Asys expert 96 elisa reader

ELISA was performed by coating ELISA plates (Nunc) with unconjugated anti-mouse IgM (Southern Biotech). After incubation overnight (4°C), washing (PBS + 2% Tween20) and blocking for 1 h with PBS containing 2% dry milk (blocking buffer), serum was added in threefold serial dilutions in blocking buffer and incubated for 1.5 h at room temperature (RT), before addition of HRP-coupled anti-IgM or IgG3 (Southern Biotech). The assay was developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL) followed by 1M H₂SO₄ and the OD values were read at 450 nm using an Asys Expert 96 ELISA reader (Biochrom).

ELISA was performed by coating ELISA plates (Nunc) with polysaccharide antigens: 500 ng/well of NP (25) conjugated with BSA (Biosearch Technologies) or the pneumococcal polysaccharide antigens Type 1 161-X™ or Type 3 169-X™ (both ATCC) or 1:100 dilution of Pneumovax (corresponding to 50 ng/well of each antigen contained in the vaccine). To measure total IgM and IgG3 levels, plates were coated with unconjugated anti-IgM or anti-IgG3 (Southern Biotech). Plates were incubated overnight (4°C). Following washing (PBS + 2% Tween20) and blocking for 1 h with PBS containing 2% dry milk, serum was added in threefold serial dilutions in blocking buffer and incubated for 1.5 h at room temperature (RT) before addition of secondary antibody HRP-coupled anti-IgM or IgG3 (Southern Biotech). The assay was developed with TMB substrate (KPL) followed by 1M H₂SO₄ and the OD was read at 450 nm using an Asys Expert 96 ELISA reader (Biochrom).

96‐well ELISA plates (Nunc MaxiSorp) were coated with freshly prepared SARS‐CoV‐2 S trimers or the RBD (100 µl of 1 ng/µl) in PBS for 15 h at 4°C. Plates were washed six times with PBS–Tween‐20 (0.05%) and blocked using PBS‐5% no‐fat milk (Sigma). Human serum samples were thawed at room temperature, diluted, vortexed and incubated in blocking buffer for 1 h (4°C) before plating to block non‐specific binding. Serum samples were incubated for 15 h at 4°C to allow low‐affinity binding interactions, before washing as before. Secondary HRP‐conjugated anti‐human antibodies were diluted in blocking buffer and incubated with samples for 1 hour at 4°C. Plates were washed a final time before development with TMB Stabilized Chromogen kept at 4°C (Invitrogen). The reaction was stopped using 1 M sulphuric acid and optical density (OD) values were measured at 450 nm using an Asys Expert 96 ELISA reader (Biochrom Ltd.). Secondary antibodies (from Southern Biotech) and dilutions used were as follows: goat anti‐human IgG (2014‐05) at 1:10,000. All assays were developed for their fixed time, and negative control samples were run alongside test samples in all assays. Anti‐SARS‐CoV‐2 S and RBD IgG were detectable at up to 1:20,000 serum dilution using this assay [8 (link)], and all study samples were here run at 1:100 dilution.