The simulated neutralization assay was performed as described by Abe et al. [35 (link)] with minor modifications. A Maxisorp ELISA microplate (Thermo Fisher Scientific, Waltham, MA, USA) was coated for 12 h at 4 °C with RBD (2 μg/mL) diluted in 50 mM carbonate buffer at pH 9.6. The plates were washed with PBS and blocked with 0.05% PBS-Tween, 2% BSA, and 3% glucose. Thereafter, the plates were incubated at room temperature for 1 h and washed with PBS. Serial dilutions of the serum were prepared in 1% PBS milk, and 50 μL of the dilution was added to the wells and incubated for 30 min at 20 °C. Four washes were performed with 0.05% PBS-Tween. Biotinylated ACE2 diluted in PBS (2 μg/mL) was added to the plates and incubated for 30 min. After washing, HRP-streptavidin (1:2000) was added and incubated for 30 min. After washing four times, TMB substrate solution (Sigma-Aldrich, St. Louis, MO, USA) was added and incubated for 20 min at 20 °C. The reaction was stopped with 50 μL of 1 M H2SO4. ACE2 was used in duplicate as a control. The results are expressed as % neutralization = (optical density problem well/optical density control well) × 100. Samples with values > 35 were considered positive for neutralizing antibodies.
Tmb substrate solution
Manufactured by Merck Group
Sourced in United States, Germany
TMB substrate solution is a laboratory reagent used in various immunoassays, such as enzyme-linked immunosorbent assays (ELISA), to detect and quantify the presence of specific target analytes. The solution contains 3,3',5,5'-Tetramethylbenzidine (TMB), a chromogenic substrate that undergoes an enzymatic reaction, resulting in a color change that can be measured spectrophotometrically. The core function of the TMB substrate solution is to provide a colorimetric readout for the detection and quantification of the target analytes in the assay.
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Lab products found in correlation
Spectramax 190 microplate reader, by Molecular Devices (3 mentions)
96 well half area microplate, by Corning (3 mentions)
Secondary streptavidin hrp, by BD (2 mentions)
Nunc plate, by Thermo Fisher Scientific (2 mentions)
Immuno plate, by SPL Life Sciences (2 mentions)
Hrp labeled antibodies, by Jackson ImmunoResearch (2 mentions)
Synergy microplate reader, by Agilent Technologies (2 mentions)
Maxisorb immunoassay plates, by Thermo Fisher Scientific (2 mentions)
96 well plate, by Corning (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hrp conjugated anti m13 antibody, by Sino Biological (1 mentions)
Anti human igm, by Merck Group (1 mentions)
Imark microplate absorbance reader, by Bio-Rad (1 mentions)
Cs from porcine heart, by Merck Group (1 mentions)
Immulon 2 plates, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Cell death detection elisa, by Merck Group (1 mentions)
Sodium caseinate, by Merck Group (1 mentions)
Sulphuric acid, by Merck Group (1 mentions)
Microplate autoreader, by Tecan (1 mentions)
39 protocols using tmb substrate solution
1
SARS-CoV-2 Neutralization Antibody Assay
2
SARS-CoV-2 RBD Phage ELISA Assay
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A polyclonal phage ELISA was performed using pools of purified phage from each library stock. A 96-Well Half-Area Microplate (Corning, Cat. 3690, New York, NY, USA) was coated overnight at 4 °C, with 30 μL per well of 1 μg/mL SARS-2 RBD (Sino Biological, Cat. 40592-V08H, Beijing, China), and each well was blocked with 5% skimmed milk in PBS (MPBS) for 1 h at room temperature. Phage pools (~1012 phage particles) were also blocked in MPBS for 1 h at room temperature and then blocked phage pools were added to the SARS-2 RBD-coated plate and incubated for 1 h at 37 °C. After washing four times with PBST, the horseradish peroxidase (HRP)-conjugated anti-M13 antibody (1:5000, Sino Biological, Cat. 11973-MM05, Beijing, China) was incubated for 1 h at 37 °C. After washing four times with PBST, a TMB substrate solution (Sigma-Aldrich, Cat. T0440, St. Louis, MO, USA) was added for 8 min, and the reaction was stopped with 1 N sulfuric acid (Merck, Cat. 100731, Darmstadt, Germany). The absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnydale, CA, USA).
