Plates (96-well, Nunc Maxisorp; Thermo Fisher Scientific, Waltham, MA, USA) were coated with human Neuropilin-1-Fc (10 ng per well, Cat# 50-101-8343, Fisher, Hampton, NH) and incubated at room temperature overnight. The following day, the plates were washed and blocked with 3% BSA in PBS to minimize non-specific adsorptive binding to the plates. SARS-CoV2 Spike protein (S1 domain aa16–685, Cat# Z03485, Genscript, Piscataway, NJ) was added at concentrations ranging from 500 to 0.07 nM. As a negative control, some wells received PBS containing 3% BSA. The plates were incubated at room temperature with shaking for 3 h. Next, the plates were washed with PBS to eliminate unbound protein. Bound SARS-CoV2 Spike was detected by anti‐His probe HRP (Cat#15165; Thermo Fisher Scientific). Tetramethylbenzidine (Cat#DY999, R&D Systems, St. Louis, MO) was used as the colorimetric substrate. The optical density of each well was determined immediately, using a microplate reader (Multiskan Ascent; Thermo Fisher Scientific) set to 450 nm with a correction wavelength of 570 nm. Data were analysed by non-linear regression analysis using GraphPad Prism 8 (GraphPad, San Diego, CA, USA).
Tetramethylbenzidine
Manufactured by R&D Systems
Sourced in United States, Macao, United Kingdom
Tetramethylbenzidine is a chromogenic substrate used in various immunoassay and enzymatic detection applications. It undergoes an oxidation reaction catalyzed by peroxidase enzymes, resulting in a colored product that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Nunc maxisorp, by Thermo Fisher Scientific (6 mentions)
Multiskan ascent, by Thermo Fisher Scientific (4 mentions)
Sars cov 2 spike protein, by GenScript (3 mentions)
Anti his probe hrp, by Thermo Fisher Scientific (3 mentions)
Prism 8, by GraphPad (3 mentions)
Human neuropilin 1 fc, by Thermo Fisher Scientific (2 mentions)
Hydrogen peroxide, by R&D Systems (2 mentions)
Neuropilin 1 fc, by Thermo Fisher Scientific (2 mentions)
Mouse anti human tgf β3, by R&D Systems (2 mentions)
Streptavidin horseradish peroxidase hrp, by R&D Systems (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Duoset enzyme linked immunosorbent assay, by R&D Systems (1 mentions)
Multiskan fc plate reader, by MultiSciences Biotech (1 mentions)
Affinipure donkey anti human igg h l, by Jackson ImmunoResearch (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Streptavidin conjugated horseradish peroxidase, by R&D Systems (1 mentions)
Ha tag antibody, by Proteintech (1 mentions)
Tnf α antibody, by R&D Systems (1 mentions)
Duoset elisa development kit, by R&D Systems (1 mentions)
Omicron ba 1, by ACROBiosystems (1 mentions)
21 protocols using tetramethylbenzidine
1
SARS-CoV-2 Spike Protein Binding Assay
2
Enzyme-Linked Immunosorbent Assay for IFN-γ and TNF-α
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DuoSet® Enzyme-linked immunosorbent assays (ELISA) were purchased from R&D systems, UK. Human IFN gamma (DY285B), and human TNF alpha (DY210) were used. ELISA were performed following manufacturer’s instructions. Nunc MaxiSorp™ immunoplates were coated with recommended dilutions of capture antibody, blocked with 0.2 µm filtered 1% w/v BSA (Sigma, UK, A7030) (reagent diluent) for 1 h, before incubation of samples and standards diluted in reagent diluent for 2 h. Recommended concentrations of detection antibody diluted in reagent diluent were added for 2 h in sealed plates. Streptavidin horse radish peroxidase (HRP) diluted in reagent diluent was incubated for 20 min before washing and addition of substrate solution (1:1 mix hydrogen peroxide and tetramethylbenzidine, R&D Systems, UK) for 20 min. Colour development was halted with 2N sulphuric acid. Optical density was determined using a Multiskan™ FC plate reader by subtracting readings at 540 nm from 450 nm to account for any imperfections in the plate.
