The feasibility of assaying plasma phosphorylated tau protein (threonine 181), denoted p-tau181, using immunomagnetic reduction (IMR) is explored. The reagent for assaying p-tau181 with IMR was synthesized, and its analytic performances were characterized. Seventy-three subjects were recruited. Each participant was examined with neuropsychological tests, magnetic resonance imaging, and IMR assay for plasma p-tau181. Using commercially available IMR kits, the plasma total tau protein (T-tau) of each subject was assayed. The dynamic range for assaying p-tau181 using IMR was 1.96×10 ^-2 pg/ml to 10 ⁴ pg/ml. There was no significant interference from total tau protein in the assay of p-tau181. The measured concentrations of plasma p-tau181 were 2.46±1.09 pg/ml for healthy controls, 4.41±1.85 pg/ml for MCI due to AD, and 6.14±1.59 pg/ml for very mild AD. Meanwhile, the measured concentrations of plasma T-tau were 18.85±10.16 pg/ml for healthy controls, 32.98±10.18 pg/ml for MCI due to AD, and 37.54±12.29 pg/ml for very mild AD. A significant difference in plasma p-tau181 was observed between healthy controls and MCI due to AD ( p < 0.001) and between MCI due to AD and very mild AD ( p < 0.001). However, for the plasma T-tau concentration, a significant difference existed only between healthy controls and MCI due to AD ( p < 0.001). This implies that the plasma p-tau181 level is correlated more to AD severity than plasma T-tau is. Additionally, p-tau181 was observed as approximately 14% of T-tau in human plasma.

Alzheimer’s disease (AD) is the most common form of dementia. The development of assay technologies able to diagnose early-stage AD is important. Blood tests to detect biomarkers, such as amyloid and total Tau protein, are among the most promising diagnostic methods due to their low cost, low risk, and ease of operation. However, such biomarkers in blood occur at extremely low levels and are difficult to detect precisely. In the early 2000s, a highly sensitive assay technology, immunomagnetic reduction (IMR), was developed. IMR involves the use of antibody-functionalized magnetic nanoparticles dispersed in aqueous solution. The concentrations of detected molecules are converted to reductions in the ac magnetic susceptibility of this reagent due to the association between the magnetic nanoparticles and molecules. To achieve ultra-high sensitivity, a high-Tc superconducting-quantum-interference-device (SQUID) ac magnetosusceptometer was designed and applied to detect the tiny reduction in the ac magnetic susceptibility of the reagent. Currently, a 36-channeled high-Tc SQUID-based ac magnetosusceptometer is available. Using the reagent and this analyzer, extremely low concentrations of amyloid and total Tau protein in human plasma could be detected. Further, the feasibility of identifying subjects in early-stage AD via assaying plasma amyloid and total Tau protein is demonstrated. The results show a diagnostic accuracy for prodromal AD higher than 80% and reveal the possibility of screening for early-stage AD using SQUID-based IMR.

It is difficult to discriminate healthy subjects and patients with Parkinson disease (PD) or Parkinson disease dementia (PDD) by assaying plasma α-synuclein because the concentrations of circulating α-synuclein in the blood are almost the same as the low-detection limit using current immunoassays, such as enzyme-linked immunosorbent assay. In this work, an ultra-sensitive immunoassay utilizing immunomagnetic reduction (IMR) is developed. The reagent for IMR consists of magnetic nanoparticles functionalized with antibodies against α-synuclein and dispersed in pH-7.2 phosphate-buffered saline. A high-Tc superconducting-quantum-interference-device (SQUID) alternative-current magnetosusceptometer is used to measure the IMR signal of the reagent due to the association between magnetic nanoparticles and α-synuclein molecules. According to the experimental α-synuclein concentration dependent IMR signal, the low-detection limit is 0.3 fg/ml and the dynamic range is 310 pg/ml. The preliminary results show the plasma α-synuclein for PD patients distributes from 6 to 30 fg/ml. For PDD patients, the concentration of plasma α-synuclein varies from 0.1 to 100 pg/ml. Whereas the concentration of plasma α-synuclein for healthy subjects is significantly lower than that of PD patients. The ultra-sensitive IMR by utilizing antibody-functionalized magnetic nanoparticles and high-Tc SQUID magnetometer is promising as a method to assay plasma α-synuclein, which is a potential biomarker for discriminating patients with PD or PDD.

