Clones: 2F10 ^ Beads: 2F10 ^ Detection Antibody: Biotinylated secondary antibody specific for tau or phosphorylated tau (Tau13) 40x biotin excess, (Tau13) 80x biotin excess, (Tau12) 40x biotin excess ^ Samples: in order; 1 blank (cal a), 4 calibrator point (cal b,c,d,h), 5 CSF samples (c1, c2, c3, c4, c5), 2 plasma samples (p1, p2) Table 23. Tau1340x biotin excess AEB1 AEB Mean S / N cal a 0.005961 0.005451 0.005706 1 cal b 0.016184 0.012584 0.014384 2.520723 cal c 0.084927 0.090983 0.087955 15.41393 cal d 0.785762 0.831558 0.80866 141.716 iqc h 0.011009 0.012484 0.011746 2.058497 c1 0.027984 0.023631 0.025808 4.52276 c2 0.063826 0.064787 0.064307 11.26964 c3 0.20674 0.211125 0.208932 36.61498 c4 0.144286 0.143005 0.143645 25.17353 c5 0.141027 0.1428 0.141914 24.87009 p1 0.004749 0.00376 0.004254 0.745586 p2 0.004175 0.003976 0.004076 0.714225 Table 24. Tau1380x biotin excess AEB1 AEB Mean S / N cal a 0.003824 0.004107 0.003966 1 cal b 0.014218 0.014896 0.014557 3.670603 cal c 0.095186 0.099281 0.097233 24.51807 cal d 0.925648 1.022242 0.973945 245.5867 iqc h 0.010366 0.01183 0.011098 2.798342 c1 0.02342 0.023605 0.023512 5.928763 c2 0.065225 0.057568 0.061397 15.48154 c3 0.209086 0.225784 0.217435 54.82766 c4 0.146806 0.143973 0.14539 36.66097 c5 0.151564 0.14472 0.148142 37.35493 p1 0.004271 0.003933 0.004102 1.03432 p2 0.003299 0.003661 0.00348 0.877447 Table 25. Tau1240x biotin excess AEB1 AEB Mean S / N cal a 0.008358 0.00696 0.007659 1 cal b 0.013523 0.014914 0.014219 1.856459 cal c 0.063233 0.062217 0.062725 8.189607 cal d 0.606909 0.688796 0.647852 84.58582 iqc h 0.008986 0.008262 0.008624 1.12596 c1 0.015495 0.016764 0.01613 2.105937 c2 0.035434 0.036481 0.035958 4.694758 c3 0.121152 0.130564 0.125858 16.43251 c4 0.088574 0.089116 0.088845 11.59995 c5 0.084419 0.085618 0.085018 11.10028 p1 0.004627 0.006987 0.005807 0.758161 p2 0.003692 0.003358 0.003525 0.460213 Tau1380x gave the lowest blank and slightly higher average number of enzymes per bead (AEB) than Tau1340x in some samples, whereas Tau1240x had a lower S / N. EXAMPLE 8 This Example optimized the concentration of streptavidin covalently coupled to ^–D–galactosidase (SBG) and detector antibody in the immunoassay. This optimization involved testing combinations of SBG and detector antibody at different concentrations, with the aim of reducing the background signal or noise and / or increase the AEB signals. Several brain- derived molecules, mostly in blood, are present at such low levels that SBG and detector titration are important to achieve successful measurements in all samples. In general, higher concentrations of detector antibody and SBG result in higher signals, while lower concentrations yield lower signals. On the other hand, higher concentrations of detector antibody and SBG result in higher background noise, while lower concentrations yield lower background. Thus, in order effectively find the best signal to noise ratio, 9 different combinations of detector antibody and SBG concentration were tested. Reagents and Antibodies ^ p-tau205 Antibody Clones: 2F10 ^ Beads: 2F10 ^ Detection Antibody: Tau13 (80x) at concentrations: 0.5, 1 and 2 µg / mL ^ SBG (enzyme): concentrations: 50 pM, 150 pM and 300 pM ^ Samples: in order; 1 blank (cal a), 4 calibrator point (cal b,c,d,h), 2 plasma samples (p1, p2), 2 CSF samples (c1, c2) Table 26. Det 0.5 / SBG 50 pM Sample AEB Mean AEB %CV S / N 0.002344 CAL A 0.00309 34.2 1 0.003839 0.004898 CAL B 0.00476 4.2 1.538 0.004614 0.025312 CAL C 0.02457 4.3 7.947 0.023826 0.205436 CAL D 0.22412 11.8 72.496 0.242813 0.002798 IQC H 0.00390 39.9 1.261 0.005 0.019636 P1 0.01505 43.0 4.870 0.010473 0.00262 P2 0.00308 21.3 0.998 0.003549 0.028865 C1 0.02923 1.8 9.455 0.029596 0.026515 C2 0.02642 0.5 8.547 0.026329 Table 27. Det 0.5 / SBG 150 pM Sample AEB Mean AEB %CV S / N 0.004234 CAL A 0.00572 36.7 1 0.007201 0.011210 CAL B 0.01045 10.3 1.828 0.009691 0.056672 CAL C 0.05803 3.3 10.148 0.059379 0.605115 CAL D 0.60610 0.2 106.004 0.607082 0.010289 IQC H 0.00957 10.6 1.674 0.008856 0.042061 P1 0.04189 0.6 7.327 0.041724 0.006610 P2 0.00735 14.2 1.285 0.008090 0.066638 C1 0.06635 0.6 11.604 0.066055 0.061141 C2 0.05827 7.0 10.192 0.055405 Table 28. Det 0.5 / SBG 300 pM Sample AEB