Ultra tmb substrate

NPC1 binding ELISAs were performed as described previously (4 (link)). Plates were coated with GP-specific monoclonal antibody (Mab) KZ52 (14 (link)) diluted to 2 μg/ml in phosphate-buffered saline (PBS). VSV-GP(WT) or VSV-GP(R64A) was normalized for GP content and then incubated at 37°C for 4 h in the presence of 500 μg/ml THL (Sigma-Aldrich, St. Louis, MO). The reaction was stopped by addition of 10 mM phosphoramidon. ELISA plates were blocked using PBS supplemented with 3% bovine serum albumin (PBSA; Thermo Fisher Scientific, Waltham, MA). THL-treated virus was added to blocked KZ52-coated plates and allowed to adsorb at 37°C for 1 h. After the plates were washed with 3% PBSA, a dilution series of FLAG-tagged NPC1-domain C was added and allowed to bind for 1 h at 37°C. Bound domain C was then detected using a horseradish peroxidase (HRP)-conjugated anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO) and the Ultra-TMB substrate (Thermo Fisher Scientific, Waltham, MA). Half-maximal effective concentration (EC₅₀) values were calculated from two independent experiments, each with three technical replicates.

Plates were coated with GP-specific mAb KZ52 (44 (link)) diluted to 2 μg/ml in phosphate-buffered saline (PBS). VSV-GP mutants were normalized for GP content and then incubated at 37°C for 1 h in the presence of 250 μg/ml thermolysin (Sigma-Aldrich, St. Louis, MO), before the addition of 10 mM phosphoramidon to stop the reaction. ELISA plates were blocked using PBS supplemented with 3% bovine serum albumin (PBSA) (Thermo Fisher Scientific, Waltham, MA). THL-treated virus was added to blocked KZ52-coated plates and allowed to adsorb at 37°C for 1 h. Following washing with 3% PBSA, a dilution series of FLAG-tagged NPC1 domain C (45 (link)) was added and allowed to bind for 1 h at 37°C. Bound domain C was then detected using a horseradish peroxidase (HRP)-conjugated anti-FLAG antibody (Ab) (Sigma-Aldrich, St. Louis, MO) and the Ultra-TMB substrate (Thermo Fisher Scientific, Waltham, MA). Fifty percent effective concentration (EC₅₀) values were calculated using nonlinear regression of data from two independent experiments with three technical replicates each.

A previously established ELISA for the detection of α-DG-N in uterine lavage was used [56 ]. In brief, half area 96-well plates were coated overnight at 4°C with 0.5 μg/ml of α-DG-N mAb (2A3, Sigma Aldrich) in 0.1 M sodium carbonate/bicarbonate buffer of pH 9.6 (Sigma Aldrich). The wells were then washed with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 1.8 mM KH₂PO₄) containing 0.05% Tween20 (PBS-T), and blocked with 1% BSA (Bovogen, VIC, Australia) in PBS for 90 min at 37°C. The wells were washed with PBS-T, incubated with recombinant α-DG-N as the standards or uterine lavage (undiluted) for 2 hr with gentle agitation. The wells were washed and incubated first with 3 μg/ml of biotinylated detection antibody (3B4, Creative Diagnostics, NY, USA) for 1 hr, then with poly streptavidin-HRP (Thermo Scientific, Waltham, MA, USA) at 1:25,000 dilution for 50 min with gentle agitation, and finally with ultra-TMB substrate (undiluted, Thermo Scientific) for 7–10 min in the dark at room temperature without agitation. The ELISA reaction was stopped with 1 M sulfuric acid (Banksia Scientific Company Pty Ltd, QLD, AUS) and the absorbance at 450 nm was measured (Envision Multilabel reader, PerkinElmer, Waltham, MA, USA). All washes and incubations were performed at room temperature.

EIA plates were coated with 0.25 μg/ml VLP in PBS before adding 2-fold serial dilutions of serum. The primary Ab incubation was followed by anti-human-IgG-HRP secondary Ab (GE Healthcare), and positive wells were color-developed with Ultra TMB substrate (Thermo Scientific). Plates were washed with PBS/0.05% Tween 20 (Thermo Scientific) between steps, and Ab dilutions were done in 5% Blotto/PBS/0.05% Tween 20. All incubations were done at room temperature. The percent maximum binding was calculated, sigmoidal dose–response curves fit to the data, and EC₅₀ values determined using GraphPad Prism version 6.02 for Windows (GraphPad Software). All samples were tested in a minimum of two independent assays. EC₅₀ titers below the lower limit of detection (100) were assigned a titer of 50.