Briefly, 50 μL of full-length spike protein at 2 μg/mL in TBS pH 7.4 was coated in the 96-well, high-binding microtiter plate (Greiner Bio-One cat # 655061) for 1 hour at 37°C. The coating solution was removed, then 100 μL of blocking solution (3% milk in TBST), was added for 1 hour at 37°C. Serum samples were diluted at 1:40, or serially diluted (1:100 – 1:8100), in the blocking solution. The blocking solution was removed, then 50 μL of diluted serum was added to the plate and incubated for 1 hour at 37°C. The plate was washed three times using wash buffer (TBS containing 0.2% Tween 20), then 50 μL of horseradish peroxidase-conjugated secondary Goat Anti-Human secondary antibody at 1:40,000 dilution in 3% milk was added for 1 hour at 37°C. For measuring isotype specific antibody, only the respective goat anti-human IgG, IgM, or IgA was used (Jackson ImmunoResearch). The plate was washed three times using wash buffer, then 50 μL of 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate (Sigma-Aldrich) was added to the plate, and absorbance was measured at 450 nm using a plate reader (Molecular Devices SpectraMax ABS Plus Absorbance ELISA Microplate Reader) after stopping the reaction with 50 μl of 1 N HCl.
Tmb liquid substrate
Manufactured by Merck Group
Sourced in United States
TMB liquid substrate is a chromogenic substrate used in enzyme-linked immunosorbent assay (ELISA) and other immunoassay techniques. It provides a blue color change upon reaction with the enzyme, which can be measured spectrophotometrically. The core function of TMB liquid substrate is to facilitate the detection and quantification of target analytes in these immunological assays.
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Lab products found in correlation
Hrp anti m13 monoclonal conjugate, by GE Healthcare (2 mentions)
Infinite f200 pro, by Tecan (2 mentions)
Goat anti mouse igg hrp, by Agilent Technologies (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Spectramax abs plus absorbance elisa microplate reader, by Molecular Devices (1 mentions)
Goat anti mouse igg hrp, by Enzo Life Sciences (1 mentions)
Non animal protein blocker, by G Biosciences (1 mentions)
Horseradish peroxidase conjugated secondary goat anti human secondary igg, by Jackson ImmunoResearch (1 mentions)
Maxisorp microtiter plate, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions)
Prism 7, by GraphPad (1 mentions)
Goat anti rabbit igg hrp, by Merck Group (1 mentions)
Horseradish peroxidase conjugated streptavidin, by BD (1 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions)
Sunrise plate reader, by Tecan (1 mentions)
Biotin conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
96 well maxisorp plates, by Thermo Fisher Scientific (1 mentions)
Hybond p pvdf membrane, by GE Healthcare (1 mentions)
24 protocols using tmb liquid substrate
1
SARS-CoV-2 Spike Protein ELISA
2
Quantifying DENV-2 Infection in Huh7 Cells
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Huh7 cells were infected with DENV-2 as mentioned above. The cells were lysed at 48 h post infection using RIPA buffer. Anti-dengue rabbit polyclonal antibody, ab26837 was diluted in 50 mM carbonate-bicarbonate buffer (1:300) and coated onto F 96 Maxisorp flat-bottom ELISA plates (Nunc-immuno, Thermo Fischer) and incubated overnight at 4 °C. Two wells were kept as only buffer blank without the antibody. Wells were blocked with 5% BSA and treated with cell lysates for 2 h before adding the detection antibody (anti-dengue mouse monoclonal ab9202). Secondary antibody was an HRP conjugated anti-mouse antibody. Wells were washed with 1X PBS thrice between each antibody addition and five times before adding the substrate. 100 μl of TMB liquid substrate (Sigma Aldrich) was added to each well and the reaction was stopped after 15 minutes using equal volumes of 1 M H2SO4. Absorbance was measured at 450 and 630 nm and the difference between the two absorbance readings were plotted as percentage values with DMSO treated infected sample taken as 100%.
