Serum was incubated with a final concentration of 0.5% Triton X-100 and 0.5 mg/ml RNase A to inactivate any potential SARS-CoV-2. SARS-CoV-2 stabilized spike glycoprotein (BEI Resources, NR-53524) was coated at a concentration of 2 μg/ml in carbonate buffer on 96-well MaxiSorp plates (Thermo Fisher) overnight at 4°C. Plates were blocked with 1% BSA in PBS for 1 h at room temperature. Serum samples were serially diluted in 1% BSA in PBS and incubated in plates for 2 h at room temperature. Antibody isotypes were detected with anti-mouse IgM-HRP or anti-mouse IgG Fc-HRP (Southern Biotech) by incubation for 1 h at room temperature. The plates were developed with TMB stabilized chromogen (Thermo Fisher), stopped with 3 N hydrochloric acid, and read at 450 nm on a microplate reader.
Tmb stabilized chromogen
Manufactured by Thermo Fisher Scientific
TMB-stabilized chromogen is a laboratory reagent used in colorimetric assays. It serves as a substrate for enzymes, such as horseradish peroxidase (HRP), resulting in a color change that can be measured spectrophotometrically. The stabilized formulation helps maintain the chromogen's stability and performance in various experimental conditions.
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Lab products found in correlation
Maxisorp plate, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse ige hrp conjugate, by Southern Biotech (2 mentions)
Anti mouse igm hrp, by Southern Biotech (1 mentions)
Goat anti mouse igg fc hrp, by Jackson ImmunoResearch (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Asys expert 96 elisa reader, by Harvard Bioscience (1 mentions)
Goat anti mouse ige, by Southern Biotech (1 mentions)
6 protocols using tmb stabilized chromogen
1
SARS-CoV-2 Spike Protein Antibody Assay
2
Whole Cell and Surface ELISA Assay
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Cells were seeded on 96-well plates simultaneously with each corresponding HTRF signaling assay, as described above. Twenty-four hours after seeding, cells were washed with HBSS (+Ca2+/+Mg2+) and fixed with 4% PFA for 20 min at room temperature. Whole cell ELISAs were performed under permeabilizing conditions in the presence of 0.1% Triton X-100 throughout, while surface ELISAs were performed under non-permeabilizing conditions in the absence of any detergent. The fixed cells were first blocked in HEK293T medium containing 10% FBS for 1 h at room temperature, and then incubated with primary antibodies diluted in HEK293T medium at concentrations indicated in the key resources table for 1 h at room temperature. After 3 thorough washes with PBS, cells were incubated with horseradish peroxidase-conjugated secondary antibodies diluted 1:500 in HEK293T medium for 1 h at room temperature. After 3 additional washes with PBS, cells were overlayed with TMB-stabilized chromogen for 10 min (Thermo Fisher, Cat# SB02) followed by an equal volume of acidic stop solution (Thermo Fisher, Cat# SB04). Absorbance was read at 450 nm using a Synergy H1 multi-mode plate reader (Biotek). Non-specific background was determined using “secondary-antibody only” control wells and subtracted from sample wells.
3
Whole Cell and Surface ELISA Assay
4
Thy1.1 Antibody Binding ELISA in CHO Cells
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An amount of 24 μg of pcDNA3.1(+)-Thy1.1 plasmid was transfected into CHO.K1 cells (10 mm Petri dish, 80% confluent) using the lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's recommendation. The following day, cells were plated into a 96-well plate (5 × 105 cells/well). Two days after transfection, an antibody binding ELISA was performed. The cell supernatant was discarded, and either anti-Thy1.1 IgE, IgG2a, or IgG1 were added in serial dilutions. After incubation at room temperature for 1 hour, goat anti-mouse IgE-HRP conjugate (Southern Biotech, 1:4,000) or goat anti-mouse IgG Fc-HRP (Jackson Immuno Research 1:5,000) in 1:1 1% BSA phosphate-buffered saline with Tween 20 (PBS/PBST) were added for 45 minutes at room temperature. Immunoreactivity was visualized with TMB Stabilized Chromogen (Invitrogen). Reactions were stopped after 15 minutes with 0.5 mol/L H2SO4, and absorbances were read at 450 and 620 nm. All samples were tested in duplicate.
5
SARS-CoV-2 Spike Protein IgG ELISA
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96‐well ELISA plates (Nunc MaxiSorp) were coated with freshly prepared SARS‐CoV‐2 S trimers or the RBD (100 µl of 1 ng/µl) in PBS for 15 h at 4°C. Plates were washed six times with PBS–Tween‐20 (0.05%) and blocked using PBS‐5% no‐fat milk (Sigma). Human serum samples were thawed at room temperature, diluted, vortexed and incubated in blocking buffer for 1 h (4°C) before plating to block non‐specific binding. Serum samples were incubated for 15 h at 4°C to allow low‐affinity binding interactions, before washing as before. Secondary HRP‐conjugated anti‐human antibodies were diluted in blocking buffer and incubated with samples for 1 hour at 4°C. Plates were washed a final time before development with TMB Stabilized Chromogen kept at 4°C (Invitrogen). The reaction was stopped using 1 M sulphuric acid and optical density (OD) values were measured at 450 nm using an Asys Expert 96 ELISA reader (Biochrom Ltd.). Secondary antibodies (from Southern Biotech) and dilutions used were as follows: goat anti‐human IgG (2014‐05) at 1:10,000. All assays were developed for their fixed time, and negative control samples were run alongside test samples in all assays. Anti‐SARS‐CoV‐2 S and RBD IgG were detectable at up to 1:20,000 serum dilution using this assay [8 (link)], and all study samples were here run at 1:100 dilution.
6
Quantification of Mouse IgE Antibodies
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96-well MaxiSorp plate (ThermoFisher Scientific) was coated with 0.15μg/ml of goat antimouse IgE in PBS (Southern Biotech, 100μL/well) overnight at 4°C. Blocking was done with 1% BSA in PBS for 1 h at 37°C. Next, supernatants containing IgE antibodies were added in serial dilutions. Initially, before serial dilutions, 2C6 hybridoma supernatant was diluted 4x, whereas FreeStyle293 supernatant containing anti-Thy1.1. IgE was initially diluted 2x with cell culture medium. Mouse IgG1 kappa was used as negative control (clone P3.6.2.8.1). Mouse IgE (Bio-Rad cat# PMP68) was used for the standard curve. The incubation was performed at room temperature for 1 h. Finally, goat anti-mouse IgE-HRP conjugate (Southern Biotech, 1:4000 in 1:1 1% BSA PBS/PBST) was added for 45 min at room temperature. Immunoreactivity was visualized with TMB Stabilized Chromogen (Invitrogen). The reaction was stopped after 15 min with 0.5 M H2SO4 and absorbances were read at 450 nm and 620 nm.
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