Zeba column

Jurkat cells expressing the MARV (Angola) GP (kindly provided by Carl Davis and Rafi Ahmed) were primed proteolytically with 0.5 mg/mL thermolysin (Pierce) in PBS for 5 minutes at 34°C, then washed with PBS containing 2% of fetal bovine serum and 2 mM ethylenediaminetetraacetic acid (pH 8.0). For staining, cells were incubated for 30 minutes at 4°C with serial 2-fold dilutions of plasma in triplicate, followed by incubation for 30 minutes with 5 µg/mL fluorescently labeled MARV GP-specific mAbs. Cells were washed, fixed with 4% paraformaldehyde in PBS, and analyzed using a high-throughput flow cytometer (iQue, Intellicyt). Data were analyzed with ForeCyt software (Intellicyt). Plasma from a donor without an exposure history to filovirus infection was used as a negative control. Background values were determined from binding of second labeled antibody to untransfected Jurkat cells. Results were expressed as the percent of MARV GP-reactive antibody binding in the presence of plasma relative to a MARV-specific mAb-only control (maximal binding), minus background. For fluorescent labeling of antibodies, we used Alexa Fluor 667 NHS ester (ThermoFisher) and followed the manufacturer’s protocol. Labeled mAbs were purified further and buffer exchanged into PBS using desalting Zeba columns (ThermoFisher), and stored at 4°C.

A spiked E. coli lysate was prepared by pelleting the equivalent of OD₆₀₀ = 8 culture, discarding the supernatant, and lysing the cells with BPER at 4 ml per g pellet, 20 min at room temperature with rotation. The insoluble material was removed by centrifugation, and the cleared lysate adjusted to a total protein concentration of approximately 1.87 mg/ml, 20% BPER in PBS, pH 7.4. The target protein was spiked into the lysate to a final concentration of 0.025 to 0.2 mg/ml, depending on the application with a highly concentrated stock so that the total protein concentration remained unchanged. The spiked lysate was then incubated with 10 μl of the nanoCLAMP resin (packed volume) in a total volume of 1.4 ml, rotating at 4 °C for 1 h. The resin was precipitated by centrifugation and transferred to a small column. The resin was washed 4 times with 400 μl PBS, pH 7.4, and then eluted with 3 M imidazole, pH 8. The eluates were buffer exchanged twice with Zeba columns (7 kD MWCO, Thermo), quantified by A₂₈₀ or fluorescence using an iD5 plate reader, and analyzed by SDS-PAGE as described above.

cDNA encoding Tie2 ectodomain fusion proteins were constructed by ligating cDNA encoding wild-type or mutant Tie2 ectodomain (residues 1–442) with a GS4 linker, fragment of human Fc immunoglobulin domain, and C-terminal His6 tag. Tie2 ectodomain mutants were created from wild-type human Tie2 ectodomain by site-directed mutagenesis using the QuikChange protocol (Agilent Technologies). All constructs were sequenced to confirm desired mutations.
For protein expression, cDNA encoding fusion proteins in mammalian expression plasmids were transfected into Hek293 cells using polyethylenimine (25 (link)). Expressed protein was allowed to accumulate in culture medium for approximately 3 days and media was then clarified by centrifugation and filtration. His6-tagged proteins were recovered by nickel chromatography and, after extensive column washing, eluted with imidazole. Purified proteins were transferred to Tris-buffered saline (TBS; 25 mM Tris, 150 mM NaCl) containing 10% glycerol using Zeba columns (Thermo Fisher). Purity of proteins was assessed by SDS–polyacrylamide gel electrophoresis and Coomassie staining, protein concentrations were determined by Bradford assay, and fusion proteins stored at 4 °C.

SARS-CoV-2 antigens used for functional and Luminex based assays included SARS-CoV-2 RBD (Sino Biological), SARS-CoV-2 spike protein (LakePharma) and SARS-CoV-2 N (Aalto Bio Reagents). Additional antigens included a mix of HA A/Michigan/45/2015 (H1N1), HA A/Singapore/ INFIMH-16–0019/2016 (H3N2), and HA B/Phuket/3073/2013 (Immunetech). Antigen was biotinylated using Sulfo-NHS-LC-LC biotin (Thermo Fisher Scientific) and desalted using Zeba Columns (Thermo Fisher Scientific).

The ADCP assay was performed as previously described (Ackerman et al., 2011 (link)). SARS-CoV-2 spike (kindly provided by Eric Fischer) and nucleocapsid (Aalto Bioreagents) was biotinylated using Sulfo-NHS-LC-LC-biotin (Thermo Fisher), desolated using Zeba columns (Thermo Fisher), and coupled to yellow-green Neutravidin beads (Invitrogen) for 2 hours at 37°C or overnight at 4°C. Coupled beads were washed twice in 0.01% BSA in PBS and resuspended at 10 ug/mL for use in the assay. Immune complexes were formed by adding coupled beads to 96-well plates with equal volume of diluted serum (1:100) or diluted breastmilk (1:10). Immune complexes were incubated for two hours at 37°C. After the incubation, the immune complexes were washed, and THP-1 cells were added to the immune complexes at 1.25x10ˆ5 cells/mL. Cells were incubated with the immune complexes overnight at 37°C. The next day, the cells were fixed in 4% PFA. Fluorescence was acquired using an iQue (Intellicyt) and analyzed using Forecyt software. A Phago score was determined using the following formula: (percentage of bead-positive cells) x (GeoMean of MFI of bead-positive cells)/10,000