Tumour necrosis factor (TNF)-α, interleukin (IL)-10, IL-2, IL-4, and Interferon (INF)-γ were measured in serum and BAL samples at days 2 and/or 10 post-infection by using commercially available enzyme-linked immunosorbent assay (ELISA) technique kits, following the manufacturer's recommendations (ELISPOT Ready-SET-Go!®, eBioscience, San Diego, USA). Anti-pneumococcal antibodies (IgA, IgM, and IgG) were determined by ELISA on day 10 post-infection as previoulsy described [8 (link)]. In brief, plates were coated with a 1:100 dilution of heat-killed S. pneumoniae overnight at 4°C and blocked with PBS containing 5% non-fat dry milk. Appropriate dilutions of the samples (serum 1:20; BAL 1:2) were incubated for 1 h at 37°C. Peroxidase conjugated anti-mouse IgG, IgA, or IgM (1:500) (Sigma- Aldrich) were added and incubated for 1 h at 37°C. The reaction was developed with TMB Substrate Reagent (Sigma-Aldrich). The concentration was measured with reference to standard curves using known amounts of the respective murine Immunoglobulin (Sigma-Aldrich).
Tmb substrate reagent
Manufactured by Merck Group
The TMB Substrate Reagent is a colorimetric reagent used in various laboratory procedures, such as enzyme-linked immunosorbent assays (ELISA), to detect and quantify the presence of specific analytes. The reagent contains a chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB), which undergoes a color change reaction in the presence of an enzyme, such as horseradish peroxidase (HRP). This color change can then be measured using a spectrophotometer, providing a quantitative analysis of the target analyte.
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Lab products found in correlation
Elispot ready set go, by Thermo Fisher Scientific (1 mentions)
Nunc immuno maxisorp plates, by Thermo Fisher Scientific (1 mentions)
Spectramax 190, by Molecular Devices (1 mentions)
Ez link sulfo nhs lc biotin kit, by Thermo Fisher Scientific (1 mentions)
Elisa technique kits, by R&D Systems (1 mentions)
6 protocols using tmb substrate reagent
1
Pneumococcal Infection Cytokine Profiling
2
Quantification of IGF1R-Ligand Binding
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We coated 96-well Nunc-Immuno Maxi-Sorp plates (Thermo Scientific) with recombinant human IGF1R ECD (Sino biological, 100 ng/well) for 16 h at 4°C. The plates were then washed with PBS-T (Tween 20, 0.05%) and blocked with 1% BSA in PBS (200 μL/well) for 2 h at 37°C. Serially diluted M30103, B30104, or A12 (400 nM–0.026 nM) was added to each well (100 μL/well) and incubated for 30 min at 37°C. PBS was used as a negative control. After washing five times with PBS-T, human IGF1 that was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotin kit (Thermo Fisher Scientific) was added into each well (100 ng/well) and incubated for 2 h at 37°C. The plates were further incubated with streptavidin-horseradish peroxidase (Pierce, Cat: #21126, 1:5,000 from 1 mg/mL stock, 20 ng/well) for 1 h at 37°C. After washing with PBS-T, the plates were incubated with 100 μL of TMB substrate reagent (Sigma) for 5 min. The calorimetric reaction was measured using a microplate reader (Molecular Device SpectraMax 190) at 450–650 nm after the addition of 50 μL of 1 N sulfuric acid.
3
Immune Response to Venus Antigen
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Blood and BAL samples were taken 10 days after 2nd boosting as described previously 39 (link).
Specific anti-Venus total immunoglobulins (IgT), IgA and IgG were determined by ELISA. Plates were coated with 3 μg recombinant His-Venus per well overnight at 4 °C and blocked with BSA. Appropriate dilutions of the samples (serum 1:20; BAL 1:2) were incubated for 1 h at 37 °C. Peroxidase conjugated anti-mouse IgT (Polyclonal rabbit anti-mouse immunoglobulins/HRP, DakoCytomation) IgG, or IgA antibodies (Sigma-Aldrich) (at a 1:500 dilution) were added and incubated for 1 h at 37 °C. The reaction was developed with TMB Substrate Reagent (Sigma-Aldrich) and measured at 450 nm in a microplate reader.