3
SARS-CoV-2 RBD Antibody ELISA Assay
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All ELISA assays were performed on a DSX automated ELISA system device (DYNEX Technologies). The in‐house assay was prepared as follows: 96‐well plates were coated overnight at 4°C with 100 µL of 1 µg/mL RBD protein in PBS. The following day, each well was blocked with 300 µL of PBS/0.15% casein at 4°C until use and at least overnight. Subsequently, plates were washed twice with PBS and 100µl sera were added at a 1:100 dilution in PBS/0.15% casein for 1 hour at RT. After five washes with 300 µL PBS/0.1% Tween, 100 µL of HRP‐labeled secondary polyclonal anti‐human IgM (Sigma, A0420) and anti‐human IgG (Sigma, A0170) antibodies was added in a 1:10’000 dilution for 30 minutes at RT. Again, the plates were washed 5 times with PBS/0.1% Tween and 100 µL of TMB substrate solution (Sigma, T4444) was added for 15 minutes at RT. The development was stopped by adding 100 µL of 0.5M H2SO4, and results were measured at OD450‐620nm. All samples with an OD > 0.5 were assigned as positive.
4
IgE Binding Assay for rTyr p 13
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The ability of rTyr p 13 to bind to IgE was assessed by ELISA. The rTyr p 13 used for ELISA was purified by Ni NTA affinity chromatography. For antigen coating, 100 µl rTyr p 13 (1 µg/ml) diluted in coating buffer (0.1 mol/l PBS)was added to a 96-well microtiter plate and incubated at 8˚C overnight. Wells were washed with 300 µl PBS-0.05%Tween-20 (PBS-T) buffer and blocked with 3% bovine serum albumin (cat. no. A1933, Sigma-Aldrich; Merck KGaA) in PBS-T for 1 h at 37˚C. Subsequently, 100 µl patient serum and 100 µl healthy donor serum (diluted at 1:4 with PBS-T) were added, followed by overnight incubation at 4˚C. Secondary antibody (50 µl; HRP-mouse anti-human IgE; 1:5,000; Sigma-Aldrich; Merck KGaA) was added and samples were incubated at 37˚C for 1 h. To detect binding, 100 µl TMB substrate solution (cat. no. P0209; Beyotime Institute of Biotechnology) was added and samples were incubated for 15 min at room temperature. A total of 2 mol/l H2SO4 (50 µl) was added to stop the reactions. Optical density (OD) was measured for triplicate samples at a wavelength of 450 nm using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Inc.). The reactivity was considered positive if the OD values of the detected serum were higher than the cutoff ELISA value (mean ELISA value of healthy donors + 3 SD).
5
Enzyme-Linked Immunosorbent Assay for Antibody Detection
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Ninety-six well polystyrene plates (NUNC) were coated with CS from porcine heart (Sigma-Merck, Munich, Germany) in 0.1 M bicarbonate buffer, pH 9.6 [3 (link)]. Following this, the saturation of nonspecific binding sites with our alternative, combined blocking buffer (0.5% polyvinyl alcohol solution combined with bovine gelatin solution, at a ratio of 2:1) at room temperature (RT) for 2 h was performed. After being washed with PBS + 0.05% Tween 20 (washing buffer; WB), sera were diluted (1:100 in WB) and incubated 50 min at 37 °C. The secondary antibodies were incubated at 37 °C for 45 min (horseradish peroxidase-conjugated antihuman IgG and IgM, polyclonal rabbit antihuman (Agilent-Dako Santa Clara, CA, US). TMB substrate solution (Sigma-Merck, Munich, Germany) was used to visualize the HRP enzymatic reaction, and the reaction was stopped by 1 M H2SO4. Reading was performed at λ = 450/620 nm using the BEP2000 Advanced automated system. Results are expressed in absorbance (OD) and in quantitative (standard curve-based) results. For data comparison, results were handled as continuous, non-normally distributed integers and the alterations of titers were considered.
6
Serum GSTA1 Antibody Quantification
7
Quantifying BIN1 and p-Tau181 in Brain Tissue
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The 96-well Nunc-Immuno MaxSorp plates (VWR Cat. # 62409–024) were coated with anti-BIN1 (1–277) antibody that was diluted at 1:2,000 in PBS (100 μl/well). After being incubated overnight at 4°C, the plates were washed with PBS/Tween-20 (PBST; 0.5%, v/v) and then blocked using 2% BSA in PBST for 2 h at room temperature. The brain tissue lysates were diluted 1:1,000 and added to the plates (100 μl/well). The plates were incubated overnight at 4°C. After washing, the detection antibody (anti-BIN1, 1:1,000) was added and incubated for 4 h at 4°C. Then, the anti-mouse HRP-conjugated secondary antibody (Thermo Fisher, 1:5,000) was added and incubated for 2 h at room temperature. After washing, the TMB substrate solution (Sigma-Aldrich, CL07) was added to each well and incubated at 37°C for 20 min. The reaction was stopped by adding 3 N HCl, and the values of each well were recorded using a microplate reader at 450 nm. The concentrations of BIN1 (1–277) were calculated using the standard curve generated by purified BIN1 (1–277). The concentrations of p-Tau181 in the brain lysates were determined using the Tau (Phospho) [pT181] human ELISA kit (Thermo Fisher, # KHO0631).
