Serum levels of anti-cBSA IgG (subclass IgG1 and IgG2a) were measured by ELISA. Briefly, Nunc-Immuno 96 MicroWell solid plates (Sigma-Aldrich Canada) were coated with 0.5 µg/well of cBSA (Chondrex, Inc.) in carbonate-bicarbonate coating buffer (50 mM Na2CO3, 50 mM NaHCO3, pH 9.6) at 4 °C overnight, and were blocked with 1% BSA (Sigma-Aldrich Canada) in PBS-T (PBS containing 0.05% Tween-20) buffer for 1 h at RT. After washing with PBS-T buffer, serum samples (100 µL/well) in triplicate, serially diluted with ELISA buffer (150 mM NaCl, 1 mM KH2PO4, 10 mm Na2HPO4, 2.6 mm KCl, 0.5% BSA, 0.1% Tween-20, pH 7.4), were incubated at 4 °C overnight. The serum anti-cBSA IgG1 and anti-cBSA IgG2a were detected by using biotinylated goat anti-mouse IgG1 (ab98691, Abcam, 1:1000 diluted) and anti-mouse IgG2 (ab98696, Abcam, diluted from 1:1000), respectively. The color was developed with streptavidin-horseradish peroxidase (HRP) and tetramethylbenzidine (Sigma-Aldrich Canada) and was stopped with 2 M H2SO4 solution. Similarly, serum complement C3 was determined by using mouse complement C3 ELISA kit (ab157711, Abcam) according to the manufacturer’s protocol. The serum titers of each substance were expressed as optical density (OD) at 450 nm (OD450). The “noise” associated with this protocol was confirmed to be low (approximately 5%) by three technical replicates.
Streptavidin horseradish peroxidase hrp
Manufactured by Merck Group
Sourced in Canada, United States
Streptavidin-horseradish peroxidase (HRP) is a conjugate of the protein streptavidin and the enzyme horseradish peroxidase. Streptavidin has a high affinity for the small molecule biotin, while HRP is a widely used enzyme for various detection and signal amplification applications in biochemical and immunological assays.
Automatically generated - may contain errors
Lab products found in correlation
O phenylenediamine dihydrochloride, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Tetramethylbenzidine, by Merck Group (1 mentions)
Ab98691, by Abcam (1 mentions)
Bovine calf serum (bcs), by Thermo Fisher Scientific (1 mentions)
Aminoethyl carbazole staining kit, by Merck Group (1 mentions)
Anti human igm antibody, by Merck Group (1 mentions)
Phosphate buffered saline ph 7.4, by Merck Group (1 mentions)
Multiscreen 96 well filter plate, by Merck Group (1 mentions)
Immunospot software, by Cellular Technology (1 mentions)
Tween 20, by Merck Group (1 mentions)
Bca protein assay kit, by R&D Systems (1 mentions)
Human phospho kinase array, by R&D Systems (1 mentions)
Opteia tmb substrate, by BD (1 mentions)
Tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Immunospot series 5 analyzer, by Cellular Technology (1 mentions)
Elispot kit, by BD (1 mentions)
8 protocols using streptavidin horseradish peroxidase hrp
1
Measuring Serum Anti-cBSA Antibodies
2
Quantification of IgM-Secreting Cells by ELISPOT
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The number of IgM-secreting cells was quantified by ELISPOT. Multiscreen 96-well filter plates (Millipore, Billerica, Massachusetts) were coated with anti-human IgM antibody (5 μg/ml) (Sigma Aldrich, St. Louis, MO) overnight and subsequently blocked with 5% bovine serum albumin (Sigma Aldrich, St. Louis, MO) for 2 h. Cells were washed with RPMI 1640 twice, resuspended in RPMI 1640 containing 10% bovine calf serum (Thermo Scientific, Lafayette, Colorado) and incubated on the primary antibody-coated plates overnight at 37°C with 5% CO2. Biotin-conjugated anti-human IgM antibody (Sigma Aldrich, St. Louis, MO) and streptavidin horseradish peroxidase (HRP) (Sigma Aldrich, St. Louis, MO) were added for one-hour and incubated at 37°C with 5% CO2. All incubations were followed by three washes with phosphate-buffered saline (pH 7.4) containing 0.1% Tween-20 (Sigma Aldrich, St. Louis, MO) and three washes with nanopure water. The spots were developed with an aminoethylcarbazole staining kit (Sigma Aldrich, St. Louis, MO). The number of spots per well between 0.0001mm2 and 9.6372mm2 were quantified via the Immunospot Software (Cellular Technology, Ltd, Shaker Heights, Ohio) and normalized to the number of viable cells collected from each well.
3
Phospho-Kinase Array Analysis of Dexamethasone Effects
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Hs68 cells were treated with vehicle or 1 µM dexamethasone and total protein was isolated using a specific buffer from the Human Phospho-Kinase Array (R&D Systems; Minneapolis, MN, USA) according to the manufacturer’s protocol. The protein concentration in the lysates was quantified using a bicinchoninic acid (BCA) Protein Assay Kit (R&D Systems) and 43 kinase phosphorylation sites were detected on phosphokinase array membranes. The array was rinsed to remove unbound proteins and incubated with biotinylated detection antibodies. Streptavidin–horseradish peroxidase (HRP, Sigma-Aldrich) and electrochemiluminescence detection reagents (Biomax, Seoul, Republic of Korea) were used, and the signals were recorded at each capture spot corresponding to the amount of phosphorylated protein bound using Light-capture (ATTO, Tokyo, Japan).