Immunomagnetic reduction (IMR), which involves the use of antibody-functionalized magnetic nanoparticles to specifically label target biomarkers, was utilized to develop an assay for total tau protein in human plasma. The analytic properties of the IMR assay on tau protein were investigated. The limit of detection was found to be 0.026 pg/ml. Other properties such as Hook effect, assay linearity, dilution recovery range, reagent stability, interference test, and spiked recovery were also characterized. The ultra-sensitive IMR assay was applied to detect the plasma tau protein levels of subjects with prevalent neurodegenerative diseases, such as Alzheimer’s disease (AD), mild cognitive impairment (MCI) due to AD, Parkinson’s disease (PD), frontotemporal dementia (FTD) and vascular dementia (VD). The concentrations of plasma tau protein in patients with VD, PD, MCI due to AD, FTD, and AD patients were higher than that of healthy controls. Using an ROC curve analysis, the cutoff value for discriminating dementia patients from healthy controls was 17.43 pg/ml, resulting in 0.856 and 0.727 for clinical sensitivity and specificity, respectively. The area under the ROC curve was 0.908. These results imply that the IMR plasma tau assay would be useful to screen for prevalent neurodegenerative diseases.

Phosphorylated α-synuclein accounts for more than 90% of α-synuclein found in Lewy bodies of Parkinson’s disease (PD). We aimed to examine whether plasma Ser129-phosphorylated α-synuclein (pS129-α-synuclein) is a surrogate marker of PD progression. This prospective study enrolled 170 participants (122 PD patients, 68 controls). We measured plasma levels of total and pS129-α-synuclein using immunomagnetic reduction-based immunoassay. PD patients received evaluations of motor and cognition at baseline and at a mean follow-up interval of three years. Changes in the Movement Disorder Society revision of the Unified Parkinson’s Disease Rating Scale motor score (MDS-UPDRS part III) and Mini-Mental State Examination (MMSE) score were used to assess motor and cognition progression. Our results showed that plasma levels of total and pS129-α-synuclein were significantly higher in PD patients than controls (total: 1302.3 ± 886.6 fg/mL vs. 77.8 ± 36.6 fg/mL, p < 0.001; pS129-α-synuclein: 12.9 ± 8.7 fg/mL vs. 0.8 ± 0.6 fg/mL, p < 0.001), as was the pS129-α-synuclein/total α-synuclein ratio (2.8 ± 1.1% vs. 1.1 ± 0.6%, p = 0.01). Among PD patients, pS129-α-synuclein levels were higher with advanced motor stage (p < 0.001) and correlated with MDS-UPDRS part III scores (r = 0.27, 95% CI: 0.09–0.43, p = 0.004). However, we found no remarkable difference between PD patients with and without dementia (p = 0.75). After a mean follow-up of 3.5 ± 2.1 years, PD patients with baseline pS129-α-synuclein > 8.5 fg/mL were at higher risk of motor symptom progression of at least 3 points in the MDS-UPDRS part III scores than those with pS129-α-synuclein < 8.5 fg/mL (p = 0.03, log rank test). In conclusion, our data suggest that plasma pS129-α-synuclein levels correlate with motor severity and progression, but not cognitive decline, in patients with PD.