8
Quantification of MPO-DNA Complexes in Sepsis
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An in-house ELISA was used to quantify MPO-DNA complexes in plasma and peritoneal fluid from sham and CLP mice 24 hours after the surgical procedure (24 (link), 27 (link)). Briefly, after overnight coating with anti-MPO capture antibody in calcium carbonate coating buffer (2 µg/ml; 0400-0002, Bio-Rad) at 4°C, a 96-well plate was blocked with 2.5% bovine serum albumin in PBS for 2 hours at room temperature. The plate was subsequently washed, before incubating for 90 minutes at room temperature with 10% peritoneal fluid in blocking buffer. The plate was washed five times, and then incubated for 90 minutes at room temperature with anti-DNA detection antibody (1:20; Cell Death detection ELISA, 11544675001, Sigma). After five washes, the plate was developed with TMB substrate solution (Sigma).
9
Synthetic Peptide Screening for CHIKV Antibodies
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Biotinylated peptide library consisting of 18-mer overlapping peptides (Mimotopes) were generated from sequence alignments of different CHIKV amino acid sequences as previously described [17] (link), [29] (link). Peptides were dissolved in dimethyl sulphoxide (DMSO) to obtain a stock concentration of approximately 15 µg/mL. All the peptides were screened in triplicates. Briefly, streptavidin-coated plates (Pierce) were first blocked with 1% sodium caseinate (Sigma-Aldrich) diluted in 0.1% PBST (0.1% Tween-20 in PBS), before coating with peptides diluted at 1∶1,000 in 0.1% PBST and incubated at room temperature for 1 hour on a rotating platform. Plates were then rinsed with 0.1% PBST before incubation with CHIKV-infected macaque serum samples (1∶2,000) diluted with 0.1% PBST for 1 hour. Plates were rinsed and then followed by incubation with the respective anti-monkey IgG antibodies conjugated to HRP diluted in 0.1% blocking buffer for 1 hour at room temperature to detect for peptide bound antibodies. Reaction was detected with TMB substrate solution (Sigma-Aldrich) and terminated with sulphuric acid (Sigma-Aldrich). Absorbance was read at 450 nm in a microplate autoreader (Tecan). Peptides are considered positive if absorbance values are higher than the mean ±3 standard deviation (SD) values of non-infected macaque serum controls. Data are presented as mean ± SEM.
10
ELISA for Aflatoxin Detection
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ELISA method was used to determine the titers of prepared antiserums and purified antibodies as well as the cross reaction of purified antibodies with other aflatoxins (AFB1, AFB2, AFG1, AFG2). The optimal dilution of antigen pre-coated on the microtiter plate was determined by a checkerboard test. One hundred microliter of antigen (AFM1-BSA or BSA at 5 µg/mL in PBS) was coated for 2 hours at 37°C. After 3 times of washing with PBS containing 0.05% (v/v) Tween 20 (PBS-T), nonspecific sites were blocked with PBS-BSA 2% (w/v) for 2 hours followed by 2 washes with PBS-T. 100 µL of 1:1000 to 1:64000 dilutions of the antiserums, purified antibodies or standard antiserums were added to each well and incubated for 1 hour at 37°C. The plates were washed 5 times with PBS-T and 100 µL of 1:10000 dilutions of secondary antibodies (goat anti-rabbit IgG conjugated with HRP, prepared in our laboratory) was added to each well. Plates were incubated for 45 minutes at 37°C and after 5 times of washing, 100 µL of TMB substrate solution (Sigma, St. Louis, MO, USA) was added. After 20 minutes of incubation in a dark place, the reaction was stopped with 100 µL of 5% solution of sulfuric acid. Absorbance of wells was read by a plate reader (Stat Fax 2100, Awareness Technology Inc. Palm City, USA) at 450 nm.
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