4
Aptamer Binding Assay for HupB Protein
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In brief, 500ng/well recombinant HupB protein was coated overnight using standard 100 mM carbonate-bicarbonate buffer, pH 9.6 at 4° C. After washing with selection buffer (SB; 10 mM Tris, pH 7.5, 10 mM MgCl2, 50 mM KCl, 25 mM NaCl) and blocking with 5% skimmed milk for 90 min at room temperature (RT), the optimized amount of prepared or commercially obtained 5′-biotin labeled ssDNA was added to wells for 60 min at RT. After washing with SB supplemented with 0.5% Tween 20 (v/v), streptavidin-horseradish peroxidase (HRP) (Sigma) was added at 1:1,500 dilution and incubated for 1 hr at RT. ALISA was developed using 100 μL OptEIA TMB substrate, (3,3′,5,5′-tetramethylbenzidine; BD Biosciences, USA), the reaction quenched using 5% H2SO4 and optical density (OD) measured at 450 nm. ΔOD 450 nm estimated by subtracting OD 450 nm of appropriate negative controls (aptamer control, antigen control, etc.) was plotted.
5
Evaluating Bacterial Vaccination Responses
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Sera from selected subjects were obtained before vaccination and at 18-45 months after initiating either MV130 or MV140. Anti-K. pneumoniae, anti-S. pneumoniae, anti-MV130 or anti-MV140 IgA and IgG were determined by ELISA. Briefly, 96-well non-tissue culture treated plates were pretreated with 100 µL of poly-L-lysine (stock at 0.01%, 1:1000 dilution) (Sigma-Aldrich) for 1 hour under UV light and coated with the appropriate whole-cell heat-inactivated bacteria or polybacterial mixture (300 Nephelometric Turbidity Units -NTU-, ~ 109 bacteria) overnight at 4°C, and subsequently incubated with human sera for 2 hours at room temperature. IgA and IgG antibodies were detected using the following reagents: Biotin rat anti-human IgA or IgG (both from Sigma-Aldrich) and Streptavidin Horseradish Peroxidase (HRP) (Sigma-Aldrich). Peroxidase activity was revealed by addition of o-phenylenediamine dihydrochloride (Sigma-Aldrich) and reaction was stopped with HCl 1N. Plates were read on an ELISA reader at 490 nm (Triturus Elisa, Grifols). Absorbance values (arbitrary units) from each individual subject were normalized to their corresponding pre-vaccination value, thus defined as 1.
6
ELISA for Quantifying Tissue Factor
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Quantification of TF in ocular fluids and in retinal tissues was performed using a specific enzyme-linked immunosorbent assay (ELISA). Briefly, goat anti-human TF polyclonal antibodies (Bio-Rad, Hercules, CA, USA) were coated on a 96-well high-binding affinity plate. Captured human TF was detected using a biotinylated goat polyclonal antibody raised against the human TF (Bio-Rad, Hercules, CA, USA) and revealed with streptavidin-horse radish peroxidase (HRP, Sigma-Aldrich, St. Louis, MI, USA) enzyme and tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, St. Louis, MI, USA). The 450-nm absorbance was measured using the Infinite F200 plate reader (Tecan, Männedorf, Switzerland). Recombinant human TF (Bio-Rad, Hercules, CA, USA) was used as a standard. The lower limit of quantification (LLoQ) in rat ocular fluids (aqueous humor and vitreous pooled together) and in rabbit aqueous humor, vitreous and retina/RPE/choroid complex was defined as 195.3 and 39 pg/mL, respectively. Concentrations of human TF were calculated using a four-parameter logistic curve fit (4-PL, MyAssays software) from the standard curve. Values were expressed as mean ± standard error of the mean (sem) in ng/mL of fluids or ng/g of tissues.
7
Immunoglobulin Levels in Serum After MV130 Therapy
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Levels of specific IgA and IgG against MV130 and against each bacteria contained in the mixture (i.e., S. pneumoniae, S. epidermidis, S. aureus, M. catarrhalis, K. pneumoniae, and H. influenzae) were determined in serum samples from blood collected before starting immunotherapy with MV130 and between 4 to 41 months after initiating the treatment. Quantification was performed by ELISA following standard procedures as previously described (26 (link)). Once collected, sera samples were stored frozen at −80°C until further processing. Briefly, 96-well non-tissue culture-treated plates were pretreated with poly-L-lysine (Sigma-Aldrich) for 1 h under UV light and coated with each of the heat-inactivated whole cell bacteria or the polybacterial mixture (300 FTU/ml ~ 109 bacteria/ml) overnight at 4°C, and, subsequently, incubated with human serum dilutions for 2 h at room temperature. Specific immunoglobulins were detected with biotin rat anti-human IgA and IgG (both from Sigma-Aldrich). Signal was developed by incubation with streptavidin-horseradish peroxidase (HRP) (Sigma-Aldrich). Peroxidase activity was revealed by the addition of o-phenylenediamine dihydrochloride (Sigma-Aldrich) and the reaction was stopped with HCl 1N. Plates were read on an ELISA reader at 490 nm (Triturus Elisa, Grifols).
8
Quantifying Antigen-Specific IFN-γ Responses
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At the indicated time points after hydrodynamic injection, liver mononuclear cells were cultured and assayed for the frequency of antigen-specific IFN-γ- secreting cells using an ELISPOT kit (BD Biosciences, San Jose, CA). Briefly, 105 liver mononuclear cells were co-cultured with 1µg/ml of rHBcAg (ID Labs, London, Canada) in 200µl RPMI 1640 supplemented with 10% fetal calf serum (FCS). Cell suspensions were incubated for 20h. Spot-forming cells were revealed with a biotin-conjugated antibody, streptavidin-horseradish peroxidase (HRP), and AEC substrates (Sigma-Aldrich), and were analyzed using the ImmunoSpot series 5 analyzer (Cellular Technology Limited, Cleveland, OH).
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