405 ts plate washer

Streptavidin-coated 96-well plates (R&D Systems, CP004) were coated with 50 ng of biotinylated HLA-A*02:01 pHLA monomers in 50 μl of blocking buffer (PBS with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide) or 25 ng of recombinant human CD3_ε/δ (Acro Biosystems, CDD-H82W6) at 4°C overnight. Plates were washed with 1X TBST (TBS + 0.05% Tween-20) using a BioTek 405 TS plate washer. Serial dilutions of scDb or IgG were incubated on plates for 1 hour at room temperature (RT) and washed. For scDbs, the plate was then incubated with 1 μg/ml recombinant protein L (Thermo Fisher Scientific, 77679) for 1 hour at RT, washed, followed by incubation with antiprotein L HRP (1:10000, Abcam, ab63506) for 1 hour at RT. For IgG, the plate was incubated with anti-human IgG HRP (1:1000, Thermo Fisher Scientific, 62-8420) for 1 hour at RT. Plates were washed, 50 μl of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (BioLegend, 421101) was added to each well, and the reaction was quenched with 50 μl 1N sulfuric acid (Thermo Fisher Scientific, SA212-1). Absorbance at 450 nm was measured with a Synergy H1 Multi-Mode Reader (BioTek).

Fifty microliters of coupled microsphere mix was added to each well of clear-bottom, 96-well, black polysty-rene microplates (Greiner Bio-One North America, Monroe, NC) at a concentration of 1000 microspheres per region per well. All wash steps and dilutions were accomplished using 1% BSA and 1× PBS assay buffer. Except when assay parameters were under development (Figs. 1–3), serum was assayed at 1:500 dilution and surveyed for Abs specific for the four-candidate Ags. After a 1-h incubation in the dark, on a plate shaker at 800 rpm, wells were washed five times in 100 μl of assay buffer using a BioTek 405 TS plate washer. Then, the secondary reagent, PE-conjugated Goat Anti-Human IgA, IgG, and/or IgM (SouthernBiotech; Birmingham, AL), was added at 3 μg/ml. After 30 min of incubation at 800 rpm in the dark, wells were washed three times in 100 μl of assay buffer, resuspended in 100 μl of assay buffer, and analyzed using a Luminex FLEXMAP 3D instrument (Luminex) running xPonent 4.3 software. Median fluorescent intensity (MFI) using combined or individual detection Abs (anti-IgA/anti-IgG/anti-IgM) was measured using the Luminex xPONENT software. The background value of assay buffer was subtracted from each serum sample result to obtain MFI minus background (net MFI).

Approximately 50 μl of coupled microsphere mix was added to each
well of 96-well clear-bottom black polystyrene microplates (Greiner Bio-One) at
a concentration of 1,000 microspheres per region per well. All wash steps and
dilutions were accomplished using 1% BSA, 1× PBS assay buffer. Sera were
assayed at 1:500 dilutions and surveyed for antibodies against N or RBD. After a
1-h incubation in the dark on a plate shaker at 800 r.p.m., wells were washed
five times in 100 μl of assay buffer, using a BioTek 405 TS plate washer,
then applied with 3 μg ml⁻¹ PE-conjugated goat
anti-human IgA, IgG and/or IgM (Southern Biotech). After 30 min of incubation at
800 r.p.m. in the dark, wells were washed three times in 100 μl of assay
buffer, resuspended in 100 μl of assay buffer and analyzed using a
Luminex FLEXMAP 3D instrument (Luminex) running xPONENT 4.3 software. MFI using
combined or individual detection antibodies (anti-IgA, anti-IgG or anti-IgM) was
measured using the Luminex xPONENT software. The background value of assay
buffer was subtracted from each serum sample result to obtain MFI minus
background (MFI-B; net MFI).