Anti human igg peroxidase conjugate

The detection of anti-SARS-CoV-2 IgG antibodies was performed using an in-house ELISA as described elsewhere by Meireles et al. [23 (link)]. Briefly, a 96 well microplate (Corning^®, New York, USA) was coated with 100 μl of N122-419 protein (2.2 μg/mL) diluted in 0.1 M sodium carbonate buffer pH 9.5 and the microplates were maintained overnight at 4°C in a humid chamber. The microplates were washed with PBS containing 0.05% Tween 20 (PBST) and blocked with 250 μL of 5% fat-free milk (Molico^®) diluted in PBST for 1h at 37°C. After washing, 100 μL of serum samples diluted 1:100 in PBST containing 5% fat-free milk (PBST-milk) were incubated with viral antigens for 1h at 37°C. The microplates were again washed and 100 μL/well of anti- human IgG peroxidase conjugate (Sigma, St. Louis, USA) diluted 1:20,000 in PBST-milk were added and incubated for 1h at 37°C. After washing, 100 μL/well of ABTS substrate solution (2, 2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) were added. The microplates were maintained in the dark for 30 min at room temperature. The reaction was stopped with 0.1M citric acid (100 μL/well). Optical density (OD) at 414 nm was measured in a microplate reader (Labsystems Multiskan MS^®, Midland, Canada). ELISA results were expressed in OD at 414 nm and the cut-off threshold was determined using the receiver operating characteristic (ROC) curve.

To further validate the microarray findings, the peptide selected was then custom synthesized by the company Biomatik (Biomatik Corporation, Kitchener, ON, Canada) for ELISA assay. For this assay, a subset of the sera samples from the microarrays was used (Negative samples n = 11; WT-YFV samples n = 9; 17DD-YFV samples n = 13). To coat the plates, 8 µg/mL of the peptide diluted in coating buffer were added to Nunc MaxiSorp 96-well ELISA plates and incubated at 4 °C overnight. Next, the wells were washed three times using a washing buffer and a blocking buffer (2.5% w/v BSA, 2.5% w/v nonfat dry milk, 1X phosphate-buffered saline with 0.05% Tween20) were added followed by incubation at 37 °C for 1 h. After washing as described serum from each group, diluted in Standard Buffer for ELISA, (1:100) was added, followed by an incubation at 37 °C for 2 h. Anti-human IgG peroxidase conjugate (Sigma-Aldrich Corporation, Saint Louis, MO, USA), diluted in standard buffer for ELISA (1:10000) was added. After 1 h of incubation at 37 °C, the wells were washed and TMB Substrate Solution (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well following by the addition of the Stop Solution. Absorbance was read on SpectraMax (Molecular Devices, San José, CA, USA) using a 450 nm wavelength.

The cytosolic fraction of the T. cruzi epimastigotes from a mixture of the I.RHO/CO/00/CAS-15.CAS and I.TRI/CO/03/MG-8.MAG Colombian strains was used as the antigen. Briefly, Nunc microtiter plates were sensitized overnight at 4°C with 100 μl of the antigen solution in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at a concentration of 200 ng/ml. The sera were diluted 1/100 in phosphate-buffered saline–Tween 20 (0.05%; PBST), and 100 μl of this mixture was added to the wells. After one hour at 37°C, the plates were washed three times with PBST, anti-human IgG-peroxidase conjugate (Sigma-Aldrich, Missouri, USA; 1/10,000 dilution in PBST) was added to the wells, and the wells were incubated at 37°C for an additional hour. After three additional washes, the immune complexes were developed with a citrate phosphate (pH 5.0), o-phenylenediamine (OPD; Sigma-Aldrich, Missouri, USA), and H₂O₂ buffer, and the absorbance was read at 450 nm. The cutoff values were calculated as the average absorbance of two negative controls plus two times the standard deviation, as was previously described by Guhl and Nicholls, 2001 [11 ].