Blood was collected from mice at specific time points pre- and postchallenge and diluted 1 in 20 in PBS. The samples were centrifuged at 8,928 × g for 5 min, and the supernatant was removed and stored at −80°C. Ninety-six-well MaxiSorp Nunc immunoplates (Nunc) were coated with 10 μg/ml of crude parasite antigen in coating buffer for 2 h at room temperature. Plates were blocked with 5% skim milk in PBS overnight at 4°C. Serum samples were used at a 1 in 20 dilution or titrated using 3-fold dilutions from 1 in 50 to 1 in 109,350. Samples were dispensed at a volume of 50 μl per well and incubated for 2 h at room temperature. For the assessment of memory B cell responses, a dilution of 1 in 1,350 of the titrated sera was chosen as a representation of the data. After five washes with PBS–0.05% Tween 20 (Merck), the plates were incubated with goat anti-mouse IgG/horseradish peroxidase-conjugated antibody (1:3,000 in blocking buffer) (Bio-Rad) for 2 h. Plates were washed a further five times and then incubated with tetramethylbenzidine substrate (BD Biosciences) for 15 min. The reaction was stopped using 1 M sulfuric acid, and the plates were read using a Victor 3 plate reader (Perkin-Elmer) at 450 nM.
Tetramethylbenzidine substrate
Manufactured by BD
Sourced in United States
Tetramethylbenzidine (TMB) substrate is a colorimetric reagent used in various immunoassays and enzyme-linked immunosorbent assays (ELISAs) to detect and quantify the presence of target analytes. It serves as a chromogenic substrate for enzymes, such as horseradish peroxidase (HRP), that catalyze its oxidation, resulting in a blue-colored reaction product. The intensity of the color can be measured spectrophotometrically, providing a quantitative assessment of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Ovalbumin (ova), by Merck Group (2 mentions)
Nbt reagent, by Merck Group (2 mentions)
Immobilon p plates, by Merck Group (2 mentions)
Ovalbumin (ova), by Biosearch Technologies (2 mentions)
Alkaline phosphatase conjugated abs to igg, by Southern Biotech (2 mentions)
Immulon 2 hb plates, by Thermo Fisher Scientific (2 mentions)
Ab97040, by Abcam (2 mentions)
Maxisorp immunoplate, by Thermo Fisher Scientific (2 mentions)
Victor3 plate reader, by PerkinElmer (1 mentions)
Maxisorp nunc immunoplates, by Thermo Fisher Scientific (1 mentions)
Enspire plate reader, by PerkinElmer (1 mentions)
96 well microplate, by Corning (1 mentions)
Horseradish peroxidase, by Cytiva (1 mentions)
Goat polyclonal anti mouse igg, by Abcam (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
Ap112p, by Merck Group (1 mentions)
Ab98693, by Abcam (1 mentions)
Ab97235, by Abcam (1 mentions)
Biotin ratanti mouse igg1 a85 1, by BD (1 mentions)
Biotin rat anti mouse ige r35 118, by BD (1 mentions)
18 protocols using tetramethylbenzidine substrate
1
ELISA Assay for Parasite-Specific Antibodies
2
UGT1A1 Antibody Detection in Mice
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Maxisorp 96 wells plates (Thermo Fisher Scientific) were coated with UGT1A1
protein in carbonate buffer at 4 °C overnight. A standard curve of rat
recombinant IgG (Sigma Aldrich) was coated to the wells in seven twofold
dilution starting from 1 μg/ml. After blocking, plasma samples
were added to plates and incubated 1 hour at 37 °C. Detection was
performed by adding to the wells
3,3′,5,5′-tetramethylbenzidine substrate (BD Biosciences, San
Diego, CA), and color development was measured at 450 and 570 nm (for
background subtraction) on an Enspire plate reader (Perkin Elmer) after
blocking the reaction with H2SO4.
To detect antibodies against the human UGT1A1 protein in mice, 20 µg of
total cell protein extract from HEK293 cells transfected with a plasmid
expressing the human UGT1A1 cDNA or from untransfected HEK293 cells were run
in 10% Sodium dodecyl sulfate polyacrylamide gel electrophoresis gel,
blotted onto a nitrocellulose membrane and blocked with 5% milk in Phosphate
buffered saline with Tween 20. Plasma from individual animals was used as
primary antibody (1:200).