Mean AEB %CV S / N 0.006561 CAL A 0.00710 10.7 1 0.007638 0.015216 CAL B 0.01575 4.8 2.219 0.016285 0.077057 CAL C 0.07859 2.8 11.070 0.080123 0.701518 CAL D 0.75273 9.6 106.023 0.803943 0.012383 IQC H 0.01231 0.9 1.733 0.012232 0.060396 P1 0.05725 7.8 8.064 0.054113 0.011202 P2 0.00996 17.6 1.403 0.008721 0.092895 C1 0.08976 4.9 12.643 0.086628 0.078844 C2 0.08044 2.8 11.331 0.082046 Table 29. Det 1 / SBG 50 pM Sample AEB Mean AEB %CV S / N 0.005453 CAL A 0.00401 50.9 1 0.002567 0.005893 CAL B 0.00590 0.3 1.472 0.005914 0.029266 CAL C 0.03238 13.6 8.076 0.035503 0.268538 CAL D 0.28951 10.2 72.198 0.310483 0.005315 IQC H 0.00664 28.2 1.656 0.007965 0.013505 P1 0.01632 24.4 4.069 0.019128 0.004469 P2 0.00379 25.3 0.945 0.003111 0.040357 C1 0.04202 5.6 10.478 0.043677 0.042196 C2 0.04283 2.1 10.682 0.043473 Table 30. Det 1 / SBG 150 pM Sample AEB Mean AEB %CV S / N 0.014766 CAL A 0.01220 29.7 1.000 0.009637 0.019759 CAL B 0.01650 27.9 1.352 0.013242 0.076352 CAL C 0.07512 2.3 6.156 0.073880 0.556809 CAL D 0.63869 18.1 52.344 0.720567 IQC H 0.013580 0.01418 6.0 1.162 0.014787 0.042029 P1 0.03427 32.0 2.808 0.026504 0.007427 P2 0.00669 15.6 0.548 0.005948 0.088433 C1 0.09671 12.1 7.926 0.104994 0.086162 C2 0.08258 6.1 6.768 0.079007 Table 31. Det 1 / SBG 300 pM Sample AEB Mean AEB %CV S / N 0.006876 CAL A 0.00657 6.6 1.000 0.006264 0.019693 CAL B 0.01785 14.6 2.717 0.016015 0.097327 CAL C 0.10489 10.2 15.964 0.112450 1.000008 CAL D 1.07292 9.6 163.301 1.145837 0.025800 IQC H 0.02273 19.1 3.459 0.019655 0.050693 P1 0.05631 14.1 8.570 0.061921 0.012737 P2 0.01132 17.7 1.723 0.009910 0.130013 C1 0.13431 4.5 20.442 0.138598 0.114536 C2 0.11440 0.2 17.412 0.114269 Table 32. Det 2 / SBG 50 pM Sample AEB Mean AEB %CV S / N 0.005635 CAL A 0.00390 63.1 1.000 0.002157 0.006601 CAL B 0.00620 9.1 1.592 0.005802 0.035593 CAL C 0.03362 8.3 8.628 0.031643 0.278266 CAL D 0.32977 22.1 84.641 0.381275 0.006030 IQC H 0.00582 5.0 1.495 0.005617 0.013851 P1 0.01428 4.3 3.666 0.014717 0.003853 P2 0.00374 4.1 0.961 0.003634 0.046225 C1 0.04600 0.7 11.805 0.045766 0.040252 C2 0.03751 10.3 9.628 0.034772 Table 33. Det 2 / SBG 150 pM Sample AEB Mean AEB %CV S / N 0.012648 CAL A 0.00936 49.6 1.000 0.006078 0.013956 CAL B 0.01423 2.7 1.519 0.014499 0.080214 CAL C 0.07033 19.9 7.511 0.060443 0.606438 CAL D 0.63480 6.3 67.797 0.663158 0.014452 IQC H 0.01299 15.9 1.387 0.011527 0.037397 P1 0.03786 1.7 4.043 0.038320 0.007073 P2 0.00733 5.0 0.783 0.007589 0.096006 C1 0.09389 3.2 10.027 0.091765 0.085571 C2 0.08907 5.6 9.513 0.092574 Table 34. Det 2 / SBG 300 pM Sample AEB Mean AEB %CV S / N 0.007734 CAL A 0.00900 19.9 1.000 0.010270 0.017371 CAL B 0.01818 6.3 2.019 0.018985 0.109646 CAL C 0.10882 1.1 12.088 0.107995 1.042475 CAL D 1.03633 0.8 115.122 1.030177 0.020440 IQC H 0.01975 4.9 2.194 0.019058 0.056883 P1 0.05604 2.1 6.226 0.055202 0.010912 P2 0.01148 7.0 1.275 0.012045 0.128728 C1 0.13561 7.2 15.064 0.142486 0.139834 C2 0.13069 9.9 14.518 0.121547 The most optimal detector antibody and SBG concentration rendering high S / N in both plasma and CSF samples were identified as 1 µg / ml detector antibody and 300 pM SBG. EXAMPLE 9 The aim was of this Example was to further test biotin excess using Tau13 as detection antibody to further increase S / N in plasma samples. We test different concentrations of biotin for Tau13 and included several plasma samples in the test. Reagents and Antibodies p-tau205 Antibody Clones: 2F10 Beads: 2F10 Detection Antibody: Tau13 (80x, 100x, 120x) Samples: in order; blanks, 2 calibrator points, 2 CSF samples, 11 plasma samples Table 35. Detection Antibody Tau13 (80x, 100x, 120x) x80 x100 x120 Mean Mean Mean Sample S / N S / N S / N AEB AEB AEB 0.0097 0.0089 0.0172 0.0135 0.0129 0.0094 Blank 0.0105 0.0103 0.0110 0.0094 0.0091 0.0093 0.0094 0.0105 0.0080 26.90 0.6361 0.6638 0.7871 0.6227 59.2 0.6698 64.8 0.7749 70.6 (pg / mL) 0.6094 0.6758 0.7627 0.42 0.0182 0.0145 0.0149 0.0185 1.8 0.0156 1.5 0.0162 1.5 (pg / mL) 0.0189 0.0166 0.0176 0.0424 0.0479 0.0482 CSF 1 0.0459 4.4 0.0482 4.7 0.0481 4.4 0.0494 0.0485 0.0480 CSF pool 0.1775 0.1793 0.1860 0.1845 17.5 0.1840 17.8 0.1823 16.6 BD1 0.1915 0.1886 0.1787 0.0146 0.0129 0.0151 QC high 0.0133 1.3 0.0133 1.3 0.0139 1.3 0.0121 0.0137 