Two novel beta synuclein specific assays for the detection of Alzheimer’s disease

Abstract

AbstractBackgroundRecent studies reported the potential use of elevated CSF β‐synuclein levels as an AD‐specific synaptic biomarker. These results were obtained with a sandwich ELISA where one of the antibodies recognizes both alpha‐and β‐synuclein1. Blood contamination can affect alpha‐synuclein concentrations in CSF, which is not observed for β‐synuclein2. Therefore, we aimed to develop a β‐synuclein‐specific assay to avoid possible interference with alpha‐synuclein.MethodsTwo novel sandwich CSF ELISA assays were established using only β‐specific synuclein monoclonal antibodies: a mid‐region and C‐terminal assay. Both assays use a monoclonal capture antibody that maps to C‐terminal. The mid‐region assay involves a detection antibody mapping to an epitope that overlaps with the SRM peptide used for CSF mass‐spectrometry quantification3. The C‐terminal assay includes a detection antibody targeting a less well‐defined C‐terminal epitope. Forty‐four remnant routine CSF samples were analyzed in duplicate (22 individuals with AD biomarker profile based on pTau181 and amyloid‐beta(1‐42), age70±7 years, 38%F and 22 control profiles, age70±7, 38%F).ResultsThe mid‐region assay showed a precision of 4%CV in clinical samples and a sensitivity of 1.25 pg/ml compared to 10%CV and 4.22 pg/ml with the C‐terminal assay (Fig. 1). CSF β‐synuclein levels were significantly elevated in patients with an AD CSF‐profile compared to individuals with negative‐AD CSF‐profiles, with AUCs of 0.77 [95%CI: 0.62‐0.90] for the C‐terminal assay and 0.71 [0.55‐0.87] for the mid‐region assay (Fig. 2). The assays showed comparable (Delong’s P = 0.53) clinical performance and their results correlated (r = 0.61, P = <0.001)(Fig. 2). Both C‐terminal and mid‐region β‐synuclein associated with CSF pTau181 (Spearman’s rho = 0.58 (p<0.001), rho = 0.51 (P = <0.001), respectively), and did not correlate with CSF Abeta(1‐42) (Fig. 3). Both assays are specific for recombinant beta‐synuclein, but the C‐terminal assay showed higher selectivity for β‐synuclein compared to the mid‐region assay, as increasing signals were observed with the latter with increasing concentrations of spiked recombinant alpha/gamma‐synuclein.ConclusionThe two novel β‐synuclein assays show clinically relevant changes in relation to AD CSF biomarker positivity. Both assays are specific to β‐synuclein. The assays will be transferred to SIMOA, which provides a wider dynamic range, and thus we will further assess the selectivity in plasma/serum.