Hrp conjugated goat anti human igg

Individual wells were coated overnight at 4 °C with 50 ng of BSA (Thermofisher Scientific, Burlington, Canada), SARS-CoV-2 nucleocapsid (#40588-V08B, Cedarlane labs, Burlington, Canada) or SARS-CoV-2 spike protein, kindly provided by Yannick Doyon. After 6 washes, wells were blocked with PBS, 5% milk for an hour. Wells were then incubated with 1/100 diluted sera for an additional hour at 37 °C. Following extensive washes, wells were incubated for an hour with 15 ng of HRP conjugated goat anti human IgG (Thermofisher Scientific). After a final round of washes, well were incubated with 50ul of ABTS substrate (Mandel Scientific, Guelph, Canada). The absorbance was then read at 405 nm.

ELISAs were conducted to detect high affinity MOG Ab binding to the extracellular MOG domain. Purified native-MOG^1–117 and native-P42S^1–117 (10 μg/mL) was coated onto 96-well Nunc MaxiSorp™ plates (ThermoFisher Scientific) in coating buffer (0.1 M Na₂CO₃, 0.1 M NaHCO₃, Milli-Q water, pH 9.6) overnight at 4 °C. Control wells were coated with bovine serum albumin (BSA) (10 μg/mL) to detect background sera binding. Wells were blocked (PBS, 1% BSA) for 3 h at RT, patient sera (1:50) was incubated for 2 h at RT, HRP-conjugated goat anti-human IgG (1:2000) (ThermoFisher Scientific) was added for 1 h at RT, followed by 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich, USA) for 15 min at RT, and 1 M HCl was added to stop the reaction. Wells were washed thoroughly with PBS, 0.05% Tween20 following each incubation. Optical density (OD) values were obtained at 450 nm and were wavelength- and blank-corrected. MOG Ab binding to native-MOG^1–117 was determined by the formula: native-MOG^1–117 ΔOD = native-MOG^1–117 OD – BSA OD. The positive threshold was set at 3SD above the control mean ∆OD . Native-MOG^1–117 Ab titers were also independently assessed in 15 serum samples by ELISA, as described above.

Plasma samples collected at baseline as well as at 2 weeks after the first and last vaccination time point were tested for the presence of IgG antibodies specific to V1, V2, V3 and MPER peptides of HIV-1 envelope gp120 using direct ELISA. Briefly, 96 well plates were coated with 5 μg/ml of peptide pools (15-mer peptides with 11 amino acid overlap) constituting the V1, V2, V3 and MPER regions, in 100 mM NaHCO_{3 (}pH 9.6), by overnight incubation at 4°C. Plates were washed with PBST (PBS containing 0.05% Tween 20) and blocked with PBS (Lonza, India) containing 1% BSA and 0.05% Tween 20 at 37°C for 2 hours. After washing, heat-inactivated plasma samples diluted 1:50 with diluent (ABL Inc., MD) were added to each well and incubated at 37°C for 1 hour. Subsequently, plates were washed and incubated with HRP-conjugated goat anti-human IgG (Thermo Fisher, Waltham, MA) at a dilution of 1:120000 at 37°C for 1 hour. Plates were developed using One-step TMB substrate (Thermo Fisher). The reaction was stopped using 1N H₂SO₄ and plates were read at 450nm using a microplate reader (ELx808-BioTek). All tests were performed in duplicates. Pooled normal healthy plasma was used as the negative control. The cut-off for a positive response was defined as mean OD of the negative samples plus 3 times the standard deviation (Table 1).