0.0126 0.0093 0.0098 0.9 0.0076 0.0084 0.8 0.0083 0.0094 0.9 Plasma pool 0.0102 0.0093 0.0104 0.0177 0.0225 0.0158 Plasma 1 0.0170 1.6 0.0210 2.0 0.0167 1.5 0.0163 0.0194 0.0177 0.0253 0.0170 0.0189 Plasma 2 0.0235 2.2 0.0149 1.4 0.0168 1.5 0.0216 0.0128 0.0148 0.0158 0.0147 0.0161 Plasma 3 0.0169 1.6 0.0160 1.5 0.0143 1.3 0.0180 0.0172 0.0125 0.0119 0.0097 0.0085 Plasma 4 0.0110 1.0 0.0120 1.2 0.0092 0.8 0.0100 0.0142 0.0099 0.0245 0.0208 0.0198 Plasma 5 0.0235 2.2 0.0231 2.2 0.0203 1.9 0.0224 0.0254 0.0209 0.0113 0.0166 0.0137 Plasma 6 0.0135 1.3 0.0151 1.5 0.0130 1.2 0.0158 0.0137 0.0122 0.0574 0.0515 0.0533 Plasma 7 0.0561 5.3 0.0544 5.3 0.0516 4.7 0.0547 0.0573 0.0499 0.0101 QC low 0.0104 1.0 NaN NaN NaN! NaN 0.0107 QC 0.0123 0.0123 1.2 NaN NaN NaN NaN medium 0.0123 There was no significant difference between the different tested biotin excessed. EXAMPLE 10 This Example optimized the buffer used in the immunoassay for diluting the samples. Some key components to optimize are the buffer pH, blockers (including both general blockers, such as BSA and heterophilic blockers), detergents and salt content. Two commercially available buffers, Tau 2.0 and Advantage buffer, both from Quanterix, were tested. Reagents and Antibodies p-tau205 Antibody Clones: 2F10 and Invitrogen p-tau205 Beads: 2F10 vs Invitrogen p-tau205 (EDC and Sulfo-NHS conjugated) Detection Antibody: Tau13 Assay diluent: 2F10 assay (Tau2.0 and Advantage buffer) vs Invitrogen p-tau205 assay (Tau2.0) Samples: in order; 4 blanks, 2 calibrator point, 2 CSF samples, 8 plasma samples Table 36. DiluentDiluentTau2.0 Advantage Tau2.0 Tau2.0Invitrogen - S-NHS Invitrogen - standard Clone 2F10 Clone 2F10 Capture conjugation conjugationSampleMean AEB S / N Mean AEB S / N Mean AEB S / N Mean AEB S / N0.0112 0.0094 0.0100 0.0063 0.0081 0.0124 0.0085 0.0071 Blank 0.0087 0.0113 0.0122 0.0131 0.0078 0.0077 0.0168 0.0071 0.0077 0.0156 0.0137 0.0320 2.90 0.7908 0.3895 8.9034 5.9565 (pg / mL 0.7700 88.3 0.3915 34.8 8.9647 732.6 5.9797 456.6 ) 0.7491 0.3935 9.0261 6.0030 0.42 0.0134 0.0177 0.1771 0.1604 (pg / mL 0.0171 2.0 0.0168 1.5 0.1840 15.0 0.1586 12.1 ) 0.0208 0.0158 0.1909 0.1568 0.0421 0.0484 0.0568 0.0356 0.0402 4.6 0.0439 3.9 0.0587 4.8 0.0346 2.6 CSF 1 0.0383 0.0394 0.0605 0.0336 CSF 0.1622 0.2047 0.1410 0.0693 pool 0.1648 18.9 0.1973 17.5 0.1412 11.5 0.0702 5.4 0.1674 0.1898 0.1414 0.0711 BD1 0.0116 0.0142 0.6973 0.3993 iQC H 0.0115 1.3 0.0173 1.5 0.6663 54.4 0.4158 31.7 0.0113 0.0204 0.6354 0.4323 0.6210 0.4073 iQC H 0.6525 53.3 0.3845 29.4 1:4 0.6840 0.3618 0.5106 0.3351 iQC H 0.4894 40.0 0.3361 25.7 1:8 0.4681 0.3370 0.3390 0.2424 iQC H 0.3584 29.3 0.2422 18.5 1:16 0.3778 0.2420 0.0151 0.0760 0.0143 0.0078 plasma 0.0155 1.8 0.0731 6.5 0.0140 1.1 0.0093 0.7 2 0.0160 0.0702 0.0138 0.0107 0.0227 0.0403 0.0229 0.0179 plasma 0.0237 2.7 0.0486 4.3 0.0224 1.8 0.0168 1.3 5 0.0248 0.0568 0.0219 0.0157 0.0565 0.1059 0.0507 0.0216 plasma 0.0548 6.3 0.1068 9.5 0.0488 4.0 0.0222 1.7 7 0.0531 0.1078 0.0469 0.0227 0.0396 0.0200 plasma 0.0392 3.2 0.0190 1.4 71:4 0.0388 0.0180 Higher signal in plasma samples was obtained with the antibody clone 2F10 than commercial Invitrogen p-tau205. There was clearly more signal in plasma samples with the antibody clone 2F10 is there clearly more signal in plasma as compared to Tau2.0, although GSK was higher with Tau2.0. The Advantage buffer was selected as the most optimal as assay diluent. EXAMPLE 11 This Example evaluated whether higher concentrations of Tau13 detection antibody and SBG concentration would render higher S / N in plasma samples. Reagents and Antibodies p-tau205 Antibody Clones: 2F10 Beads: 2F10 Detection Antibody: Tau13 concentrations: 1 µg / mL and 2 µg / mL SBG concentrations: 300 pM and 400 pM Assay diluent: Advantage buffer Samples: in order; 1 blank, 7 calibrator point, 1 CSF sample, 15 plasma samples Table 37. Detector antibody and SBC concentrations. HB 1 2 SBG conc, pM 300 400 Detector conc, 1 g / ml 2 µg / ml sample AEB Mean AEB CV % AEB S / N AEB Mean AEB CV % AEB S / N 0.0113 0.0098 Blank 0.0152 36.3 0.0108 13.2 0.0191 0.0118 1.3921 1.6953 Cal 100 1.4029 1.09 92.1 1.7849 7.1 165.3 1.4137 1.8744 0.7062 0.9709 Cal 50 0.7678 11.35 50.4 0.9386 4.9 86.9 0.8294 0.9062 0.3711 