3
Quantitative ELISA Protocol for Antibody Detection
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MaxiSorp ELISA plates (Nunc Serving Life Science, Denmark) were coated with antigens (5 ng/well) in 0.1 M NaCO3 buffer (pH 9.6) and incubated overnight at 4°C. The plates were washed with phosphate-buffered saline (PBS) containing 0.05% (vol/vol) Tween 20 and blocked with 3% (wt/vol) bovine serum albumin (BSA) at room temperature (RT) for 1 h. After additional washing steps, 50 μL of 1:800 diluted serum samples were added to each well and incubated for 45 min at RT. After washing, 50 μL/well of peroxidase-conjugated AffiniPure donkey anti-human IgG (H+L) (Jackson Immunoresearch Laboratories, USA) (1:10,000) was added and incubated at RT for 1 h. The reaction was activated with hydrogen peroxide, developed with tetramethylbenzidine (R&D Systems, USA), and stopped with 2 N sulfuric acid after 10 min of incubation in the dark. Optical density (OD) was measured at 450/570 nm using an ELISA microreader (FLUOstar Omega, BMG Labtech). All ELISAs have been repeated twice with serum samples in duplicate.
4
HA-SDC4 Capture and Detection
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Microplates (96-well; R&D Systems) were coated overnight with 5 μg/ml HA-tag antibody (1:1000; 51064, Proteintech) in PBS at RT. The plates were blocked in PBS containing 0.05% Tween-20 (0.05% PBS-T) with 1% BSA for 1 h at RT. HA-SDC4 was captured by 2 h incubation of 100 μl cell lysate (1 μg/μl) at RT. Unbound material was removed by extensive washing with 0.05% PBS-T. Wells were incubated with streptavidin-conjugated horseradish peroxidase (R&D Systems) in 0.05% PBS-T containing 1% BSA for 1 h at RT. Following further washing, biotinylated SCD4 was detected with tetramethylbenzidine (R&D Systems).
5
Quantifying Microglial Cytokine Profiles
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Cytokine concentrations were assessed in supernatant samples from cultured microglia [21 (link)]. Briefly, 96-well plates (Nunc-Immuno plate with Maxisorp surface, Denmark) were coated with capture antibody (rat anti-mouse IL-1β or TNFα antibody (R&D Systems, USA) and incubated (overnight, room temperature). Duplicate samples or standards (50 μl/well) were added and plates were incubated (24 h, 4 °C) and washed before addition of detection antibody (biotinylated goat anti-mouse; 1 h, room temperature). Plates were washed, incubated with streptavidin-horseradish peroxidase conjugate (20 min, room temperature) and washed before addition of substrate solution (50 μl; 1:1 hydrogen peroxide (H2O2): tetramethylbenzidine; R&D Systems, USA). After colour development, the reaction was stopped by adding 1 M sulphuric acid (H2SO4; 25 μl) and plates were read at 450 nm (Labsystem Multiskan RC, UK). Cytokine mRNA expression was assessed in harvested microglia by RT-PCR as previously described [22 (link)].
6
Quantifying Cytokine Levels in Mouse Tissue
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Swiss mice had the left ear sample removed on day 4 for the analysis of cytokines. The samples were stored at −70°C until required for the assay. The collected tissue was homogenized and processed [40 ]. The concentrations of IL-6 and neutrophil KC were determined using an enzyme-linked immunosorbent assay (ELISA) [41 (link)]. Briefly, microtiter plates were coated with an antibody against mouse IL-6 and neutrophil KC (4 μg/mL, DuoSet ELISA Development kit, R&D Systems) overnight at 4°C. After blocking the plates, the sample and standard were added at various dilutions in duplicate and incubated at 4°C for 2 h. The plates were washed three times with buffer. After washing the plates, biotinylated goat anti-mouse (diluted 1 : 1000 with assay buffer 1% BSA, R&D Systems, USA) was added to the wells. After a further incubation at room temperature for 2 h, the plates were washed, and 100 μL of streptavidin-HRP diluted 1 : 200 was added. To the plate, 100 μL of substrate solution (1 : 1 mixture of H2O2 and tetramethylbenzidine; R&D Systems, USA) was added, and the plate was incubated in the dark at room temperature for 20 min. The enzyme reaction was stopped with H2SO42 N, and the absorbance was measured at 450 nm. The results are expressed as pg/g of tissue and reported as mean ± SEM.
