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. 2025 Jun;21(6):e70241.
doi: 10.1002/alz.70241.

Development and validation of a novel Simoa assay for NPTX2 in Alzheimer's disease and Down syndrome

Mathias Sauer  1 , Bárbara Fernandes Gomes  1 , Parasto Shahrouki  1 , Juan Lantero-Rodriguez  1 , Laia Montoliu-Gaya  1 , Elena Camporesi  1 , Olivia Belbin  2   3 , Daniel Alcolea  2   3 , Ashish Kumar  4 , Mitu Sharma  4 , Sangeeta Singh  4 , Gunnar Brinkmalm  1 , Johan Gobom  1 , María Carmona-Iragui  2   3   5 , Michael Schöll  1   6   7   8 , Shorena Janelidze  9 , Gagan Deep  4 , Kaj Blennow  1   10   11   12 , Henrik Zetterberg  1   10   13   14   15   16 , Ann Brinkmalm  1 , Alberto Lleó  2   3 , Juan Fortea  2   3   5 , Oskar Hansson  9   17 , Nicholas J Ashton  1   18   19 , Johanna Nilsson  1
Affiliations

Development and validation of a novel Simoa assay for NPTX2 in Alzheimer's disease and Down syndrome

Mathias Sauer et al. Alzheimers Dement. 2025 Jun.

Abstract

Introduction: Synaptic dysfunction and loss are pathological hallmarks of neurodegenerative diseases. Neuronal pentraxin 2 (NPTX2), a presynaptic protein involved in synaptic plasticity, has been linked to cognitive decline in Alzheimer's disease (AD) and other neurodegenerative disorders.

Methods: We developed and validated a novel single molecule array (Simoa) for NPTX2 in cerebrospinal fluid, which was evaluated in two independent cohorts.

Results: CSF NPTX2 concentration was lower (fold change [FC] 0.82, p < 0.01) in AD patients and Down syndrome individuals (FC 0.56, p < 0.001), compared with cognitively unimpaired patients (CU). It was also associated with Mini-Mental State Examination (MMSE) score (β = 2.51, p < 0.001), tau-PET (β = -0.21, p < 0.01), and cortical thickness (β = 0.08, p < 0.001).

Discussion: We describe the first assay for NPTX2 on the Simoa platform, where we continue to highlight the valuable addition of NPTX2 to routine diagnostics of suspected cognitive impairment in patients as it associates better with cognition than other, more established AD biomarkers.

Highlights: Novel method validated for measuring CSF NPTX2 on semi-automated Simoa platform. Method validated for use in CSF, where it shows a significant decrease in both AD and DS patients. Associated with cognition, neurofibrillary tangles, and cortical thickness in AD patients. Associations with cognition are shown to be stronger than those of pTau and NFL.

Keywords: Alzheimer's disease; Simoa; biomarker; cerebrospinal fluid; down syndrome; neuronal pentraxin 2; synaptic biomarker.

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Conflict of interest statement

H.Z. has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZPath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, LabCorp, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures sponsored by Alzecure, BioArctic, Biogen, Cellectricon, Fujirebio, Lilly, Novo Nordisk, Roche, and WebMD, and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). K.B. has served as a consultant and on advisory boards for Abbvie, AC Immune, ALZPath, AriBio, BioArctic, Biogen, Eisai, Lilly, Moleac Pte. Ltd., Neurimmune, Novartis, Ono Pharma, Prothena, Roche Diagnostics, Sanofi and Siemens Healthineers; has served at data monitoring committees for Julius Clinical and Novartis; has given lectures, produced educational materials and participated in educational programs for AC Immune, Biogen, Celdara Medical, Eisai and Roche Diagnostics; and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program, outside the work presented in this paper. D.A. participated in advisory boards from Fujirebio‐Europe, Roche Diagnostics, Grifols S.A., and Lilly, and received speaker honoraria from Fujirebio‐Europe, Roche Diagnostics, Nutricia, Krka Farmacéutica S.L., Zambon S.A.U., Neuraxpharm and Esteve Pharmaceuticals S.A. D.A. declares a filed patent application (WO2019175379 A1 Markers of synaptopathy in neurodegenerative disease). G.D. is the founder of LiBiCo LLC, which has no influence or contribution to the work presented in this manuscript. Author disclosures are available in the Supporting Information.

