The presence of IgG antibodies against PvAMA-1 protein and all four peptides were determined through a conventional enzyme-linked immunosorbent assay (ELISA) that was performed as previously described [10 (link), 25 (link)]. Briefly, the microplates (Costar®) were coated with the antigens (concentrations of 0.5μg/mL and 2μg/well for PvAMA-1 and peptides respectively) and incubated overnight at 4°C for PvAMA-1 and at 37°C for peptides. Subsequently, the plates were washed and blocked (PBS 1x pH 7.2 + 3% BSA). All samples were diluted 1:100 and evaluated for total IgG using peroxidase-conjugated anti-human IgG antibodies at a concentration of 1:10.000 (Catalog No. W4031) (Promega Corporation). Monoclonal antibody binding was detected with OPD substrate tablets (Thermo Fisher Scientific, USA). The cut-off value was obtained by testing 15 different negative control sera from individuals never exposed to malaria from Belo Horizonte, Brazil. The mean optical density value at 492 nm ± 3 SD (VersaMaxTM Microplate Reader) for duplicate determinations in negative sera was used as the cut-off value for different peptides.
Opd substrate tablets
Manufactured by Thermo Fisher Scientific
Sourced in United States
OPD Substrate Tablets are a laboratory reagent used in enzyme-linked immunosorbent assay (ELISA) procedures. The tablets contain o-Phenylenediamine dihydrochloride (OPD), which is an enzyme substrate that produces a colorimetric reaction when cleaved by the enzyme horseradish peroxidase (HRP). This color change can be measured spectrophotometrically to quantify the presence of the target analyte in the ELISA.
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Lab products found in correlation
Tween 20, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Synergy 2 plate reader, by Agilent Technologies (1 mentions)
Goat anti human igg fc, by Fortis Life Sciences (1 mentions)
Peroxidase affinipure donkey anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Igg3 clone hp 6050, by Merck Group (1 mentions)
Igg4 clone hp6025, by Merck Group (1 mentions)
5 protocols using opd substrate tablets
1
Quantifying anti-PvAMA-1 antibodies by ELISA
2
ELISA for Quantifying IgG Antibodies
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For quantification of transfection supernatants and sera, ThermoFisher MaxiSorp 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 5 µg mL−1 goat anti-human IgG-Fc (Bethyl Laboratories, Montgomery, TX, USA) overnight at 4 °C. The following day, each plate was washed with PBS (Corning Inc., Corning, NY, USA) + 0.01% Tween-20 (ThermoFisher, Waltham, MA, USA) (PBS-T) four times (4×). Plates were then blocked with 5% milk in PBS for 2 h at RT. Upon completion of blocking, plates were washed again 4× with PBS-T and samples diluted in 1% NCS in PBS-T were transferred to plates for a 1 h incubation at RT. A standard curve was generated using purified human IgGκ or IgGλ (Bethyl Laboratories, Montgomery, TX, USA). Plates were subsequently washed and goat anti-human IgGκ or IgGλ HRP-conjugated secondary antibody (Bethyl Laboratories) was diluted to 1:10,000 and transferred onto plates for 1 h at RT. After secondary incubation, plates were washed 4× and developed using OPD Substrate Tablets (Thermo Scientific) for 10 min in the dark and stopped with 2 N H2SO4. The Biotek Synergy 2 plate reader was used to read absorbance at 450 nm.
3
Quantifying SARS-CoV-2 Nucleocapsid Protein
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Vero A/T cells were prepared as in Section 2.3 and infected with SARS-CoV-2 (England-2) at an MOI of 5 for 30 min on a platform rocker at 37 °C, 5% CO2. Cells were subsequently treated with 6.4 mg of product, as in Section 2.3 . At 24 and 48 hpi, supernatant was removed and cells were fixed via the addition of 0.5 mL/well 4% (v/v) formaldehyde for 30 min. Cells were washed twice with PBS and 250 µL/well of NP-40 was added for 15 min to permeabilise cells. Cells were washed once with PBS-Tween (PBST) and blocked in 3% milk for 1 h in the dark. Block was removed and cells were stained with 250 µL/well of anti-nucleocapsid (N) primary antibody (SARS-CoV-2 Nucleocapsid Protein (1C7) Monoclonal Antibody; bsm-441411M; Generon, Slough, UK) and diluted in 1% milk in PBST for 1 h in the dark. Cells were washed once with PBST and 250 µL/well of secondary antibody (Peroxidase AffiniPure Donkey Anti-Mouse IgG (H + L); Jackson ImmunoResearch Europe Ltd., Ely, UK) was added in 1% milk in PBST for 1 h in the dark. Cells were washed once with PBST and levels of N were determined by the OD at 492 nm following the addition of OPD substrate as per manufacturers’ instructions (OPD Substrate Tablets; ThermoFisher Scientific, Waltham, MA, USA).
4
Enzyme-Linked Immunosorbent Assay for HEV
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Microtiter plates (Greiner 96-well flat bottom) were coated with 100 µL/well of serial dilutions of purified protein in duplicate wells (100 ng/well to 12.5 ng/well) in PBS (pH 7.4) and incubated overnight at 4 °C. After three washes with PBST, plates were incubated with 200 µL/well of blocking solution (PBST 5% (w/v) dry milk) and incubated for 1 h at room temperature. Then, 100 µL anti-HEV IgG positive and negative swine serum diluted 1:100 in blocking buffer was added and incubate for 1 h at 37 °C. Plates were washed again with PBST before the addition of 100 µL/well of an HRP-conjugated anti-swine secondary antibody 1:10,000 (Thermo Fisher Scientific, Waltham, MA, USA). After incubation with the secondary antibody, plate wells were washed three times before adding 50 µL/well of the substrate solution of OPD Substrate Tablets (Thermo Fisher Scientific, Waltham, MA, USA). Plates were incubated, in the dark, at room temperature from 10 to 20 min, and then the reaction was stopped by the addition of 50 µL/well of 1M H2SO4, and the plates were read at 492 nm in a plate reader Epoch Microplate Spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). Previously characterized negative swine sera by using commercial kits from PrioCheck HEV Ab (Thermo Fisher Scientific, Waltham, MA, USA) were used as controls.
5
ELISA for Human IgG Subclasses
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The ELISA to detect the IgG subclasses was performed as previously described [10 (link)]. The sera were diluted 1:100 and evaluated for each IgG subclass using mouse monoclonal antibodies to human IgG subclasses (IgG1 clone 8c/6-39; IgG2 clone HP-6014; IgG3 clone HP-6050; IgG4 clone HP-6025) (Sigma, St. Louis, MO) according to the manufacturer instructions. Specifically, the secondary antibody dilutions were 1:1.000, 1:15.000, 1:40.000 and 1:60.000 for IgG 1, IgG 2, IgG 3 and IgG 4 respectively. The cut-off value was obtained by testing 6 different negative control sera from individuals never exposed to malaria from Belo Horizonte, Brazil. Monoclonal antibody binding was detected with OPD substrate tablets (Thermo Fisher Scientific, USA). The mean optical density value at 492 nm±3 SD (VersaMaxTM Microplate Reader) for duplicate determinations in negative sera was used as the cut-off value for different subclasses and peptides.
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