A positive control plasma from mice immunized with recombinant human UGT1A1
was used (1:2,000). A band corresponding to the UGT1A1 protein is detected
if antibodies against UGT1A1 are present in the plasma of the AAV-treated
animals.
protein in carbonate buffer at 4 °C overnight. A standard curve of rat
recombinant IgG (Sigma Aldrich) was coated to the wells in seven twofold
dilution starting from 1 μg/ml. After blocking, plasma samples
were added to plates and incubated 1 hour at 37 °C. Detection was
performed by adding to the wells
3,3′,5,5′-tetramethylbenzidine substrate (BD Biosciences, San
Diego, CA), and color development was measured at 450 and 570 nm (for
background subtraction) on an Enspire plate reader (Perkin Elmer) after
blocking the reaction with H2SO4.
To detect antibodies against the human UGT1A1 protein in mice, 20 µg of
total cell protein extract from HEK293 cells transfected with a plasmid
expressing the human UGT1A1 cDNA or from untransfected HEK293 cells were run
in 10% Sodium dodecyl sulfate polyacrylamide gel electrophoresis gel,
blotted onto a nitrocellulose membrane and blocked with 5% milk in Phosphate
buffered saline with Tween 20. Plasma from individual animals was used as
primary antibody (1:200).
A positive control plasma from mice immunized with recombinant human UGT1A1
was used (1:2,000). A band corresponding to the UGT1A1 protein is detected
if antibodies against UGT1A1 are present in the plasma of the AAV-treated
animals.
3
Quantification of Serum CLEC18 Levels
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CLEC18 levels in serum were quantified using ELISA according to the standard operating procedure. Briefly, the CLEC18 antibody (clone 3F10F8) was coated onto 96-well microplates (50 μl/well, Corning) and incubated for 16–18 h at 4 °C, and then incubated with 200 μl of blocking buffer (Tris-buffered saline [TBS] with 1% polyvinyl alcohol) for 1 h at room temperature before washing twice with TBST (TBS with 0.05% Tween 20). Each well was incubated with 50 μl of the patient’s serum or standard for 2 h at room temperature before washing with TBST three times. After washing, the well was incubated with 50 μl of biotin-conjugated CLEC18 monoclonal antibody (clone 3A9E6) for 2 h at room temperature before washing with TBST three times. Finally, each well was incubated with 50 μl of streptavidin-HRP for RT 20 min, followed by the addition of 100 μl of tetramethylbenzidine substrate (BD Biosciences) for 15 min; the reaction was stopped by addition of 1 N sulfuric acid prior to analysis using an ELISA reader. All of the samples and standards were measured in duplicate, and concentrations were determined from a standard curve using mean optical density values. Serum CLEC18 levels were expressed as ng/mL.
4
ELISA and ELISPOT Assays for Antibody Detection
5
Measuring Anti-LG3 Titers by ELISA
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Anti-LG3 titers were measured using a locally developed ELISA. Recombinant perlecan fragment LG3 was produced and purified as previously described [21 (link)]. The purity of the recovered LG3 protein was assessed by reducing SDS–PAGE and Coomassie Blue R-250 staining. Recombinant mouse LG3 (5 ng/μL) was first coated onto 96-well Immulon II HB plates (0.5 μg per well; Thermo Electron). Of note, mouse and human LG3 fragments are 3 highly homologous at the amino acid level (87%). After dilution of the sera (1:100), 100 μL of diluted sera was added to each well. The plates were washed, and bound IgG was detected using horseradish peroxidase coupled with anti-mouse IgG (1:5000) (Amersham). Reactions were revealed with 100 μL of tetramethylbenzidine substrate (BD Biosciences) and stopped with 50 μL of 1 M H2SO4 sulfuric acid. Spectrophotometric analysis was performed at 450 nm, and the results are expressed as optical density × 1000.
6
M2e Epitope Binding Evaluation by ELISA
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To confirm identity and accessibility of M2e to antibodies, ELISA plates were coated overnight at 4 °C with serial dilutions of Flg-4M or Flg proteins in sodium bicarbonate buffer pH 8.5. Plates were treated with a blocking buffer supplemented with 0.1 % (w/v) BSA for 2 h at 37 °C and washed three times using PBS with Tween. Plates were probed with monoclonal antibody specific for M2e or 6-His tag for 1 h at 37 °C. As a conjugate, rat monoclonal anti-mouse IgG (Imtek, Russia) labelled with a horseradish peroxidase was used at a 1:10,000 dilution. After adding tetramethylbenzidine substrate (BD Bioscience) and monitoring color development, the reaction was stopped by H2SO4, and OD at 450 nm was measured on a microplate spectrophotometer.