Figures

FIGURE 1
FIGURE 1
Spearman's rank correlation between neuronal pentraxin 2 (NPTX2) measurements of paired cerebrospinal fluid (CSF) and plasma samples, analyzed with mass spectrometry (MS) and single molecule assay (Simoa). (A) NPTX2 in CSF measured with immunoprecipitation mass spectrometry (IP‐MS) and Simoa. (B) NPTX2 in plasma was measured with IP‐MS and Simoa. (C) NPTX2 was measured in CSF with IP‐MS and in plasma measured with Simoa. (D) NPTX2 was measured in CSF and plasma with IP‐MS
FIGURE 2
FIGURE 2
(A–B) Schematic illustration of the neuronal pentraxin 2 (NPTX2) sequence, including the peptides captured by the capture antibody (A, EPR15618, Abcam) and the detection antibody (B, ERP24020‐38, Abcam), where cerebrospinal fluid (CSF) is illustrated by fill color blue, brain by frame color red, plasma by rhombus shape, and trypsin sites are denoted by a red letter. (C) Representative Western blot images of NPTX2 in CSF and brain. cognitively unimpaired (CU), n = 3, Alzheimer's disease (AD), n = 3. (D) Tris buffered saline (TBS) NPTX2 in CU (n = 3) and AD (n = 3)
FIGURE 3
FIGURE 3
(A–C) BioFINDER Pilot cohort (cerebrospinal fluid [CSF]). (A) Neuronal pentraxin 2 (NPTX2) in cognitively unimpaired (CU) (n = 51) and Alzheimer's disease (AD) (n = 48) patients. (B) Receiver operating characteristics curve (ROC) curve for NPTX2 in AD compared to CU. (C) Correlations for CSF biomarkers in the full cohort. (D–F) DABNI cohort (CSF). (D) NPTX2 in CU (n = 20), prodromal AD in Down syndrome (pDS) (n = 23), asymptomatic AD in Down syndrome (aDS) (n = 83) and dementia AD in Down syndrome (dDS) (n = 91). (E) ROC curve for NPTX2 in the three DS groups compared to CU. (F) Correlations for CSF biomarkers in the full cohort. p‐values are indicated by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.001
FIGURE 4
FIGURE 4
Associations of cerebrospinal fluid (CSF) neuronal pentraxin 2 (NPTX2) in BioFINDER Pilot cohort, adjusted for age and sex. Association with (A–C) Mini‐Mental State Examination (MMSE) score in (A) cognitively unimpaired (CU) + Alzheimer's disease (AD) (n = 95), (B) CU (n = 49), and (C) AD (n = 46); (D–F) tau‐PET in (D) CU + AD (n = 95), (E) CU (n = 49), (F) AD (n = 46); (G–I) amyloid‐PET in (G) CU + AD (n = 78), (H) CU (n = 49), (I) AD (n = 29); (J–L) cortical thickness in (J) CU + AD (n = 96), (K) CU (n = 49), and (L) AD (n = 47)
FIGURE 5
FIGURE 5
L1CAM+ NDE, (A) neuronal pentraxin 2 (NPTX2) in cognitively unimpaired (CU) (n = 23) and Alzheimer's disease (AD) (n = 24) patients. (B) Receiver operating characteristics curve (ROC) curve for NPTX2 in AD compared to CU. (C) Correlations for cerebrospinal fluid (CSF) biomarkers and NPTX2 from NDE in all patients. p‐values are indicated by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.001
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