0.4433 Cal 25 0.3489 10.12 23.1 0.4405 0.9 40.8 0.3267 0.4376 0.1727 0.2176 Cal 12.5 0.17455 1.53 11.5 0.2397 13 22.2 0.1764 0.2617 0.0819 0.1169 Cal 6.25 0.082 0.22 5.4 0.1209 4.7 11.2 0.0821 0.1249 0.0471 0.0649 Cal 3.13 0.04815 3.08 3.2 0.0683 7 6.3 0.0492 0.0717 0.0388 0.0632 Cal 1.56 0.03405 19.75 2.2 0.0601 7.4 5.6 0.0293 0.0569 0.2701 0.339 CSP pool BD1 0.263 3.8 17.3 0.3568 7 33.1 0.2559 0.3746 0.0602 0.0945 Plasma (3) 0.0582 4.8 3.8 0.1024 10.8 9.5 0.0562 0.1102 0.0272 0.0454 Plasma (4) 0.02625 5.28 1.7 0.0468 4.1 4.3 0.0253 0.0481 0.0141 0.0182 Plasma 1 0.01245 18.93 0.8 0.0180 1.6 1.7 0.0108 0.0178 0.0079 0.0118 Plasma 2 0.01255 52.45 0.8 0.0392 98.8 3.6 0.0172 0.0666 0.0477 0.0753 Plasma 3 0.045 8.4 3 0.0746 1.3 6.9 0.0423 0.0739 0.0071 0.0121 Plasma 4 0.00685 5.74 0.4 0.0122 1.4 1.1 0.0066 0.0123 0.0214 0.0352 Plasma 5 0.0231 10.54 1.5 0.0359 2.7 3.3 0.0248 0.0366 0.0352 NaN Plasma 6 0.04465 29.9 2.9 0.0343 ### 3.2 0.0541 0.0343 0.0181 0.0265 Plasma 7 0.0167 11.63 1.1 0.0254 6.1 2.4 0.0153 0.0243 0.0069 0.0117 Plasma 8 0.00875 29.98 0.6 0.0113 5 1 0.0106 0.0109 Plasma 9 0.0232 0.0232 0.14 1.5 0.0502 0.0496 1.9 4.6 0.0232 0.0489 0.0079 0.0124 Plasma 10 0.0084 8.25 0.6 0.0122 2.3 1.1 0.0089 0.012 0.0639 0.1048 Plasma 11 0.06305 1.82 4.1 0.0996 3.7 9.5 0.0622 0.0944 0.0288 0.0565 Plasma 12 0.02785 4.78 1.8 0.0536 7.8 5 0.0269 0.0506 0.0097 NaN Plasma 13 0.00975 0.53 0.6 0.0138 ### 1.3 0.0098 0,0138 The increase in Tau13 detection antibody concentration from 1 µg / ml to 2 µg / ml and the increase in SBG concentration from 300 pM to 400 pM resulted in a pronounced increase in S / N across plasma samples. Accordingly, a Tau13 detection antibody concentration of 2 µg / ml and a SBG concentration of 400 pM were selected. EXAMPLE 12 This Example tested the use of helper beads in the immunoassay. Helper beads are naked beads used in order to simultaneously reduce the amount of conjugated beads as well as to improve the S / N. Reagents and Antibodies p-tau205 Antibody Clones: 2F10 Beads: 2F10 Detection antibody: Tau13, 2 µg / mL SBG concentrations: 400 pM Assay diluent: Advantage buffer Helper beads: 0%, 50% and 70% Table 38. Helper beads 0% Sample AEB Mean AEB CV% AEB S / N 0.016 Blank 0.012 40.96% 1.000 0.009 0.721 Cal 50 (pg / mL) 0.743 4.12% 60.776 0.764 Cal 12.50 (pg / mL) 0.191 0.195 2.73% 15.926 0.198 0.062 Cal 3.13 (pg / mL) 0.063 3.55% 5.193 0.065 0.025 Cal 0.78 (pg / mL) 0.024 5.68% 1.957 0.023 0.322 CSF pool BD1 0.323 0.77% 26.466 0.325 0.076 Plasma 3 6.321 0.078 0.077 2.18% 0.031 Plasma 5 0.030 2.86% 2.467 0.030 0.058 Plasma 6 0.049 25.35% 3.999 0.040 0.048 Plasma 9 0.046 5.38% 3.749 0.044 0.102 Plasma 11 0.102 0.87% 8.308 0.101 0.054 Plasma 12 0.056 5.70% 4.565 0.058 0.156 Plasma 2 12.639 0.153 0.154 1.48% 0.049 Plasma 15 0.052 8.22% 4.255 0.055 Table 39. Helper beads 50% Sample AEB Mean AEB CV% AEB S / N 0.009 Blank 0.010 12.61% 1.000 0.011 0.942 Cal 50 (pg / mL) 0.881 9.88% 87.027 0.819 0.229 Cal 12.50 (pg / mL 0.217 7.98% 21.434 0.205 Cal 3.13 (pg / mL) 0.069 0.065 10.25% 6.412 0.061 0.026 Cal 0.78 (pg / mL) 0.024 10.43% 2.378 0.022 0.383 CSF pool BD1 0.385 0.93% 38.075 0.388 0.079 Plasma 3 0.079 0.03% 7.830 0.079 0.033 Plasma 5 0.032 2.73% 3.198 0.032 0.038 Plasma 6 0.036 9.55% 3.545 0.033 0.041 Plasma 9 0.041 5.42% 4.100 0.042 0.102 Plasma 11 0.101 2.35% 9.940 0.099 0.052 Plasma 12 0.053 2.30% 0.054 5.210 0.164 Plasma 2 0.162 1.73% 0.16016.0360.049 Plasma 15 0.052 7.83% 5.166 0.055 Table 40. Helper beads 70% Sample AEB Mean AEB CV% AEB S / N 0.010 Blank 0.011 15.65% 1.000 0.012 0.866 Cal 50 (pg / mL) 0.896 4.74% 82.332 0.926 0.220 Cal 12.50 (pg / mL) 0.229 5.65% 21.088 0.239 0.076 Cal 3.13 (pg / mL) 0.071 8.70% 6.530 0.066 Cal 0.78 (pg / mL) 0.030 0.029 5.33% 2.642 0.028 0.391 CSF pool BD1 0.399 2.80% 36.662 0.407 0.078 Plasma 3 0.079 1.88% 7.300 0.080 0.038 Plasma 5 0.036 7.15% 3.328 0.034 0.046 Plasma 6 0.044 6.92% 4.013 0.042 0.047 Plasma 9 0.045 0.23% .142 0.043 0.099 Plasma 11 0.097 2.85% 8.943 0.095 0.062 Plasma 12 0.058 9.59% 5.356 0.054 0.168 Plasma 2 0.166 1.28% 15.298 0.165 0.054 Plasma 15 0.053 3.97% 4.865 0.051 Table 41. Summary helper beads HB 0% HB 50% HB70% AEB 0.743 0.881 0.896 Cal 50 (pg / mL) S / N 60.776 87.027 82.332 AEB 0.195 0.217 