Molecular Biomarkers in the Cerebrospinal Fluid (CSF) Assays

The quantitative Western blot analysis was used to detect sPDGFRβ in human CSF (ng/mL), as we previously reported⁸. Standard curves were generated using recombinant human PDGFRβ (Cat. No. 385-PR-100/CF, R&D Systems, Minneapolis, MN).

CSF levels of the astrocytic cytokine, S100B, were determined using ELISA (Cat. No. EZHS100B-33K, EMD Millipore, Billerica, MA).

Meso Scale Discovery (MSD) multiplex assay was used to determine CSF levels of interleukin-2 (IL-2), IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-1β, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) (Cat. No. K15049G, MSD, Rockville, MD).

MSD multiplex assay (Cat. No. K15200E, MSD, Rockville, MD) was used to determine CSF levels of Aβ38, Aβ40 and Aβ42. The CSF Aβ42 cutoff level of 190 pg/mL was applied as previously reported for the MSD Aβ peptide assay¹⁹.

CSF Aβ oligomers were measured by ELISA (protocol modified from IBL Cat. No. 27725 and Holtta et al⁴⁶). Aβ_1–16 peptide dimer was used as the standard protein prepared at 0, 1, 2.5, 5, 7.5, 10, 15, 20 pM, and 100 μL of prepared standards and neat CSF were added to each well on an uncoated 96-well plate along with 20 μL/well of HRP-conjugated anti-human Aβ (N) (82E1) mouse IgG monoclonal antibody; the plates were incubated overnight at 4°C on an orbital plate shaker at 600 rpm. 100 μL/well was transferred to a 96-well plate precoated with anti-human Aβ (N) (82E1) mouse IgG monoclonal antibody and incubated for 1 hour at 4°C with shaking. Plates were washed (Tris buffered saline with 0.1% Tween-20, TBST) and ELAST ELISA amplification was performed (Perkin Elmer). Briefly, 100 μL/well of biotinyl-tyramide (1:100 dilution) was incubated for 15 minutes at room temperature with shaking. Plates were washed and 100 μL/well of streptavidin-HRP (1:500 dilution) was incubated for 30 minutes at room temperature with shaking. Plates were washed and 100 μL/well of tetramethylbenzidine (TMB) substrate (Kirkegaard & Perry Laboratories Cat. No. 53–00-01) was incubated in the dark for 60 minutes, then 100 μL/well of 2N HCl was added, and the plates were read at 450 nm.

MSD assay was used to determine CSF levels of total tau (Cat. No. K15121G, MSD, Rockville, MD). Phosphorylated tau (pT181) was determined by ELISA (Cat. No. 81581, Innotest, Belgium). The CSF pTau₁₈₁ cutoff level of 78 pg/mL was applied as previously reported²⁰.

CSF tau oligomers were measured by direct ELISA using tau oligomer-specific antibody (T22)⁴⁷. Briefly, 12 μL CSF was diluted in a total volume of 50 μL 0.05 M carbonate-bicarbonate buffer, added to a 96-well MaxiSorp plate (Nunc) and incubated overnight at 4°C on an orbital plate shaker at 600 rpm. Plates were washed (TBST) and blocked with 300 μL of 10% nonfat dry milk (BioRad) for 2 hours room temperature with shaking. Plates were washed and incubated with 100 μL/well of T22 antibody (1:250 diluted in 5% nonfat milk) and incubated for 1 hour at room temperature with shaking. Plates were washed and incubated with HRP-conjugated anti-rabbit IgG antibody (1:3000 diluted in 5% nonfat dry milk) and incubated for 1 hour at room temperature with shaking. Plates were washed and incubated in the dark with 100 μL/well TMB substrate for 14 minutes, then 100 μL/well 2N HCl was added, and the plates were read at 450 nm.

CSF levels of neuron specific enolase (NSE) were determined using ELISA (Cat. No. E-80NEN, Immunology Consultant Laboratories, Portland, OR).