BACKGROUND Phosphorylated tau (p-tau) is a specific blood biomarker for Alzheimer’s disease (AD) pathology. Multiple p-tau biomarkers on several analytical platforms are poised for clinical use. The Alzheimer’s Association Global Biomarker Standardisation Consortium plasma phospho-tau Round Robin study engaged assay developers in a blinded case-control study on plasma p-tau, aiming to learn which assays provide the largest fold-changes in AD compared to non-AD, have the strongest relationship between plasma and cerebrospinal fluid (CSF), and show the most consistent relationships between methods (commutability) in measuring both patient samples and candidate reference materials (CRM). METHODS Thirty-three different p-tau biomarker assays, built on eight different analytical platforms, were used to quantify paired plasma and CSF samples from 40 participants. AD biomarker status was categorised as “AD pathology” (n=25) and “non-AD pathology” (n=15) by CSF Aβ42/Aβ40 (US-FDA; CE-IVDR) and p-tau181 (CE-IVDR) methods. The commutability of four CRM, at three concentrations, was assessed across assays. FINDINGS Plasma p-tau217 consistently demonstrated higher fold-changes between AD and non-AD pathology groups, compared to other p-tau epitopes. Fujirebio LUMIPULSE G, UGOT IPMS, and Lilly MSD p-tau217 assays provided the highest median fold-changes. In CSF, p-tau217 assays also performed best, and exhibited substantially larger fold-changes than their plasma counterparts, despite similar diagnostic performance. P-tau217 showed the strongest correlations between plasma assays (rho=0.81 to 0.97). Plasma p-tau levels were weakly-to-moderately correlated with CSF p-tau, and correlations were non-significant within the AD group alone. The evaluated CRM were not commutable across assays. INTERPRETATION Plasma p-tau217 measures had larger fold-changes and discriminative accuracies for detecting AD pathology, and better agreement across platforms than other plasma p-tau variants. Plasma and CSF markers of p-tau, measured by immunoassays, are not substantially correlated, questioning the interchangeability of their continuous relationship. Further work is warranted to understand the pathophysiology underlying this dissociation, and to develop suitable reference materials facilitating cross-assay standardisation. FUNDING Alzheimer’s Association (#ADSF-24-1284328-C) Evidence before this study Phosphorylated tau (p-tau) in cerebrospinal fluid is an established biomarker of Alzheimer’s disease (AD), and several studies of plasma phosphorylated tau (p-tau) biomarkers now show evidence for its diagnostic accuracy in AD. We reviewed the literature on plasma p-tau in AD by searching without language restrictions in PubMed for articles from 1^st January 2014 to 1^st March 2024 using the terms ((("Alzheimer Disease"[Mesh]) OR ((Alzheimer[Title/Abstract]) OR (Alzheimer’s [Title/Abstract))) AND ((("Blood"[Mesh]) OR ("Plasma"[Mesh])) OR (plasma[Title/Abstract]))) AND ((phospho tau[Title/Abstract]) OR (phosphorylated tau[Title/Abstract]) OR (p-tau[Title/Abstract]) OR (P-tau[Title/Abstract]) OR (pTau[Title/Abstract])), filtering for studies in humans. Several studies examined plasma p-tau181, p-tau217 and p-tau231 in well-characterised research cohorts including cognitively impaired and unimpaired individuals, demonstrating high diagnostic accuracy when utilising CSF or amyloid PET imaging “gold standard” biomarkers, and in relation to post-mortem amyloid and tau pathology. Most studies utilised single-method assays to measure biomarkers of interest, including immunoprecipitation-mass spectrometry and a variety of immunochemical methods including single molecule array, enzyme-linked immunosorbent assays, electrochemiluminescence, and immunomagnetic reduction. Some later studies have performed cross-method and cross-phospho-form comparisons (p-tau181, p-tau217 and p-tau231), including six single molecular array assays in the Amsterdam Dementia Cohort, nine assays in the BIODEGMAR memory clinic-based cohort from Spain, ten assays in individuals with mild cognitive impairment in the Skåne University memory clinic cohort from Sweden, and have generally reported the various individual methods for p-tau217 to have the highest diagnostic accuracy for the presence of cerebral β-amyloid deposition. No study to date has compared assays for p-tau217 on semi- and fully automated platforms, described comparisons with p-tau212 and p-tau205 in plasma, or assessed commutability of candidate reference materials between assays in comparison to patient samples. Added value of this study Our study included the largest number of methods for assaying plasma p-tau to date, with engagement from 12 assay developers, allowing for blinded cross-assay comparisons of 33 plasma assays (for p-tau181, p-tau205, p-tau212, p-tau217, and p-tau231). Among these were included several semi-automated and fully automated methods that have potential for widespread clinical application. This is also the first study, to our knowledge, in which the commutability of candidate reference materials was assessed for plasma p-tau, as a first step in efforts to standardise between assays. The study design capitalised on a large volume of paired CSF and plasma drawn from well-characterised attenders to a specialist cognitive disorders service, allowing for 26 intra-method comparisons between CSF and plasma measurements within the same individuals. We found that CSF p-tau217 assays consistently outperformed other CSF p-tau assays in terms of fold-change between individuals with and without Alzheimer’s pathology, and the top ten assays in terms of fold change in plasma were also p-tau217 assays. This high discriminative value of plasma p-tau217 contrasted with overall only moderate correlations between CSF and plasma within individuals, which did not persist within the AD group alone. We also did not observe commutability of the four types of candidate reference materials we investigated, for any of the assay pairs. Implications of all the available evidence Our study adds to the growing evidence for plasma p-tau217 as a candidate biomarker for translation into clinical practice by virtue of its superior discriminative ability between AD vs non-AD in symptomatic individuals, when compared head-to-head with other phosphorylated tau forms. The observed high and clinically interpretable fold-changes are pivotal for this biomarker’s possible future success. Further studies should examine whether blood biomarker-supported diagnosis will extend access to disease-specific (and potentially disease-modifying) treatments and impact patient-relevant outcomes such as quality of life, particularly in diverse and resource-limited settings. Standardization between different assays will also be important for real-world applications, and this will require further concerted efforts in developing reference materials.