0.229 Cal 12.50 (pg / mL) S / N 15.926 21.434 21.088 AEB 0.063 0.065 0.071 Cal 3.13 (pg / mL) S / N 5.193 6.412 6.530 AEB 0.024 0.024 0.029 Cal 0.78 (pg / mL) S / N 1.957 2.238 2.642 AEB 0.012 0.010 0.011 Blank S / N 1 1 1 CSF pool BD1 AEB 0.323 0.385 0.399 S / N 26.466 38.075 36.662 AEB 0.077 0.079 0.079 Plasma 3 S / N 6.321 7.830 7.300 AEB 0.030 0.032 0.036 Plasma 5 S / N 2.467 3.198 3.328 AEB 0.049 0.036 0.044 Plasma 6 S / N 3.999 3.545 4.013 AEB 0.046 0.041 0.045 Plasma 9 S / N 3.749 4.100 4.142 AEB 0.102 0.101 0.097 Plasma 11 S / N 8.308 9.940 8.943 AEB 0.056 0.053 0.058 Plasma 12 S / N 4.565 5.210 5.356 AEB 0.154 0.162 0.166 Plasma 2 S / N 12.639 16.036 15.298 AEB 0.052 0.052 0.053 Plasma 15 S / N 4.255 5.166 4.865 Helper beads improved both AEB and S / N in plasma samples as compared to not using helper beads. Both 50% and 70% helper beads perform equally well. EXAMPLE 13 This Exampled validated the utility of the p-tau205 immunoassay to detect Alzheimer´s disease (AD) pathology in human CSF and blood. Reagents and Antibodies ^ p-tau205 Antibody Clones: 2F10 ^ Beads: 2F10 ^ Detection Antibody: Tau13 concentrations: 2 µg / mL ^ SBG concentrations: 400 pM ^ Assay diluent: Advantage buffer ^ Helper beads: 50% Plasma and CSF samples were obtained from AD and control patients. The discovery cohort (Example 1) was comprised of patients with biologically defined AD (n=20) and neurological controls (n=20) clinically assessed in the Sahlgrenska University Hospital, Gothenburg, Sweden. AD cases were admitted for clinical evaluation for suspected AD, underwent lumbar puncture, and core AD CSF biomarkers were measured. Patients classified as AD displayed the typical AD CSF biomarker profile (CSF Aβ1–42 < 530 ng / L, p-tau181 > 60 ng / L, t-tau > 350 ng / L, all measured using INNOTEST ELISA). Neurological controls included patients with cognitive complains but no CSF biomarkers abnormalities. Individuals with other neurological disorders or with concomitant inflammatory diseases were not included. The results from the measurements are presented in Figure 22. It was concluded that the p-tau205 immunoassay using the antibody 2F10 as capture antibody could be used to distinguish AD pathology both in CSF samples and in blood plasma samples. EXAMPLE 14 This Exampled compared the performance of p-tau205 2F10 versus p-tau205 ThermoFisher immunoassays when detecting Alzheimer´s disease (AD) pathology in human CSF and blood. Reagents and Antibodies ^ p-tau205 Antibody Clones: 2F10 and ThermoFisher ^ Beads: 2F10 and ThermoFisher ^ Detection Antibody: Tau13 concentrations: 2 µg / mL (2F10) and 1 µg / mL (ThermoFisher) ^ SBG concentrations: 400 pM (2F10) and 150 pM (ThermoFisher) ^ Assay diluent: Advantage buffer (2F10) and Tau2.0 (ThermoFisher) ^ Helper beads: 50% for both 2F10 and ThermoFisher Plasma and CSF samples were obtained from AD and control patients. The discovery cohort (Example 1) was comprised of patients with biologically defined AD (n=20) and neurological controls (n=20) clinically assessed in the Sahlgrenska University Hospital, Gothenburg, Sweden. AD cases were admitted for clinical evaluation for suspected AD, underwent lumbar puncture, and core AD CSF biomarkers were measured. Patients classified as AD displayed the typical AD CSF biomarker profile (CSF Aβ1–42 < 530 ng / L, p-tau181 > 60 ng / L, t-tau > 350 ng / L, all measured using INNOTEST ELISA). Neurological controls included patients with cognitive complains but no CSF biomarkers abnormalities. Individuals with other neurological disorders or with concomitant inflammatory diseases were not included. The results from the comparison are presented in Figures 23A-23C (CSF samples) and Figures 24A-24B (plasma samples). It was concluded that the p-tau205 immunoassay using the antibody 2F10 as capture antibody outperformed p-tau205 immunoassay using the antibody ThermoFisher as capture antibody when discriminating AD pathology in CSF and plasma samples. The 2F10 immunoassay displayed larger fold changes (6.75 vs.2.45) as well as a perfect diagnostic accuracy in CSF samples (AUC=100%), Figures 23A-23C. In plasma, the 2F10 immunoassay was able to successfully measure p-tau205 in all plasma samples. When using the ThermoFisher