Anti human igg hrp

Maxisorp plates (Nalgene-Nunc, Grand Island, NY, USA) were coated with lysate from RVFV-infected Vero-E6 cells or with lysate from uninfected Vero-E6 cells (negative control) diluted 1:2000 in phosphate-buffered saline (PBS)^{36 (link)} and allowed to adsorb overnight at 4 °C. Plates were blocked in 5% fetal bovine serum (FBS) and 5% skim milk in PBS 1 h at 37 °C. Patient serum samples were assayed in duplicate and were serially diluted in blocking buffer and then incubated on blocked plates for 1 h at 37 °C. After three washes in PBS with 0.1% Tween 20 (PBST), plates were incubated for 1 h at 37 °C with anti-human IgG HRP (Jackson ImmunoResearch Inc, West Grove, PA, USA) diluted 1:5000 in blocking solution with 0.1% Tween 20. Following 3 PBST washes, the plates were incubated in TMB substrate (KPL) for 10 min; reactions were stopped with the addition of 1% HCl, and plates were read at 450 nm. Data were analyzed using Excel (Microsoft Corp, Redmond, WA, USA) and Prism (GraphPad Software Inc, La Jolla, CA, USA) software. Raw OD values from the negative control Vero-E6 plate were subtracted from those of the RVFV lysate plate. The endpoint titer was defined as the dilution of the serum that gave a value at least three standard deviations above the average value obtained from the negative control serum.

Corning 3690 half-well 96-well plates were coated overnight at 4°C with 50 µl/well of 2 µg/ml eOD-GT8, eOD-GT8-KO, 426cTM₄ΔV1-3, 426cTM₄ΔV1-3-KO, 426c Env N276D trimer, 426c Env N276D-KO trimer or murine iv8 Fab in PBS. Plates were washed six times in PBS 0.05% Tween 20 (wash buffer). Plates were blocked with 100 µl/well PBS 5% milk (blocking buffer) for 2 h at room temperature (RT). Sera or monoclonal antibodies were prepared at 1:30 dilution or a concentration of 10 µg/ml in fresh blocking buffer, respectively, and further diluted in threefold or fivefold serial dilutions, respectively. A total volume of 50 µl was added to the plates and incubated for 2 h at RT. Binding was revealed by either anti-mouse IgG-HRP (Jackson ImmunoResearch) or anti-human IgG-HRP (Jackson ImmunoResearch) diluted 1:5,000 in wash buffer. Plates were incubated for 1 h at RT. Plates were washed six times in wash buffer. HRP activity was determined using ABTS (2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) substrate solution (Life Technologies), adding 50 µl/well. Plates were read at 405 nm on a FLUOstar Omega microplate reader (BMG Labtech). Data were analyzed with Microsoft Excel and GraphPad Prism 6.0.

SARS-CoV-2 ELISAs were performed as previously described (14 (link), 21 (link)). Briefly, 96-well plates were coated with 0.5 μg/mL of recombinant SARS-CoV S1 and S2 (catalog 40589-V08B1, SinoBiological Inc) or recombinant SARS-CoV-2 N (catalog 40588-V08B, SinoBiological Inc) overnight at 4°C. The next day, plates were blocked with 3% BSA in PBS for 1 hour at room temperature. Plates were washed and the sera diluted at 1:1000 in PBS containing 0.05% Tween-20 (PBST) and added to the plates in duplicates before being incubated for 1 hour at room temperature. Plates were washed and 8 M urea (catalog ZU10001, Invitrogen, Thermo Fisher Scientific) or PBST only was added for 15 minutes. Plates were washed and anti-human IgG-HRP (catalog 109-035-008, Jackson ImmunoResearch) at 1:2000 dilution was added and developed by KPL SureBlue (catalog 5120-0077, Seracare) TMB Substrate. Readouts were recorded as the OD at 450 nm. Avidity index was calculated as follows: (normalized OD of urea-treated sera/normalized OD urea-untreated sera) × 100.

Approximately 1 μg of each fusion mAb or the recombinant protein were resolved on 7 or 12% SDS-PAGE gels under non-reducing or reducing conditions, respectively. Gels were either stained with Coomassie Blue (Amresco) or transferred to nitrocellulose membranes (GE Healthcare). Coomassie Blue stained gels were then scanned using a Lexmark 3600–4600 series scanner and transformed into grayscale using AdobePhotoshop CC software (Adobe Systems Incorporated 2013). nitrocellulose membranes were blocked for an 1 hour at room temperature (rt) in 0.05% PBS-Tween 20 (PBS-T), 5% non-fat milk and 1% BSA, and then incubated with serum (1:2,000 dilution) derived from a patient previously infected with P. vivax (kindly provided by Dr. Claudio R.F. Marinho, University of São Paulo, Brazil). After a 2-hour incubation at rt the membranes were washed twice and incubated for an additional hour using an anti-human IgG-HRP (1:5,000, Jackson Laboratories). After two additional washes, the membranes were developed using quimioluminescence (ECL kit, GE Healthcare) and captured on Kodak film. The films were then scanned and submitted to the same processing described above for Coomassie Blue gels.