Sulfo tag nhs ester

Multi-Array High Bind plates (96-well plates; MSD, Meso Scale Discovery, Rockville, MD) were coated with the capture antibody 2F12 diluted (6.7 ng μl⁻¹) in Tris-buffered saline with 0.05% Tween-20 (TBS-T) in 30 μL per well and incubated at 4 °C overnight. Liquid was removed and plates were blocked for 1 h at RT in blocking buffer (5% MSD Blocker A; TBS-T). After 3 washes with TBS-T, samples diluted in TBS-T with 1% MSD Blocker A and 0.5% NP40 were incubated at 4 °C overnight. Sulfo-tagged SOY1 mAb (for detection) was generated using Sulfo-Tag-NHS-Ester (MSD), diluted in blocking buffer (6.7 ng μl⁻¹), added to the plate (30 μl per well) and shaken for 1 h at RT. After three washes, MSD Reader buffer was added, and plates were immediately measured using a MSD Sector 2400 imager.

Aβ peptides were detected by electrochemiluminescence assays using anti-Aβ antibodies and a Sector Imager 2400 Analyzer (Meso Scale Discovery, MSD, Gaithersburg, MD, USA) as previously described [34 (link)] with minor modifications. The 4G8 murine monoclonal antibody (Senetek PLC), which specifically recognizes an epitope within the 18–24 amino-acid region of Aβ, was ruthenylated with MSD Sulfo-TAG NHS ester, according to the supplier’s instructions (MSD). It was thereafter used in conjunction with a 6E10-biotinylated murine monoclonal antibody (Senetek PLC), which binds to an epitope within the 2–11 amino-acid region of Aβ. This assay can detect both Aβ40 and Aβ42 peptides and all shorter forms with a core region comprised between position 2 and 24 of Aβ, thereafter termed pan-Aβ. To specifically measure Aβ42 levels, 6E10 was replaced by a murine monoclonal antibody, 22F9, which binds to the C-terminus of Aβ42 (with no cross-reaction with Aβ40). The levels of Aβ peptide were expressed as μg/g of wet tissue weight. For quantification of the soluble and insoluble fraction, the Aβ panel kit (MSD; reference K15200G) was used to quantify Aβ38, Aβ40, and Aβ42 subspecies and values are reported normalized to the protein content of the initial homogenate.