Tmb single solution substrate

SARS-CoV-2 spike specific IgM and IgG antibody responses were determined using an in-house enzyme-linked immunosorbent assay (ELISA). Briefly, 96 well plates (Corning Inc, Corning, NY, USA) were pre-coated with full length spike or EBOV GP (negative control) overnight and blocked with phosphate-buffered saline (PBS) containing 5% skim milk for one hour. A serial dilution of mouse serum was carried out in triplicate in PBS containing 5% skim milk before being applied to the plate, incubated for one hour, and then washed three times in PBS plus 0.1% Tween 20 using a BioTek plate washer. Mouse IgG was detected with a Goat anti-Mouse IgG (H + L) Secondary Antibody conjugated with HRP (Catalog # 31430; 1:10,000 dilution, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Mouse IgM was detected with a Goat anti-Mouse IgM (H + L) Secondary Antibody conjugated with HRP (Catalog # 31440; 1:10,000 dilution, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Both IgG and IgM secondary antibodies were incubated for one hour Following a second set of washes, the plate was incubated with 3,3′,5,5′-tetramethylbenzidine (TMB) Single Solution substrate (Thermo Fisher Scientific, Waltham, MA, USA). The reaction was stopped with 1N sulfuric acid and the absorbance was read at 450 nm using a BioTek Synergy HT plate reader (BioTek, Winooski, VT, USA).

IgG and IgM levels in NHP serum were measured by enzyme-linked immunosorbent assay (ELISA) as described elsewhere (54 (link)). Briefly, 96-well plates (Corning) were coated with recombinant EBOV GPΔTM (IBT Bioservices), and blocked with phosphate-buffered saline (PBS) containing 5% skim milk. NHP serum samples and the in-house standards were diluted in PBS containing 2% skim milk before being applied to the plate, incubated, and then washed three times in PBS plus 0.1% Tween 20 using a BioTek plate washer. Goat anti-monkey IgG or goat anti-monkey IgM secondary antibodies conjugated to horseradish peroxidase were used at a concentration of 1 μg/ml, and following a second set of washes, the plate was incubated with 3,3′,5,5′-tetramethylbenzidine (TMB) Single Solution substrate (Thermo Fisher Scientific). Absorbance was read at 650 nm using a BioTek Synergy HT plate reader (BioTek), and samples for which all dilutions fell outside the limits of detection were reevaluated at higher or lower dilutions, as appropriate. Data were fit to the standard curve and are displayed as arbitrary antibody units (AU) per milliliter. Note that the therapeutic antibodies CA45 and FVM04 possess human Fc domains and therefore do not cross-react with the secondary antibodies used in this assay (30 (link), 31 (link)).