Optiplate 384

A comprehensive panel of sandwich immunoassays listed in Table 1 was developed to measure different forms of tau. In general, analytes were captured by a monoclonal antibody that was biotinylated and bound to streptavidin coated donor beads (PerkinElmer). Detection was accomplished either by a monoclonal antibody conjugated to the acceptor beads directly or by a polyclonal antibody in combination with anti-rabbit IgG-conjugated acceptor beads (PerkinElmer).
Assay reactions (25 μl) were carried out in OptiPlate-384 microplates (PerkinElmer) that contained 5 μL of analyte at the specified protein concentration, 10 μL of biotinylated capture antibody (final concentration 2 nM) and 10 μL of detection antibody-conjugated acceptor beads (final concentration 20 μg/mL). After overnight incubation at 4°C, 25 μL of streptavidin donor beads were added under subdued light conditions (final concentration 40 μg/mL) and the reactions were incubated at room temperature for 60 min with gentle shaking. The fluorescent signal was detected on an Envision Plate Reader at 615 nm (PerkinElmer).

The AlphaScreen assay was used to assess the binding between recombinant HA and its receptor mimic, biotinylated sialyllactose polymer α2–3-sialyllactose-polyacrylamide (PAA) (Glycotech) as follows^{17 (link)}. Various concentrations of tetravalent peptides were incubated with HA (10 μg/ml) in individual wells of an OptiPlate-384 (PerkinElmer) for 30 min at room temperature. After the addition of α2–3-sialyllactose-PAA (16 nm), the plate was further incubated for 1.5 h. The samples were then incubated with nickel chelate acceptor beads (20 μg/ml; PerkinElmer) for 30 min and then with streptavidin donor beads (20 μg/ml; PerkinElmer) for 1 h at room temperature in the dark. The plate was then subjected to excitation at 680 nm, and emission from the wells was monitored at 615 nm with an EnVision system (PerkinElmer). Data were obtained as the AU of signal intensity (count per second) used by the EnVision system.

Cells in 96w plate were lysed in 40μ/well RIPA buffer with protease-and phosphatase inhibitors (Roche). After 20–30 minutes of gentle shaking at RT, 5μl sample was mixed with 20 μl biotinylated and acceptor bead-conjugated antibodies in OptiPlate-384 (all Perkin Elmer). After 2 hours of incubation at RT, 25μl of Streptavidin donor beads were added at RT for 30 minutes followed by detection with the Envision plate reader. Raw values were normalized to transduced (no fibril) control samples per plate.

The inhibitory effects of a series of shorter MMβA-mono-derived peptides on binding between the Stx2a A-subunit and MMβA-mono were measured using the AlphaScreen assay, as described previously^{18 (link)}. Briefly, biotinylated MMβA-mono (30 nM) was incubated with Stx2a A-subunit (20 nM) in the presence of indicated concentrations of a single shorter peptide and specific anti-Stx2a A-subunit monoclonal antibody (originally obtained) in individual wells of an OptiPlate-384 (PerkinElmer, Waltham, MA, USA) for 30 min at room temperature. Samples were then incubated with anti-IgG (protein A) acceptor beads (20 µg/ml; PerkinElmer) for 30 min, followed by incubation with streptavidin donor beads (20 µg/ml; PerkinElmer) for 1 h at room temperature in the dark. The plate was then subjected to excitation at 680 nm, and emission from wells was monitored at 615 nm with the EnVision system (Perkin Elmer). Data were obtained as arbitrary units (AUs) of signal intensity (counts per second). IC50 values were determined by using Image J software ver. 1.53 k.

The AlphaLISA assay was setup by identifying suitable antibodies and optimizing antibody concentrations according to the manufacturer’s instructions (see results for details). For the measurement of [SMOX] in A549 cells, cell pellets containing 6 million cells as determined from automated trypan blue cell counting using a Luna (Logos Biosystems) cell counter were resuspended and lysed in 500 μl AlphaLISA buffer followed by centrifugation at 14,000 g. The supernatant was aliquoted and stored at −80 ˚C until use. In the final assay, a SMAB2/ His₆-tagged Fab33 mixture was prepared by diluting SMAB2 to 0.5 nM and and His₆-Fab33 to 5 nM in 1x AlphaLISA buffer. A volume of 5 μL of this mixture was added to 10 μL of 10x diluted lysate sample (for determinations of SMOX concentration) or 10 μl AlphaLISA buffer containing a known amount of SMOX (for calibration) in a 384 well white assay plate (OptiPlate-384, Perkin Elmer) and incubated for one hour at RT. Then 5 μL 50 μg/ml acceptor beads (AL178C, Perkin Elmer) was added followed by incubation for one hour at RT. For the last step of the assay, 5 μL donor beads (200 μg/mL) (AS105D, Perkin Elmer) was added per well. After one hour incubation at RT in the dark, the Alpha signal was read using an Alpha compatible reader (Synergy NEO, BioTek). Lysate and calibration samples were measured in duplicate.

Compounds were examined on
5-HT₆R using their ability
to inhibit cAMP production induced by 1 μM (EC₈₀)
5-carboxamidotryptamine (5-CT). The level of cAMP was measured in
1321N1 cells expressing the h5-HT₆R (PerkinElmer,
#ES-316-CF). According to the manufacturer’s instructions,
total cAMP was measured using the LANCE cAMP detection kit (PerkinElmer,
#TRF0263). Cells were incubated with a mixture of compounds for 30
min at room temperature (RT) in a white polystyrene OptiPlate-384
(PerkinElmer, #6007299) microplate. After incubation, the reaction
cells were lysed by the addition of 10 μL of cAMP detection
buffer, including Eu-cAMP tracer and ULight-anti-cAMP working solution.
The plate was incubated at RT for 1 h before measuring the signal
with a Tecan multimode plate reader (Infinite M1000 Pro). Compounds
were tested in triplicate at eight concentrations in the range from
10^–11 to 10^–4 M. K_b constants were calculated from Cheng–Prusoff
equation^{63 (link)} adapted to functional assays.