Recombinant protein a g

For the initial
investigation of the metal deposition, recombinant protein A/G (ThermoFisher
Scientific) was immobilized as the antigen. This was incubated for
60 min with 200 nM mouse anti-human IgG Fc-Horse Radish Peroxidase
(HRP) (SouthernBiotech) in 100 μL volume. Beads were washed
3 times in deionized water before the silver deposition was performed
with EnzMet (Nanoprobes). 50 μL each of EnzMet solutions A,
B, and C were incubated with beads for 4, 4, and 8 min, respectively.
Note that these times are obtained from assay optimization performed
in our prior work with enzymatic metallization.^{9 (link)}

Serology was tested with slight modifications to the previously published field ELISA [16 (link)]. Briefly, the plates were coated with 200 ng of the truncated SNV nucleocapsid protein and IgG was detected using recombinant Protein A/G (ThermoFisher Scientific, Waltham, MA, USA). Plates were read on a Microplate reader at 405 nm (BioTek, USA). Samples were considered positive if they were 3× the standard deviation plus the mean of the negative controls at a dilution ≥1:400.

After reconstitution in bicarbonate buffer (50 mM, pH 8.0), a solution of Recombinant Protein A/G (Thermo Scientific, cat # 77677) was prepared at a concentration at 2 mg/mL. MSD-Gold SULFO-TAG NHS-Ester (MSD, cat # R91AO-2) was suspended in dry DMF/water mixture, added promptly to the protein solution at a molar input ratio of about 12:1 (SULFO-TAG to Protein A/G), and allowed to react at ambient temperature in a sealed polypropylene vessel with gentle inversion to allow mixing. After about 2 h, the reaction was quenched by adding a small volume of amine-containing buffer, followed by gentle mixing for an additional 20 min. The SULFO-TAG labelled Protein A/G was purified by size exclusion chromatography using a HiPrep™ 26/40 column packed with Sephadex G-50 (fine) (GE, cat # 17-0042-01) and a mobile phase consisting of PBS supplemented with 5% sucrose and 0.05% Tween-20, pH 7.4. Detailed characterization of the purified conjugated product included concentration measurement by BCA Protein Assay, purity assessment by analytical HPLC-SEC and SULFO-TAG label molar incorporation ratio determination by Abs measurement at 455 nm. The conjugated protein was diluted into PBS containing 0.2% BSA buffer and stored in 0.1 mL aliquots in tightly sealed polypropylene vials at − 80 °C.