Goat anti rabbit igg conjugated to hrp

ELISA was performed as previously described (22 (link)). Briefly, ninety-six well MaxiSorp flat bottom plates (Thermo Fischer Scientific, USA) were coated in 0.5 µg/ml antigen in phosphate buffered saline (PBS) overnight at 4°C. The following day, plates were blocked in 1% casein in PBS (w/v) and then incubated with test antibody samples. Human plasma samples were tested at 1/1000 dilution in buffer (0.1% casein in PBS, w/v), and antigen-specific antibodies were detected using goat anti-human IgG (Invitrogen, USA) and goat anti-human IgM (Millipore, USA) antibodies conjugated to HRP. Human plasma samples were also tested at 1/500 dilution for IgG subclass detection using mouse anti-human IgG1/IgG3 (Invitrogen), followed by goat anti-mouse IgG conjugated to HRP (Millipore). Purified rabbit IgG were tested at 0.001-10 µg/ml and detected using goat anti-rabbit IgG conjugated to HRP (Millipore). After the addition of HRP, plates were incubated with ABTS substrate (to detect human IgM and rabbit IgG) or TMB substrate (to detect human IgG and IgG subclasses) and absorbance was measured at an optical density (OD) of 405 nm and 450 nm, respectively. Note that all antibodies were prepared in 0.1% casein in PBS and plates were washed thrice using PBS-Tween20 0.05% between each incubation step.

Ninety-six well flat-bottom MaxiSorp Nunc plates (ThermoFischer Scientific) were coated with 5 μg/ml Avidin in PBS overnight, blocked, and then incubated with either two-fold dilution of biotinylated RBD (69 (link)) or 2.5 μg/ml in 0.1% casein for 1 hour at RT. The ACE2-Fc proteins were then added over the indicated concentration range. In experiments to measure C5b-C9 fixation, the plates were incubated with 10% fresh human serum for 30 minutes at RT followed by 1/2000 dilution of rabbit anti-C5b-C9 (Millipore) for 1 hour at RT, washed and then incubated with goat anti-rabbit IgG conjugated to HRP (Millipore) at 1/2000 dilution for 1 hour at RT, followed by TMB substrate for 15-20 minutes at RT (70 (link)). Reactivity was stopped using 1 M sulfuric acid and absorbance was measured at 450 nm. Test samples and reagents were prepared in PBS 0.1% (w/v) casein and plates washed thrice between each step using PBS, 0.05% (v/v) Tween 20. Samples were tested in duplicate and corrected for background reactivity using negative control wells from which ACE2-Fc proteins were omitted. The mean and SEM from independent experiments are shown.

To detect the binding of C2 to rSjTOR-ed1, the same concentration of rSjTOR-ed1 and Albumin Bovine V (10 μg/ml) in carbonate-bicarbonate buffer (pH 9.6) was coated on a 96-well plate (Costar, Kennebunk, USA) overnight at 4 °C. The plate was washed with PBST and blocked with 0.5% gelatin (Amresco, Solon, USA) in PBST for 1 h at 37 °C. After washing, decreasing amounts of human complement C2 (Sino Biological Inc., Beijing, China) was diluted in 20 mM Tris buffer (containing 1 mM MgCl₂ and 1 mM CaCl₂) and incubated for 2 h at RT. Then, plates were washed and incubated with rabbit monoclonal antibody to C2 (Sino Biological Inc.) diluted 1:2000 in PBST for 70 min at 37 °C. After additional washes, plates were incubated with goat anti-rabbit IgG conjugated to HRP (Sigma-Aldrich), 1:4000 in PBST for 1 h at 37 °C. Plates were washed, and 3,3′5,5′-tetramethyl benzidine dihydrochloride was added as a substrate solution. Finally, the reaction was stopped with 50 μl 2 M H₂SO₄, and absorbance was measured at 450 nm.