3
ELISA for Zaire GPΔTM antibody detection
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ELISA plates were coated with 100 ng of Zaire GPΔTM in 0.1 M NaHCO3 (pH 9.6) per well overnight at 4°C. Plates were washed and blocked with 5% milk and 0.5% BSA in PBS containing 0.05% Tween 20. Mouse sera were serially diluted in blocking buffer and incubated on the ELISA plate for 1 hr at 37°C. After washing, goat anti-mouse IgG HRP (Enzo Life Sciences, Plymouth Meeting, PA, USA) was added for 1 hr at 37°C. Bound mAb was detected using TMB Liquid Substrate (Sigma) and stopped with 1N H2SO4. Spectrophotometric readings were performed at 450 nm with a 570 nm reference subtraction. Purified proteins were used for the standard curve.
4
Phage Library Screening for CD83 Binding
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Pools of purified phage from the library stock (Sheets), and the Round 1, Round 2, Round 3 and Round 4 amplified stocks from the CD83-GFP biopanning were tested for binding to soluble CD83 using ELISA. Nunc Maxisorp 96-well plates were coated with 5 μg/mL CD83 in PBS overnight at room temperature. The wells were washed three times with PBS and then blocked for 1 hr with MPBS. Phage pools (~1011 phage particles) were also blocked in MPBS. Dilutions of the blocked phage were then added to the CD83 coated plate and incubated for 1 hr at room temperature. The wells were washed three times with PBS containing 0.1% (v/v) Tween-20 (PBST), and then 200 μL secondary antibody added (1/5000 dilution of HRP/Anti-M13 Monoclonal Conjugate-GE Healthcare) and incubated for 1 hr at room temperature. The wells were washed three times with PBST, followed by addition of 100 μL of 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate (Sigma-Aldrich). The reaction was stopped after 10 mins using 100 μL 1 M sulphuric acid. Absorbance was measured at 450 nm using BioTek Powerwave HT Microplate Reader (Millenium Science).
5
Quantification of Serum Antibody Levels
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Blood was collected from the mice and serum was obtained from it by centrifugation. Total IgG, IgG1 and IgG2a serum antibody concentrations were measured by ELISA. Briefly, 5 mg/mL of OVA in PBS was coated onto 96 well-plates and then incubated at 4 °C overnight. After washing with PBST (PBS containing 0.1% Tween 20), 2% BSA in PBS was added as the blocking agent at 37 °C for 2 h. The serum was diluted in 2% BSA, followed by reaction at 37 °C for 2 h. The HRP-labelled anti-IgG1, IgG2a or IgG antibodies were reacted with the antigen at 37 °C for 2 h. TMB Liquid Substrate (Sigma-Aldrich, St. Louis, MO, USA) was added, the plates were incubated at 37 °C for 30 min, and the reaction was stopped by adding 2 M HCl. Antibody–antigen reactions were assessed by measuring their optical density at 450 nm.
6
Quantitative ELISA for Influenza HA
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For co-expression studies, to discriminate the expression of S139/1 and 9H10, both recognizing H3 epitopes, S139/1 was detected using the HA1 subunit and 9H10 was detected with H10N8. ELISA plates coated with 200 ng of H1N1 (A/WSN/33) HA, 50 ng of H3N2 (A/Aichi/2/1968) HA1 or HA subunit, or 200 ng of H10N8 (A/Jiangxi-Donghu/346/2013) HA (Sino Biologicals, Beijing, China) were blocked and incubated with serially diluted mouse serum. After washing, goat anti-mouse IgG HRP was added for 1 hr at 37°C. Bound mAb was detected using TMB Liquid Substrate (Sigma) and stopped with 1N H2SO4. Spectrophotometric readings were performed at 450 nm with a 570 nm reference subtraction. Purified proteins were used for the standard curve.