Spleens were collected, mechanically disaggregated and the cell suspensions were kept as previously described 11 (link). Cells (4 × 106 cells/well) were cultured in 24-well plates in the presence of 0.5 μg of His-Venus for 24 h. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4 and IL-17 concentrations in spleen supernatants were measured with commercially available ELISA kits following the manufacturer's recommendations (R&D Systems).
Specific anti-Venus total immunoglobulins (IgT), IgA and IgG were determined by ELISA. Plates were coated with 3 μg recombinant His-Venus per well overnight at 4 °C and blocked with BSA. Appropriate dilutions of the samples (serum 1:20; BAL 1:2) were incubated for 1 h at 37 °C. Peroxidase conjugated anti-mouse IgT (Polyclonal rabbit anti-mouse immunoglobulins/HRP, DakoCytomation) IgG, or IgA antibodies (Sigma-Aldrich) (at a 1:500 dilution) were added and incubated for 1 h at 37 °C. The reaction was developed with TMB Substrate Reagent (Sigma-Aldrich) and measured at 450 nm in a microplate reader.
Spleens were collected, mechanically disaggregated and the cell suspensions were kept as previously described 11 (link). Cells (4 × 106 cells/well) were cultured in 24-well plates in the presence of 0.5 μg of His-Venus for 24 h. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4 and IL-17 concentrations in spleen supernatants were measured with commercially available ELISA kits following the manufacturer's recommendations (R&D Systems).
4
Measuring Anti-PCV Antibodies by ELISA
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IgA and IgG-specific anti-PCV antibodies were measured by ELISA. One μg of PCV per well was used to coat plates overnight at 4 °C. Plates were then blocked with albumin. Appropriate dilutions of the samples (serum 1:20; BAL 1:2) were incubated for 1 h at 37 °C. Peroxidase conjugated anti-mouse IgG, or IgA antibodies (1:500) (Sigma-Aldrich, St. Louis, MO, USA) were added and incubated for 1 h at 37 °C. TMB substrate reagent (Sigma-Aldrich) was used to reveal the reaction. The concentration of antibodies was measured with reference to standard curves using known amounts of the respective mouse immunoglobulin (Sigma-Aldrich).
5
Serological Analysis of Rotavirus Antibodies
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Blood samples were obtained through cardiac puncture seven days after the last immunization (day 35) and collected in heparinized tubes. Intestinal fluid samples were obtained as described previously (11 (link)). Briefly, the small intestine was flushed with 5 ml of PBS and the fluid was centrifuged (10,000×g 4°C for 10 min) to separate particulate material. The supernatant was kept frozen until use. Specific anti-rotavirus antibodies (IgA and IgG) were determined by ELISA. Plates were coated with 1.5 μg of Rotateq per well overnight at 4°C and blocked with serum bovine albumin. Appropriate dilutions of the samples (serum 1:20; intestinal fluid 1:2) were incubated for 1 h at 37°C. Peroxidase conjugated anti-mouse IgG, or IgA antibodies (1:500) (Sigma-Aldrich) were added and incubated for 1 h at 37°C. The reaction was developed with TMB Substrate Reagent (Sigma-Aldrich) and measured at 450 nm in a microplate reader.
6
Antibody and Cytokine Profiling in PSPF Infection
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Specific anti-PSPF antibodies (IgA, IgM, and IgG) were determined by ELISA as described previously [10 ]. In brief, plates were coated with 1.5 μg of PSPF per well overnight at 4°C and blocked with albumin. Appropriate dilutions of the samples [serum 1:20; BAL 1:2] were incubated for 1 h at 37 °C. Peroxidase conjugated anti-mouse IgG, IgA, or IgM antibodies (1:500) (Sigma-Aldrich) were added and incubated for 1 h at 37 °C. The reaction was developed with TMB Substrate Reagent (Sigma-Aldrich). The concentration was measured with reference to standard curves using known amounts of the respective murine Ig (Sigma-Aldrich).
Tumour necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4, and IL-10 concentrations in BAL were measured with commercially available enzyme-linked immunosorbent assay (ELISA) technique kits following the manufacturer’s recommendations (R&D Systems, MN, USA).
Tumour necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4, and IL-10 concentrations in BAL were measured with commercially available enzyme-linked immunosorbent assay (ELISA) technique kits following the manufacturer’s recommendations (R&D Systems, MN, USA).
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