p-tau205 immunoassay, more than half of the plasma samples were below the blank. Figures 24A-24B show only measurable plasma samples for the ThermoFisher p-tau205 immunoassay, i.e., leaving out the plasma samples that were below the blank. Although the plasma samples below blank were left out, the ThermoFisher p-tau205 immunoassay did not significantly discriminate AD from control samples, whereas the 2F10 immunoassay was able to significantly discriminate AD from control samples in blood plasma. EXAMPLE 15 This example generated rabbit monoclonal antibodies. Rabbit monoclonal antibodies were generated by immunizing four New Zealand Rabbits with the peptide CSGYSSPGSPG{pThr}PGSR (SEQ ID NO: 32). The negative screening peptide was CSGYSSPGSPGTPGSR (SEQ ID NO: 32). The rabbits were immunized three times. After the third immunization, a test for the sera titer was performed using ELISA. According to the ELISA results, all the animals showed promising titer against the antigen phosphorylated peptide, which had lower titer against non-phosphorylated peptide. Based on the results, two animals with higher titer against antigen were selected to proceed to the B cell cloning to select the specific antibody. After cell B cloning, the supernatant of 40 clones were selected for screening with ELISA. With the results from the screening, six clones were selected for sequencing and binding validation: 19G12, 37A2, 38C8, 2D1, 31A6 and 2F10. The binding validation resulted in five positive clones, and one negative (19G12), see Table 42. Table 42. Binding validation of antibody clones CSGYSSPGSPG{pThr}PGSR CSGYSSPGSPGTPGSR Antibody clone Non-dilution（10x）Non-dilution（10x）2F10 -2 2.865 2.721 0.202 0.062 31A6-1 2.875 2.816 0.358 0.086 2D1-1 2.858 2.79 0.224 0.064 38C8-1 2.902 2.827 0.145 0.089 37A2-1 2.928 2.837 0.161 0.067 Then, the 5 positive clones were purified to obtain the recombinant monoclonal antibodies. EXAMPLE 16 This Example involved sequencing of the monoclonal antibody 2F10-2. Total RNA was isolated from B cells (GenScript) following the technical manual of RNA-easy Isolation Reagent (Vazyme). Total RNA was then reverse-transcribed into cDNA using either isotype-specific anti- sense primers or universal primers following the technical manual of PrimeScriptTM1stStrand cDNA Synthesis Kit (Takara). Antibody fragments of heavy chain and light chain were amplified using specific primers according to the standard operating procedure (SOP) of GenScript. Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. The consensus sequence was provided. Heavy chain: DNA sequence (405 bp) – SEQ ID NO: 18 Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTC GGTGGAGGAGTCCGGGGGAAACCTGGTCACGCCTGGAGGATCCCTGACACTCACCTGCACAG TCTCTGGATTCTCCCTCAGTAGCAATGTAATGAGCTGGGTCCGCCAGGCTCCAGGGGAGGGG CTGGAATGGATCGGAACCATTAATACTCGTGGTATCACATACTACGCGAGCTGGGCGAAAGG CCGACTCACCATCTCCAAAACCTCGACCACGGTGGTTCTGAAAATGACCAGTCTGACAACCG AGGACACGGCCACCTATTTCTGTGCCACAGCTGGTAGTGGTAGTATTAGTTACTTTAACTTG TGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA Heavy chain: Amino acid sequence (135 aa) – SEQ ID NO: 19 Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 METGLRWLLLVAVLKGVQCQSVEESGGNLVTPGGSLTLTCTVSGFSLSSNVMSWVRQAPGEG LEWIGTINTRGITYYASWAKGRLTISKTSTTVVLKMTSLTTEDTATYFCATAGSGSISYFNL WGPGTLVTVSS Light chain: DNA sequence (411 bp) – SEQ ID NO: 20 Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTATTGCTCTGGCTCCCAGGTGCCAG ATGTGCTGACATTGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAG TCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATGATAACAACCGCTTATCCTGGTATCAG CAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGAATCTGGGGT CCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCGACCTGG AGTGTGGCGATGCTGCCACTTACTACTGTCAAAGCTATTCGTATAGTGATAGTGGTGGTGCT GGTTTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA Light chain: Amino acid sequence (137 aa) – SEQ ID NO: 21 Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 MDTRAPTQLLGLLLLWLPGARCADIVMTQTPASVEAAVGGTVTINCQSSQSVYDNNRLSWYQ QKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECGDAATYYCQSYSYSDSGGA GFAFGGGTEVVVK The isotype of the monoclonal antibody 2F10-2 was determined as IgG / kappa. The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible.