7
SARS-CoV-2 RBD Antibody ELISA Assay
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OC43 and NL63 RBD ELISA were carried out as previously described.80 (link),81 (link) Briefly, coating was performed by Streptavidin (Invitrogen) at 4 μg/mL in Tris-Buffered Saline (TBS) pH 7.4 for 1 h at 37°C followed by blocking with Non-Animal Protein-BLOCKER (GBiosciences). Then biotinylated spike RBD antigens for OC43 and NL63 were added at 1 μg/mL at 37°C for 1 h. All plasma samples were heat-inactivated before usage to minimize risk of residual virus in serum and then incubated at serial dilution followed by multiple washes and incubation with horseradish peroxidase-conjugated secondary Goat Anti-Human secondary IgG (Cat No: 109-035-008, Jackson ImmunoResearch) at 1:40,000 dilution in 3% milk at 37°C for 1 h. The resulting plate was washed and 3,3′,5,5’ -Tetramethylbenzidine (TMB) Liquid Substrate (Sigma-Aldrich) was added for optical density (OD) measurement at 405 nm after stopping the reaction with 50 μL of 1 N HCl.
8
Enzyme-Linked Immunosorbent Assay for Pfs230 and Pfs25
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Chimeric VLPs or monomeric recombinant proteins were coated onto Maxisorp microtiter plates (Nunc) at 1μg/ml in PBS and incubated overnight at 4°C. Plates were blocked with 1% casein in PBS (Sigma-Aldrich) for 2h at 37°C before primary antibodies were added (polyclonal mouse anti-Pfs230 or anti-Pfs25 antibodies; or rabbit antibodies against Pfs230 or Pfs25). Secondary HRP-conjugated antibodies (polyclonal goat anti-mouse IgG at 1/1000 or anti-rabbit IgG at 1/2500 from Millipore) were used to detect antibody binding. Colour detection was developed using ABTS or TMB liquid substrate (Sigma-Aldrich), which was subsequently stopped using 1% SDS (for ABTS) or 1M sulphuric acid (for TMB). PBS was used as a negative control and plates were washed thrice using PBS with 0.05% Tween in between antibody incubation steps. The level of antibody binding was measured as optical density at 405nm (for ABTS) or 450nm (for TMB).
9
Quantification of Antibody Binding Kinetics
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ELISAs were performed as previously described [77 (link)]. Briefly, ELISA plates were coated with anti-human IgG1 Fc antiserum (The Binding Site, cat. #AU004) or UG37 gp140 (5 µg/ml in PBS) (CFAR, USA) and blocked with PBS + 5% skimmed milk powder. N6 antibody was titrated two-fold, along with a positive control (500 ng/ml) in PBS + 5% skimmed milk powder and incubated for a minimum of two hours at 37 °C. Primary antibody was anti-human IgG1 light chain kappa antiserum conjugated with HRP (The Binding Site, cat. #AP015), diluted in PBS + 5% skimmed milk powder. Developing solution (3,3 5,5-Tetramethylbenzidine (TMB) Liquid Substrate, Sigma, cat. #T0440) was added and briefly incubated until colour development was complete before stopping with 2 N H2SO4. Plates were read on the Tecan Infinite F200 Pro. Data were analysed and concentrations calculated with Graphpad Prism 7 using the Michaelis Menton equation for line fitting.
10
Quantifying Antibody Complement Fixation
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The capacity of human antibodies to fix complement C1q was measured using previously optimised methods19 (link),20 (link). Antigen coating and blocking were performed as above for standard ELISA. After incubation with human antibody samples, purified human C1q (10 μg/ml; Millipore) was added as a source of complement for 30 min at room temperature, followed by a rabbit anti-C1q (1/2000; in-house, previously generated and validated69 (link)) detection antibody and finally, a goat anti-rabbit IgG HRP (1/2500; Millipore cat# AB97051) for 1 h at room temperature. The level of C1q-fixation was developed using TMB liquid substrate (Sigma-Aldrich), reactivity was stopped using 1 M sulfuric acid and measured as optical density at 450 nm.
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