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Eitan et al. Extracell Vesicles Circ Nucleic Acids 2023;4:133-150 https://dx.doi.org/10.20517/evcna.2023.13 Page 135

NDEVs. L1CAM expression levels are relatively low in non-neuronal cells, but its presence in other tissues
raises concerns regarding NDE origin and purity.

In the present study, we selected a combination of two from multiple antibodies against neuronal-specific
markers that target two neuron-specific antigens: an axonal marker, growth-associated protein 43 (GAP43),
and a neuron cell surface marker, neuroligin 3 (NLGN3) [17,18] . Our results indicate that the resulting isolation
[19]
procedure adheres to MISEV guidelines and demonstrates its efficiency in recovery experiments where
EVs released by iPSC-derived neurons are spiked into plasma samples. The specificity toward neuronal
material is confirmed by measurements of multiple neuron-specific proteins and mRNA. We evaluated the
performance of this technology for the assessment, detection, and quantification of biomarkers for AD in
NDEVs, including p181-Tau, Aβ, pro-BDNF, and synaptic proteins, retrospectively, in samples from two
cohorts of patients with early-stage AD and controls. These exploratory findings demonstrate the robust
performance of the method and the potential to add novel, non-invasive, and scalable biomarkers to inform
AD drug development, which deepens insight into neuronal pathology in AD.

METHODS
Standard protocol approvals, registrations, and consents
This study relied on de-identified samples from commercial or Government [National Institute of Aging
(NIA, Baltimore, MD)] biobanks. Commercial samples were purchased from two companies, BioIVT
(Westbury, NY) and precision for medicine (PMED) (Elk Grove, CA), which adhere to the most current
regulations for sample collection and use. The regulatory requirements met include Institutional Review
Board (IRB) approval, privacy officer authorization, appropriate government licenses, and industry
accreditations, as applicable. They also adhere to the General Data Protection Regulations. Samples from the
NIA were collected through NIH IRB-approved protocols, and their use in this research is allowed based on
the subjects’ original consent. Regarding patients with AD, legally authorized representatives or patients
consented to participate in the original studies, depending on the assessment of consent capacity at the time
of blood draws. All samples were de-identified prior to their transfer to NeuroDex for processing under the
terms of a Collaborative Research and Development Agreement.

Human plasma specimens: Human EDTA-K2 plasma was used in all experiments. Pooled human EDTA-
K2 plasma (BioIVT, HMN69947-X, ten donors per pool) was used for quality control experiments and as
internal references. Samples procured from BioIVT and PMED comprised a single cohort for most analyses.
The NIA cohort comprised 20 individuals with high probability (early-stage) AD as established by the NIA-
AA criteria and 19 healthy, cognitively normal age- and sex-matched controls, participating in NIH IRB-
[20]
approved studies (NIA Clinical Unit; Baltimore, MD, USA). AD diagnosis was based on clinical criteria and
[21]
abnormal CSF levels of amyloid-beta (Aβ) peptide 1-42 (Aβ42 < 192 pg/mL) and p181-Tau > 23 pg/mL .
Demographic and clinical data for all cohorts are summarized in Table 1. All NeuroDex employees involved
in sample processing and analysis were blinded to the identity of the samples, and only internal sample IDs
were used.

NDEV isolation: NDEV enrichment was performed using the NeuroDex ExoSORT kit (Cat. No
NDX_ESNeuro, NeuroDex, Natick, MA). Briefly, plasma samples were precipitated with 1/2 plasma volume
of a NeuroDex total EV isolation reagent (Cat. No. NDX_TPC) in 1/2 plasma volume, and pellets were
resuspended in a binding buffer. Magnetic beads were conjugated with NeuroDex proprietary antibodies
against GAP43 and NLGN3 and blocked with the NDX Blocking Reagent. The beads were incubated
overnight with plasma samples at 4 °C with slow rotation. The following day, beads-NDEVs complexes
were collected using a magnetic separator, washed three times using ExoSORT wash buffer, and transferred