Claims

CLAIMS 1. A monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to human p- tau205, wherein the monoclonal antibody, or the antigen-binding fragment thereof, has a heavy chain variable region, VH, complementarity determining region 1, CDR1, consisting of SNVMS as defined in SEQ ID NO: 12; a VH CDR2 consisting of TINTRGITYYASWAKG as defined in SEQ ID NO: 13; a VH CDR3 consisting of AGSGSISYFNL as defined in SEQ ID NO: 14; a light chain variable region, VL, CDR1 consisting of QSSQSVYDNNRLS as defined in SEQ ID NO: 15; a VL CDR2 consisting of RASTLES as defined in SEQ ID NO: 16; and a VL CDR3 consisting of QSYSYSDSGGAGFA as defined in SEQ ID NO:

17.

2. The monoclonal antibody, or the antigen-binding fragment thereof, according to claim 1, wherein the VH comprises, preferably consists of, the amino acid sequence as defined in SEQ ID NO: 19 or 33.

3. The monoclonal antibody, or the antigen-binding fragment thereof, according to claim 1 or 2, wherein the VL comprises, preferably consists of, the amino acid sequence as defined in SEQ ID NO: 21 or 34.

4. A nucleic acid molecule encoding a monoclonal antibody, or an antigen-binding fragment thereof, according to any one of claims 1 to 3.

5. An expression vector comprising a promoter and a nucleic acid molecule according to claim 4 operatively controlled by the promoter.

6. A host cell comprising an expression vector according to claim 5.

7. An immunoassay kit for determining an amount of p-tau205 in a sample, wherein the immunoassay kit comprises: a first antibody, or an antigen-binding fragment thereof, immobilized to a support or intended to be immobilized to the support; and a second antibody, or an antigen-binding fragment thereof, wherein one of the first antibody, or the antigen-binding fragment thereof, and the second antibody, or the antigen-binding fragment thereof, has specificity for p-tau205; andthe other of the first antibody, or the antigen-binding fragment thereof, and second antibody, or the antigen-binding fragment thereof, has specificity for tau.

8. The immunoassay kit according to claim 7, wherein the one of the first antibody, or the antigen- binding fragment thereof, and the second antibody, or the antigen-binding fragment thereof, having specificity for p-tau205 binds specifically to an epitope in p-tau205 that comprises threonine residue 205 in phosphorylated form.

9. The immunoassay kit according to claim 8, wherein the one of the first antibody, or the antigen- binding fragment thereof, and the second antibody, or the antigen-binding fragment thereof, having specificity for p-tau205 binds specifically to a peptide of consecutive amino acid residues in tau and where the peptide comprises threonine residue 205, preferably selected from the group consisting of SEQ ID NO: 2 to 11.

10. The immunoassay kit according to any one of claims 7 to 9, wherein the other of the first antibody, or the antigen-binding fragment thereof, and second antibody, or the antigen-binding fragment thereof, having specificity for tau binds specifically to an epitope in tau not encompassing threonine residue 205.

11. The immunoassay kit according to claim 10, wherein the other of the first antibody, or the antigen- binding fragment thereof, and second antibody, or the antigen-binding fragment thereof, having specificity for tau binds specifically to an epitope selected from the group consisting of an epitope in the N-terminal part of tau corresponding to amino acid residues 1-44 in SEQ ID NO: 1, an epitope in N1 corresponding to amino acid residues 45-74 in SEQ ID NO: 1, an epitope in N2 corresponding to amino acid residues 75-103 in SEQ ID NO: 1, an epitope in an amino acid sequence between N2 and P1 corresponding to amino acid residues 104-150 in SEQ ID NO: 1, an epitope in P1 corresponding to amino acid residues 151-198 in SEQ ID NO: 1, an epitope in R1 corresponding to amino acid residues 244-274 in SEQ ID NO: 1, an epitope in R2 corresponding to amino acid residues 281-305 in SEQ ID NO: 1, an epitope in R3 corresponding to amino acid residues 312-337 in SEQ ID NO: 1, an epitope in R4 corresponding to amino acid residues 338-370 in SEQ ID NO: 1 and an epitope in the C-terminal part of tau corresponding to amino acid residues 371-441 in SEQ ID NO:

1.