7
ELISA-based Antibody Titer Quantification
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Antibody titers in BAL and sera samples were measured using enzyme-linked immunosorbent assay (ELISA). The synthetic M2e peptide G37 (SLLTEVET PIRNEWGCRCNDSSD) or the influenza virus A/Aichi/2/68(H3N2) in PBS (pH 7.2) were used to coat 96-well microtiter plates (Greiner, Germany). Goat polyclonal anti-mouse IgG (Abcam, Cambridge, UK) labeled with a horseradish peroxidase was used as a conjugate. After adding tetramethylbenzidine substrate (BD Bioscience, Franklin Lakes, NJ, USA) and color development, the reaction was stopped by H2SO4, and OD450 was measured on a microplate spectrophotometer.
8
Quantifying FimH Glycoprotein Binding
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Immulon 4HBX 96-well plates were coated overnight with 1 μg of human glycoproteins reported to be ligands of FimH (secretory IgA, laminin, collagen IV, and THP). All glycoproteins were ordered from Sigma except crude THP, which was isolated by ammonium sulfate precipitation of urine donated by healthy volunteers. Coated wells were then incubated with 200 μl of blocking buffer [phosphate-buffered saline (PBS) + 2% bovine serum albumin (BSA)] for 2 hours at 23°C, followed by incubation with 100 μl of FimGNteH variants diluted in blocking buffer to 1 μg/ml for 1 hour at 37°C. After washing three times with PBS + 0.05% Tween 20, 100 μl of polyclonal rabbit antibody raised against FimH residues 1 to 165 (from E. coli strain J96) with a C-terminal six-histidine tag (Sigma Genesis; 1:5000 dilution in PBS + 2% BSA) was added to each well for 1 hour at 37°C. After washing three times with PBS + 0.05% Tween 20, 100 μl of polyclonal goat anti-rabbit antibody conjugated to horseradish peroxidase (KPL; 1:5000 dilution in PBS + 2% BSA) was added to each well for 1 hour at 37°C. After a final round of washing, plates were developed with 100 μl of tetramethylbenzidine substrate (BD Biosciences) and quenched within 1 min with 50 μl of 1 M H2SO4, and absorbance was measured at 450 nm.
9
Quantitative ELISA for Extracellular Matrix
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ELISA plates (96 wells) were coated with 5 ng/well of fibronectin-1 (Novoprotein), laminin B2 (Novoprotein), or collagen Vα1 (Abcam) overnight at 4°C. After washing with PBS containing 0.05% Tween-20 (PBST), the wells were incubated with blocking buffer (100 μL/well; Chondrex) for 1 hour. Afterward, serum samples (1:100, diluted in the blocking buffer) were added in duplicate wells and incubated for 1.5 hours, followed by HRP-conjugated goat anti-human IgG (1:2,000; AP112P, Sigma-Aldrich) and then tetramethylbenzidine substrate (BD). All incubations and washes were performed at room temperature. For internal control standardization, serum samples from 3 positive controls (high, intermediate, and low-positive) and 10 healthy control subjects were performed on each ELISA plate. OD values were determined at OD450 (iMarK microplate reader, Bio-Rad). OD values that were more than 3 SD above the mean values in healthy control subjects were considered positive.
10
ELISA-Based Antibody Quantification
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Antigen-specific levels of antibodies were determined by ELISA performed in 96-well microtiter plates coated overnight at 4 °C with the synthetic peptide (5 μg/mL) in PBS (pH 7.2). The plates were blocked with PBS containing 5% fetal calf serum (FCS) for 1 h at room temperature. The plates were washed 3 times in PBS containing Tween. The diluted samples were added in volumes of 100 μL per well and the plates were incubated for 1 hour at room temperature. As a conjugate, HRP-labelled goat anti-mouse IgG (ab97040, Abcam), IgG1 (ab98693, Abcam), IgG2a (ab98698, Abcam), and IgA (ab97235, Abcam) were used at 1:5000 to 1:20,000 dilutions. After adding tetramethylbenzidine substrate (BD Bioscience) and monitoring the color development, the reaction was stopped withy H2SO4, and the OD at 450 nm was measured on a microplate spectrophotometer.
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