7
Quantification of Activated Monkey C3
8
SARS-CoV-2 S Protein Antibody Assay
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We diluted the antigens for antibody detection in PBS and coated the plates (Nunc MaxiSorp, Thermofisher Scientific) with 250 ng/mL WT (AcroBiosystems) or Omicron BA.1 (AcroBiosystems) SARS-CoV-2 S protein for IgG and IgG avidity assessment and 500 ng/mL ancestral (Sinobiological) or Omicron BA.1 (AcroBiosystems) S for FcγRIIIa-binding detection. ORF8 protein of 300 ng/mL was coated at 37 for 2 hours. The plates were blocked with 1% FBS in PBS for 1 hour, followed by incubation with 1:100 HI sera diluted in 0.05% Tween-20/0.1% FBS in PBS for 2 hours for IgG detection, and 1:50 for 1 hour at 37 for FcγRIIIa-binding detection, prior to rinsing. For avidity, plates with 8M were washed with urea 3 times. IgG was measured after 2 hours of incubation period with anti-IgG-HRP (1:5000; G18-145, BD), HRP revealed with addition of stabilized hydrogen peroxide and tetramethylbenzidine (R&D systems) for 20 minutes. The reaction was terminated with 2N H2SO4, which was then analyzed at 450 nm wavelength with an absorbance microplate reader (Tecan Life Sciences). Similarly, FcγRIIIa-binding antibodies were assessed after incubation with biotinylated FcγRIIIa-V158 at 100 ng/mL for 1 hour at 37 after streptavidin-HRP (1:10000, Pierce).
9
Quantification of Mucin Concentrations
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Mucin concentrations were quantified using an enzyme‐linked lectin assay based on the protocol previously described (McCoy Jr. et al.,1984 ). Briefly, samples were sonicated for 10 min and added to high‐bind elisa plates coated in lectin from Triculum vulgaris. A standard curve was prepared using serially diluted mucin of known concentration from bovine submaxillary gland (Cat #M3895, Sigma‐Aldlich, Dorset, UK). Following incubation at 37°C for 30 min, the plates were washed three times with a wash buffer before the addition of detection reagent (containing HRP‐conjugated Glycin max soybean lectin) for a further 30 min at 37°C. Following a further wash cycle, a substrate solution containing H2O2 and tetramethylbenzidine (R&D Systems, Minneapolis, MN, USA) was added and allowed to develop for 5 min. The reaction was terminated using 2 N H2SO4 and absorbance read immediately at 450 nM with 570 nM as reference. Standard curves were created using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA), and these were used to calculate the concentration of mucin in all samples.
10
Quantifying Mucin Levels via ELLA
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Mucin concentrations were quantified using an enzyme-linked lectin assay (ELLA) based on the protocol previously described (29 (link)). Briefly, samples were sonicated for 10 min and added to high-bind ELISA plates coated in lectin from Triculum vulgaris. A standard curve was prepared using serially diluted mucin of known concentration from bovine submaxillary gland. Following incubation at 37°C for 30 min, the plates were washed three times with a wash buffer before the addition of detection reagent (containing HRP-conjugated Glycine max soybean lectin) for a further 30 min at 37°C. Following a further wash cycle, a substrate solution containing H2O2 and tetramethylbenzidine (R&D Systems, Minneapolis, MN) was added and allowed to develop for 5 min. The reaction was terminated using 2 M H2SO4, absorbance was read immediately at 450 nm with 570 nm as reference. Standard curves were created using GraphPad Prism and these were used to calculate the concentration of mucin in all samples.
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