We used a multiplex Xmap technology that uses magnetic microspheres internally coded with two fluorescent dyes to measure markers of neurodegeneration (Millipore, Cat#: HNABTMAG-68K). All samples including placebo and resveratrol at baseline and 52 weeks were analyzed in parallel using the same reagents. Through precise combinations of these two dyes, multiple proteins are measured within the sample. Each of these spheres is coated with a specific capture antibody. The capture antibody binds to the detection antibody and a reporter molecule, completing the reaction on the surface of the bead. CSF or plasma (25 μl) was incubated overnight at 4 °C with 25 μl of a mixed bead solution, containing human total tau, p-tau181, Aβ42, and Aβ40 (CSF Aβ40 is diluted 1:10). After washing, samples were incubated with 25 μl detection antibody solution for 1.5 h at room temperature. Streptavidin-phycoerythrin (25 μl) was added to each well containing the 25 μl of detection antibody solution. Samples were then washed and suspended in 100 μl of sheath fluid. Samples were then run on MAGPIX with Xponent software. The median fluorescent intensity (MFI) data was analyzed using a 5-parameter logistic or spline curve-fitting method for calculating analyte concentrations in samples. We also performed multiplex ELISA (Millipore, CAT#: HCYTOMAG-60K) to profile a panel of plasma and CSF markers that are indicative of inflammation, including human EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFNγ, GRO, IL-10, MCP-3, IL-12P40, MDC, IL-12P70, PDGF-AA, IL-13, PDGF-AB/BB, IL-15, sCD40L, IL-17A, IL-1RA, IL-1α, IL-9, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, TNFα, TNFβ, and VEGF.
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