Hrp conjugated goat anti mouse antibody

The Gb3/Gb4-LPS receptor-binding assays were performed as previously described [39 (link)]. Briefly, formaldehyde-fixed E. coli cells either expressing Gb3-LPS, Gb4-LPS, or control E. coli cells (CWG308) were diluted to 0.1 OD₆₀₀ in carbonate buffer (0.1 M NaCO₃, pH 9.6) and 100 µL was added to the wells of a 96-well ELISA plate and dried overnight at 50 °C. Wells were then blocked with 300 µL/well of 5% non-fat dry milk (NFDM) in PBS with 0.05% Tween-20 (PBST) for 1 h at room temperature. Wells were washed twice with PBST and then incubated with 100 µL of pure toxin (50 ng/mL) for 1 h at room temperature. After washing six times with PBST, wells were incubated with 100 µL of mAb Stx2e-3 (100 ng/mL) [38 (link)] for 1 h at room temperature. After washing six times with PBST, wells were incubated with 100 µL of horse radish peroxide (HRP)-conjugated goat anti-mouse antibody (Promega, Madison, WI, USA) (1:5000) for 1 h at room temperature. Signals were developed with 100 µL/well of SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) and luminescence (CPS) was read on a Victor3 plate reader (Perkin Elmer, Waltham, MA, USA) for 0.1 s. All treatments were performed in triplicate and repeated three times.

Wells on 384-well polystyrene high-binding plates were coated with 1086.C gp120 (20 μg/mL) and blocked with superblock solution (4% whey, 15% goat serum, 0.5% Tween 20 in 1xPBS) for 1 h. Mouse sera were diluted in a tenfold series with superblock to yield six dilutions from 1:10 to 1:10⁶. The coated plates were incubated with the diluted serum samples at room temperature for 1 hour, then were washed 4 times with superwash buffer (0.1% Tween 20 in 1xPBS). HRP-conjugated goat anti-mouse antibody (Promega) in 1:5000 dilution was incubated in the same condition, followed by 4 washes. SureBlue TMB microwell peroxidase substrate was used for color development, with 5 min of incubation applied before the reaction was terminated with an equal volume of TMB stop solution. OD values at 450 nm was read with a SpectraMax Plus 384 plate reader. Serum antibody avidity was measured as previously described^{62 (link)}. Plates were coated and treated with serum as described above, then treated with urea solution (7M in water) for 5 min, followed by 4 washes and an 1-hour incubation of detection antibody. Avidity indices were calculated according to the following formula: (mean OD in urea-treated wells/mean OD in PBS-treated wells) × 100.