12. The immunoassay kit according to claim 10 or 11, wherein the other of the first antibody, or the antigen-binding fragment thereof, and second antibody, or the antigen-binding fragment thereof, havingspecificity for tau binds specifically to an epitope in the N-terminal part of tau corresponding to amino acid residues 1-44 in SEQ ID NO:

1.

13. The immunoassay kit according to claim 12, wherein the other of the first antibody, or the antigen- binding fragment thereof, and second antibody, or the antigen-binding fragment thereof, having specificity for tau binds specifically to an epitope comprising, preferably consisting of, amino acid residues 6-18 in SEQ ID NO: 1 or amino acid residues 15-25 in SEQ ID NO:

1.

14. The immunoassay kit according to any one of claims 7 to 13, wherein the one of the first antibody, or the antigen-binding fragment thereof, and the second antibody, or the antigen-binding fragment thereof, having specificity for p-tau205 is a capture antibody, or an antigen- binding fragment thereof; and the other of the first antibody, or the antigen-binding fragment thereof, and second antibody, or the antigen-binding fragment thereof, having specificity for tau is a detection antibody, or an antigen-binding fragment thereof.

15. The immunoassay kit according to claim 14, wherein the capture antibody, or the antigen-binding fragment thereof, is attached to the solid support, preferably beads, and more preferably magnetic beads.

16. The immunoassay kit according to claim 14 or 15, wherein the detection antibody, or the antigen- binding fragment thereof, is attached to one of i) biotin and ii) streptavidin or avidin.

17. The immunoassay kit according to claim 16, further comprising an enzyme attached to the other of i) biotin and ii) streptavidin or avidin; and a substrate that can be converted by the enzyme into a detectable product.

18. The immunoassay kit according to any one of claims 7 to 17, further comprises the solid support, preferably selected from the group consisting of a microtiter plate and magnetic beads.

19. The immunoassay kit according to any one of claims 7 to 18, wherein the one of the first antibody, or the antigen-binding fragment thereof, and the second antibody, or the antigen-binding fragment thereof, having specificity for p-tau205 is according to any one of claims 1 to 3.

20. A method for determining an amount of p-tau205 in a sample, comprising:contacting the sample with a first antibody, or an antigen-binding fragment thereof, and a second antibody, or an antigen-binding fragment thereof, of an immunoassay kit according to any one of claims 7 to 19; detecting an amount of bound second antibody, or the antigen-binding fragment thereof; and determining an amount of p-tau205 in the sample based on the detected amount of bound second antibody, or the antigen-binding fragment thereof.

21. The method according to claim 20, further comprising correlating the detected amount of second antibody, or the antigen-binding fragment thereof, bound to p-tau205 to an amount of p-tau205 using a pre-defined correlation between detected amount of second antibody, or the antigen-binding fragment thereof, bound to a reference p-tau205 protein, preferably in vitro phosphorylated recombinant tau having an amino acid sequence as defined in SEQ ID NO: 1, and concentration of the reference p-tau205 protein.

22. The method according to claim 20 or 21, wherein the sample is a body fluid sample, preferably a body fluid sample selected from the group consisting of a cerebrospinal fluid sample, a brain interstitial fluid, a blood sample and a serum sample, more preferably selected from the group consisting of a cerebrospinal fluid sample, a blood sample and a serum sample.

23. A method for diagnosing Alzheimer’s disease in a human subject, comprising: determining an amount of p-tau205 in a body fluid sample from the human subject with an immunoassay kit according to any one of claims 7 to 19 or a method according to any one of claims 20 to 22; and diagnosing the human subject with Alzheimer’s disease based on the determined amount of p- tau205 in the body fluid sample.

24. A method for monitoring Alzheimer’s disease progression in a human subject, comprising: determining an amount of p-tau205 in a body fluid sample obtained from the human subject with an immunoassay kit according to any one of claims 7 to 19 or a method according to any one of claims 20 to 22; and monitoring Alzheimer’s disease progression in the human subject with AD based on the determined amount of p-tau205 in the body fluid sample.

25. A method for monitoring brain atrophy and / or cognitive performance in a human subject, comprising:determining an amount of p-tau205 in a body fluid sample obtained from the human subject with an immunoassay kit according to any one of claims 7 to 19 or a method according to any one of claims 20 to 22; and monitoring brain atrophy and / or cognitive performance in the human subject with AD based on the determined amount of p-tau205 in the body fluid sample.

26. A method for diagnosing and / or monitoring tau brain pathology in a human subject, comprising: determining an amount of p-tau205 in a body fluid sample obtained from the human subject with an immunoassay kit according to any one of claims 7 to 19 or a method according to any one of claims 20 to 22; and diagnosing and / or monitoring tau brain pathology in the human subject based on the determined amount of p-tau205 in the body fluid sample.

27. A method for determining efficiency of a treatment of Alzheimer’s disease in a human subject, comprising: determining a first amount of p-tau205 in a body fluid sample obtained from the human subject before the start of the treatment or in connection with the start of the treatment with an immunoassay kit according to any one of claims 7 to 19 or a method according to any one of claims 20 to 22; determining a second amount of p-tau205 in a body fluid sample obtained from the human subject during or after the treatment with an immunoassay kit according to any one of claims 7 to 19 or a method according to any one of claims 20 to 22; and determining the treatment of Alzheimer’s disease to be an efficient treatment of Alzheimer’s disease if the second amount of p-tau205 is lower than the first amount of p-tau205 and otherwise determining the treatment of Alzheimer’s disease to not be an efficient treatment of Alzheimer’s disease.

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