Spectramax pro

A capillary-based assay (Palleroni, 1976 (link)) was performed to provide quantitative measurement of the bacterial cell motility, as described previously (Sun et al., 2009 (link)). Briefly, 10 ml cultures grown to an OD of approximately 0.4–0.5 (mid-log) were spun down at ~5500 × g for 8 min at room temperature and resuspended in an equal volume of phosphate buffered saline (PBS). Each channel of the Palleroni chamber was filled with 550 μl of resuspended cells. The capillary (32 mm length, 1.1 mm inner diameter) was filled with one of the following solutions: 30 mM sulfate, 60 mM lactate or 1 × PBS (control) and placed horizontally into the Palleroni chamber. After the 15 min incubation period, contents from the capillary were dispensed into 135 μL of 1 × PBS. The micro-bicinchoninic acid (micro-BCA) assay (Pierce, Rockford, IL, USA) was used as per manufacturer's instruction to measure the protein from the cells, and served as a measure of the cell mass that entered the capillary during the assay. Absorbance was measured by the SpectraMax Pro microplate reader (Molecular Devices, Sunnyvale, CA). Dilutions of bovine serum albumin in 1 × PBS were used to prepare a standard curve.

To test the neutralizing potential of SARS-CoV-2 specific antibodies in the sera of vaccinated individuals, SARS-CoV-2 antibody neutralizing immunoassay kits (ThermoFisher Scientific) were used according to the manufacturer’s instructions. Briefly, 100 µL of controls and 1:5 diluted sera from fully vaccinated or unvaccinated individuals were added to the wells of microplates pre-coated with SARS-CoV-2 receptor-binding domain (RBD) protein. The plates were then incubated for 1 hour to allow neutralizing antibodies present in the samples to bind to RBD specifically. After 3 washes with the provided wash solution, 100 µL of biotinylated 1x ACE2 and samples were incubated for an additional 1h. After incubation, samples were washed 3 times, and 100 µL of 1x Streptavidin-HRP was added to each well. Plates were then incubated for an additional 30 min, and 50µL of stop solution containing 2N H2SO4 was added to stop the reaction. Signal development is indirectly proportional to the amount of specific neutralizing antibodies present. Plates were measured at 450 nm with the SpectraMax Pro plate reader (Molecular Devices), and neutralization potential was calculated according to the provided controls.

Microtiter plates (96 wells) were coated with 100 μL/well of 50 mM sodium carbonate containing 82E1 (IBL) and allowed to adhere overnight at 4 °C. Plates were washed with PBS and blocked for overnight at 4 °C with 200 μL/well of 1% (w/v) bovine serum albumin in PBS containing 0.05% NaN₃. After two washes with PBS containing 0.02% Tween-20 (PBS-T), 100 μL of CSF was serially diluted in 1% bovine serum albumin in PBS-T before being added in triplicate to wells before incubation overnight at 4 °C. After four washes with PBS-T, each well was treated with 100 μL of horseradish peroxidase-conjugated 24B3 for 1 h at 4 °C. ELISA signals were detected by chemiluminescence using an enhanced chemiluminescent substrate (SuperSignal ELISA Femto Maximum Sensitivity, Thermo Scientific), and then measured with a microplate luminometer (SpectraMax Pro, Molecular Devices).
Synthetic E22P-Aβ40 dimer24 (link) was used as a standard protein because the E22P-Aβ40 dimer also containing the toxic turn at Glu22 and Asp23 was more stable than the E22P-Aβ42 dimer. The concentration of the E22P-Aβ40 dimer was determined using the Bradford assay (Bio-Rad). The amount of total Aβ42 in CSF was determined by sandwich ELISA with a human β Amyloid ELISA Kit of Aβ42 [Cat# 27711, human amyloid β (1–42) including (X-42)] (IBL) according to the manufacturer’s protocols.