Spectramax abs plus absorbance microplate reader

S protein receptor-binding domain (S RBD)-specific antibody titers in serum and BALF were determined using ELISAs. The recombinant His-tagged SARS-CoV-2 S RBD protein (aa 319–541) was produced in Expi293F cells using a transient expression system (Thermo Fisher) according to the manufacturer's instructions and purified using Nuvia™ IMAC Resin (Bio-Rad). NUNC-MaxiSorp™ 96-well plates (Thermo Scientific) were coated with 1 μg/ml of recombinant SARS-CoV-2 S RBD protein overnight at 4 °C and blocked with 5% fetal bovine serum (FBS) in DBPS for 1 h at RT. Serially diluted samples were added to wells and incubated overnight at 4 °C. After washing four times with ELISA washing buffer (DPBS containing 0.05% Tween-20), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) or anti-mouse IgA (Southern Biotech, Birmingham, AL, USA) antibodies were used to detect S RBD-specific antibodies in the samples. Plates were incubated for 1 h at RT and washed four times with ELISA washing buffer. TMB (eBioscience, San Diego, CA, USA) was used as the substrate, and relative titers were calculated based on the absorbance at 450 nm using a SpectraMax® ABS Plus Absorbance Microplate Reader (Molecular Devices, San Jose, CA, USA).

SARS-CoV-2 Surrogate Virus Neutralization Test Kits (GenScript) were used to determine neutralizing antibody titers in serum according to the manufacturer's protocol. Briefly, serially diluted samples were incubated with HRP-conjugated RBD for 30 min at 37 °C, and then the sample mixtures were added to plates coated with human angiotensin-converting enzyme 2 (ACE2) protein. After incubation for 15 min at 37 °C, plates were washed four times with wash buffer and TMB substrate was used for development. Binding inhibition values were calculated based on the absorbance at 450 nm using a SpectraMax® ABS Plus Absorbance Microplate Reader (Molecular Devices).