High-binding 96 well ELISA plates (Corning #9018) were coated with 1 µg/mL RBD in PBS ×1 overnight at 4 °C. The following day, the coating was discarded, the wells were washed with “washing buffer” (PBS ×1 and 0.05% Tween20) and were blocked for 2 h at RT with 200 µL of “blocking buffer” (PBS ×1, 3% BSA (MP Biomedicals), 20 mM EDTA, and 0.05% Tween20 (Sigma)). Antibodies were added at a starting concentration of 4 µg/mL, and seven additional 4-fold dilutions in blocking buffer, and incubated for 1 h at RT. The plates were then washed 3 times with washing buffer before adding secondary, anti-human IgG (Jackson ImmmunoResearch) antibody conjugated to horseradish peroxidase (HRP) diluted 1:5000 in blocking buffer, and incubation for 1 h at RT. Following four additional washes, 100 µL of TMB (Abcam) was added to each well and the absorbance at 650 nm was read after 20 min (BioTek 800 TS).
3 3 5 5 tetramethylbenzidine (tmb)
Manufactured by Abcam
Sourced in United Kingdom
TMB (3,3',5,5'-Tetramethylbenzidine) is a chromogenic substrate used in enzyme-linked immunosorbent assays (ELISA) and other immunoassays. It is a colorless solution that turns blue-green upon oxidation, which can be detected and quantified using a spectrophotometer. TMB is commonly used as a sensitive detection reagent in various applications where a colorimetric read-out is required.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin hrp conjugate, by Thermo Fisher Scientific (3 mentions)
Tmb stop solution, by Abcam (3 mentions)
Fibrinogen, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Bovine serum albumin (bsa), by MP Biomedicals (2 mentions)
Deoxycholic acid, by Merck Group (2 mentions)
Cremophor el, by Merck Group (2 mentions)
Purified αvβ3 and anti αvβ3 conjugated with biotin, by Bioss Antibodies (2 mentions)
Cy5 nhs dye, by Lumiprobe (2 mentions)
Formic acid, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Ethyl acetate, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Maxisorb elisa plate, by Thermo Fisher Scientific (1 mentions)
Anti human igg hrp, by Jackson ImmunoResearch (1 mentions)
Maxisorb, by Thermo Fisher Scientific (1 mentions)
Rabbit anti goat igg conjugated with horseradish peroxidase, by Southern Biotech (1 mentions)
Oxyblot protein oxidation detection kit, by Merck Group (1 mentions)
Synergy h1 spectrophotometer, by Agilent Technologies (1 mentions)
Duoset elisa assay, by R&D Systems (1 mentions)
14 protocols using 3 3 5 5 tetramethylbenzidine (tmb)
1
SARS-CoV-2 RBD-specific Antibody ELISA
2
Quantification of Gut Bacteria Antibodies
3
Sandwich ELISA for Flavivirus NS1 and Cytokine Quantification
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For NS1 measurement, sandwich ELISA was used. Coating anti-NS1 antibody clone 4C4 (Biosan, Novosibirsk, Russia) in carbonate buffer were sorbed onto a high-binding 96-well plate (Grainer). Blocking was performed in PBS with 0.05% Tween-20 and 5% BSA. Detection of NS1 was performed with anti-NS1 clone 29G9 (Biosan, Novosibirsk, Russia) conjugated to biotin followed by incubation with streptavidin-HRP conjugate. TMB (Abcam, Cambridge, UK) was use as a substrate.
Cytokines levels (IFN-γ, IL-10, TNF-α and IL-6) in mice sera were measured by ELISA kits (Invitrogen) and C3a in mice sera was measured by an ELISA kit (Cloud-Clone Corp., Wuhan, China) according to the manufacturer’s instructions. For tests, mice sera were pooled (4–5 mice) and run in duplicate.
Cytokines levels (IFN-γ, IL-10, TNF-α and IL-6) in mice sera were measured by ELISA kits (Invitrogen) and C3a in mice sera was measured by an ELISA kit (Cloud-Clone Corp., Wuhan, China) according to the manufacturer’s instructions. For tests, mice sera were pooled (4–5 mice) and run in duplicate.
4
Polyclonal IgG Response to OMVs
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The level of polyclonal IgG isotype to OMVs was determined by ELISA using a 96-well ELISA microplate (MaxiSorb; Nunc, Wiesbaden, Germany). Each well was coated with 1 µg of OMVs in carbonate/bicarbonate buffer and blocked with 5% (w/v) skim milk in PBS for 2 h and then washed with PBS. Serial dilutions of serum obtained from all treated groups were added to wells and incubated for 2 h at 37 °C. After washing, the anti-mouse antibody conjugated with HRP (Biolegend, San Diego, USA) was added to the wells and incubated for 2 h at 37 °C. The chromogenic HRP substrate TMB (Abcam, United Kingdom) was added and the reaction was stopped by 3N sulfuric acid, then the absorbance was measured at 450 nm. The absorbance was measured at 450 nm to determine the levels of polyclonal IgG isotype to OMVs in each sample. This analysis allowed us to evaluate the humoral immune response and the production of specific antibodies against the OMVs. The data presented throughout the study are mean values obtained from three separate readings and P value < 0.05 were statistically considered significant.
