Sureblue tmb 1 component microwell peroxidase substrate

We modified and optimized an ELISA (26 (link)) to detect PDCoV-specific IgY antibodies in serum from PDCoV-inoculated chicks and poults. We added 50 µL of serum diluted 1:1,000 to the PDCoV antigen-coated and mock antigen-coated wells and incubated for 90 m at 37°C, then added 100 µL of biotin-conjugated antichicken IgY (Invitrogen Goat anti-Chicken IgY [H+L] Secondary Antibody, Biotin; ThermoFisher, https://www.thermofisher.com) or biotin-conjugated antiturkey IgY (Goat Anti-Turkey IgY (H+L) Biotin pAb; Cell Sciences, https://www.cellsciences.com) at a dilution of 1:10,000 and incubated at 37°C for 1 h. We added 100 µL of HRP-Conjugated Streptavidin (ThermoFisher) to each well at a dilution of 1:5,000 and incubated at 37°C for 1 h. We washed wells with phosphate buffered saline solution with 0.05% Tween-20 (×5) between each step. We added 3,3′,5,5′-tetramethylbenzidine substrate (SureBlue TMB 1-Component Microwell Peroxidase Substrate; Seracare, https://www.seracare.com), then added 100 µL of 0.3 mol/L sulfuric acid to stop the reaction. We read plates at an absorbance of 450 nm by using a SpectraMax F5 (Molecular Devices, https://www.moleculardevices.com) plate reader. We conducted statistical analysis by using Prism software (GraphPad, https://www.graphpad.com). We used analysis of variance to compare multiple groups and a 1-tailed Student t-test to compare groups of 2.

ELISA analyses of the humoral immune responses to SIV envelope protein were tested at baseline, ~ 24 wpi, and necropsy as previously described [45 (link)] with modifications. A reference plasma with strong anti-Env antibody concentrations was aliquoted and stored at − 80 °C. A batch of EIA/RIA high binding plates were coated overnight with 0.08 μg/ml of rgp130 SIV mac251 (ImmunoDx, Woburn, MA) in PBS (pH 7.4) using 100 μl/well at 4 °C. Plates were blocked with 200 μl B3T buffer (150 mM NaCl, 50 mM Tris–HCl, 1 mM EDTA, 3.3% fetal bovine serum, 2% bovine serum albumin, 0.07% Tween 20) for 1 h at 37 °C. An aliquot of the reference plasma and test plasmas were serially diluted in B3T buffer and added to the plate in duplicate at 100 μl/well for 1 h at 37 °C. 100 μl of horseradish peroxidase-conjugated goat anti-monkey IgG (Rockland Immunochemicals, Inc., Limerick, PA) at 1:10,000 was added for 1 h at 37 °C. Plates were washed 6× with 0.1% Tween 20 in PBS after each step then developed using SureBlue TMB 1-Component Microwell Peroxidase Substrate (SeraCare Life Sciences, Milford, MA) for approximately 25 min. A TMB Stop Solution (SeraCare) was added and plates were read at 450 nm. The OD values for the reference plasma were used to interpolate relative values of anti-Env antibody concentrations using Prism 7 software (GraphPad Software, Inc.).

Purified VSV pseudotypes were treated with either a phosphate buffer at pH 6.0 or 7.0, or acetate buffer at pH 5.5 or 5.0 at 37 °C or on ice for 30 min. The virions were then brought back to a neutral pH, as described above, at their respective temperatures and bound to an ELISA plate. The plate was then blocked with a 3% BSA solution for 1 h and washed with PBS three times. The immobilized virions were then probed with non-neutralizing (26.5E, 24.6C) or neutralizing antibodies (12.1F, 37.2D, 37.7H) for 1 h, washed three times with PBS, and probed with an HRP-conjugated rabbit anti-human IgG1 antibody (Abcam, Cambridge, UK) [32 (link)]. The plate was developed with SureBlue™ TMB 1-Component Microwell Peroxidase Substrate (SeraCare, Milford, MA, USA) for twenty minutes and read in a Synergy H1 Biotek plate reader.