Hrp conjugated streptavidin

Recombinant HA proteins possessing the Y98F mutation to attenuate sialic acid binding (HA∆SA) were dialyzed into 10 mM Tris-HCl (pH 8.0) and total protein concentration adjusted to ~2 mg/mL after BCA estimation. HA∆SA proteins were biotinylated with biotin protein ligase (BirA) (Avidity Biosciences, La Jolla, CA, USA) following manufacturer recommendations and confirmed by direct ELISA. High binding ELISA plates (CoStar, Washington, DC, USA) were coated overnight with 1 to 10 ng/well of BirA conjugated HA∆SA protein in carbonate buffer (pH 9.4) at 4 °C. ELISA plates were blocked and biotinylated HA∆SA proteins detected with HRP-conjugated streptavidin (Southern Biotech) followed by addition of the ABTS substrate. Biotinylated protein standard and a negative control protein were purchased from Avidity Biosciences (La Jolla, CA, USA; Cat. No. BIS-300). Fluorescently-conjugated HA∆SA probes were generated by stepwise addition of high concentration (1 mg/mL) PE- or APC-conjugated streptavidin (SA-PE or SA-APC) (BioLegend) to a final 8-fold molar excess, in ice-cold PBS with Protease Inhibitor Cocktail Set I (Calbiochem, San Diego, CA, USA; Cat. No. 539131). HA∆SA probes were stored at 4 °C in the dark up to two days.

Plates were coated with goat anti-mouse total IgG antibodies (Jackson labs). Supernatant IgG was detected with AP-conjugated goat anti-mouse IgG1, IgG2b, IgG2c, IgG3 or total IgG (Jackson Immune Research) as indicated. For IFNγ detection, BD OptEIA mouse IFN-γ ELISA Sets (BD) was used according to the manufacturer’s suggestions. To measure antibodies against Friend Virus (FV) ELISA plates were coated with FV lysate prepared as previously described [17 (link)]. Serum IgG was detected with biotin-conjugated goat anti-mouse IgG2c (SouthernBiotech) followed by HRP-conjugated streptavidin (SouthernBiotech).

Purified prefusion SARS-CoV-2 spike antigen (LakePharma) was adsorbed onto 96-well MaxiSorp immuno microplate (Thermo-Fisher). The positive control was a serum pool from three COVID-19 convalescent individuals (Nexelis). Total IgA purified from human serum was used as a standard (Sigma-Aldrich). To generate the IgA standard curve anti-human IgA capture antibodies, Mab MT57 (Mabtech), were absorbed on plates instead of spike antigen. Following incubation, the microplate was washed four times with 250 µL PBST and blocked with 1% BSA in PBST. Purified human IgA standard, controls, or sample dilutions were then added and incubated in the coated microplate to allow binding. The plates were washed and a biotinylated goat anti-human IgA antibody (Mabtech) with cross-reactivity to monkey antibodies was added to all wells. Excess biotinylated anti-IgA antibody was removed by washing and streptavidin-conjugated HRP (Southern Biotech) was added. TMB was added and color development was stopped by addition of stop solution from Invitrogen. The absorbance of each well was measured at 450 nm. The standard total IgA antibody assayed on each test plate was used to calculate the concentration of IgA antibodies against spike protein in the AGM samples expressed in the arbitrary units ELU/mL. The measurements were performed in duplicate and average values are reported with standard deviations.