5
Quantifying Antibody Response to Vaccinations
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Serum was collected from immunized mice ten days after each immunization (Fig. 1 a). In the experiment using a single dose of DTaP, mice were bled two and four weeks after DTaP immunization (Supplementary Fig. S1b). The specific IgG titers were determined by ELISA. Immunoplates were coated with inactivated DT (1 µg/mL), TT (1 µg/mL), PT (1 μg/mL) or wP (105 CFU/mL). Goat anti-mouse IgG (1:10,000; Cat. No.1036–01, Southern Biotech, AL, USA) was used as a secondary antibody and rabbit anti-goat IgG conjugated with horseradish peroxidase (1:20,000; Cat. No. 6160–05, Southern Biotech, AL, USA) as the tertiary antibody. Colorimetric development was performed using 3,3′,5,5′-tetramethylbenzidine (TMB, DTaP experiments; Abcam), or in citrate buffer with H2O2 and o-phenylenediamine dihydrochloride (OPD, DTwP experiments). Standard curves for the determination of IgG concentration were established using either the mouse reference serum from Bethyl Technologies (DTaP experiments) or recombinant IgG (DTwP experiments).
6
Protein Carbonylation Quantification
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Diluted plasma (25x in PBS) and TRIzol-extracted heart proteins were derivatized with 4 mM DNPH (in 2 M HCl) as per manufacturer’s instructions using Oxyblot Protein Oxidation Detection Kit (Merck Millipore, MA, USA). We left 2 µg of proteins in 200 µL 100 mM pH 9.6 bicarbonate buffer to bind to the ELISA plate overnight at 4 °C. The plate was washed 5 times with PBS and blocked with 100 µL Pierce Protein-Free T20 buffer for 1 h. Plate was washed with PBS-T (0.1% v/v Tween-20) and incubated with 100 µL 1:200 primary antibody (Oxyblot kit) for 2 h with shaking. Plate was then washed and incubated with 100 µL 1:400 secondary antibody (Oxyblot kit) for 1 h. Plate was washed and 100 µL of TMB (Abcam, Cambridge, UK) was added. The reaction was stopped with 100 µL of 2 M H2SO4 and absorbance was determined at 450 nm using the Synergy H1 spectrophotometer (BioTek; Agilent Technologies, Santa Clara, CA, USA). Carbonylation levels were quantified against a standard curve.
7
CXCL10/IP-10 Quantification in A549 Cells
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A DuoSetELISA assay (R&D) was used to detect the presence of human CXCL10/IP-10 in the supernatants of infected or stimulated A549 cells using TMB (Abcam) as the substrate solution and 0.3 M H2SO4 as the stop solution.
8
IgG Antibody Quantification Against Malaria Proteins
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Plasma IgG levels against four recombinant P. falciparum proteins and a crude parasite lysate were measured using indirect ELISA, as previously described (Lopez-Perez et al., 2021 (link)). Briefly, 96-well flat-bottom microtitre plates (Nunc MaxiSorp; Thermo Fisher Scientific) were coated with 50 μL of recombinant protein (0.5 μg/mL) or crude antigen lysate (5 × 105 IEs/mL) in PBS and incubated overnight at 4 °C. After blocking with 1% BSA/PBS, plasma samples (1:300) were added in duplicate, followed by horseradish peroxidase-conjugated rabbit anti-human IgG (1:3000; Dako, Denmark). Bound plasma antibodies were detected by adding TMB (abcam, UK), stopping the reaction with 0.2 M H2SO4. The optical density (OD) was read at 450 nm (Varioskan LUX; Thermo Fisher, USA). The specific antibody levels were calculated in arbitrary units (AU), using the equation [(ODSAMPLE - ODBLANK)/ (ODPOSITIVE CONTROL - ODBLANK)] × 100 (Guitard et al., 2008 (link)). Plasma samples from malaria-unexposed Danish individuals and malaria-exposed Ghanaian children were included as negative and positive controls, respectively.
9
SARS-CoV-2 RBD Serum ELISA Protocol
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For serum ELISA, high-binding 96-well ELISA plates (Corning, c9018) were coated with 1 μg/mL of RBD antigen in PBS 1× and held overnight at 4°C. The following day, the coating was discarded and washed with “wash buffer” containing PBS 1× and 0.05% Tween20, after which the plates were blocked for 2 hours at room temperature (RT) with 200 μL of “blocking buffer” containing PBS 1×, 3% BSA (MP Biomedicals, Santa Ana, Calif), 20 mM EDTA, and 0.05% Tween20 (Sigma-Aldrich, St Louis, Mo). Plasma samples were diluted 4-fold in blocking buffer, starting from 1:100 with 7 consecutive dilutions (1:100, 1:400, 1:1600, 1:6400, etc), and incubated for 1 hour at RT. The plates were then washed 3 times with washing buffer before the addition of secondary anti-IgG antibodies (Jackson ImmmunoResearch, West Grove, Pa, 109-035-088) conjugated to horseradish peroxidase diluted 1:5000 in blocking buffer and incubated for 45 minutes at RT. Following 4 additional washes with wash buffer and a final wash with PBS 1×, 100 μL of 3,3’,5,5’-tetramethylbenzidine (Abcam, Cambridge, United Kingdom) diluted 1:2 in double deionized water (DDW) was added to each well; absorbance was read after 25 minutes at 650 nm (using a BioTek 800 TS absorbance reader). Positive and negative controls were added to each plate, and the signal was normalized to the controls for each plate.
10
Quantifying Serum HMGB1 Levels
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Serum samples were collected before surgery in gold top BD vacutainers and were analysed for CRP as described.5 High mobility group box 1 was measured by ELISA. Briefly, sample and antigen prepared in 1× phosphate‐buffered saline were added to a 96‐well plate in duplicate and incubated at 4°C overnight. The primary antibody (SAB1403925; Sigma‐Aldrich Canada, Oakville, Canada) was added based on manufacturer's instructions at 3 mg/mL and incubated for 1 h at 37°C. The plate was washed and incubated for 30 min at 37°C with a 1:10 000 dilution of horseradish peroxidase‐conjugated secondary antibody. The plate was washed a final time in the chromogen 3,3′,5,5′‐tetramethylbenzidine (Abcam Inc., Toronto, Canada). Absorbance was read at 450 nm.
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