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Horseradish peroxidase hrp conjugated streptavidin

Manufactured by R&D Systems
Sourced in United States

Horseradish peroxidase (HRP)-conjugated streptavidin is a protein complex consisting of streptavidin, a tetrameric protein that binds to the vitamin biotin, and horseradish peroxidase, an enzyme that catalyzes a redox reaction. This conjugate is commonly used in various immunoassay and detection techniques due to the high affinity of streptavidin for biotin and the enzymatic activity of HRP.

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7 protocols using horseradish peroxidase hrp conjugated streptavidin

1

Sandwich ELISA for MDA-LDL Detection

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A sandwich ELISA protocol was adopted using LO1, an in-house generated IgG monoclonal mouse autoantibody, to detect MDA-LDL (13 (link)). ELISA plates were coated with 10 μg/mL of LO1 as the capture antibody prior to washing and blocking with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Goat biotinylated polyclonal anti-ApoB antibody (1:2000) (Abcam, Cambridge, MA, USA) and horseradish peroxidase (HRP)-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) at 1:200 dilution were used for detection. Subsequently, for this assay and all other assays, 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma Aldrich, Poole, UK) was added and the reaction stopped with 0.5M H2SO4. Plates were read at 450 nm optical density using a Synergy HT microplate reader (BioTek, VT, USA).
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2

Quantification of MDA-LDL and Anti-oxLDL Antibodies

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All samples were anonymised and personnel conducting assays were fully blinded to the patient treatment allocation. ELISAs to detect MDA-LDL and anti-oxLDL antibodies were performed as previously described [13 (link)]. Levels of MDA-LDL were measured using 10 μg/mL LO1, a novel laboratory-developed monoclonal IgG3κ murine antibody, as the capture antibody in a sandwich enzyme-linked immunosorbent assay (ELISA). LO1 reacts with MDA-LDL but minimally with native LDL [14 (link)]. A biotinylated anti-ApoB antibody (Abcam, Cambridge, MA, USA) at 1:2000 dilution and horseradish peroxidase (HRP)-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) at 1:200 dilution were used for detection. Subsequently, for this and all other ELISAs, 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma Aldrich, Poole, UK) was added and the reaction stopped with 0.5 M H2SO4. Plates were read at an optical density (OD) of 450 nm using a Synergy HT microplate reader (BioTek, Winooski, VT, USA).
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3

Measurement of MDA-LDL and ApoB by Sandwich ELISA

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Sandwich ELISA protocols were established to measure the majority of components within our study. MDA-LDL was measured using LO1 (10 μg/mL) as the capture antibody for MDA-LDL [18 (link)], whilst polyclonal goat anti-human anti-ApoB (Abcam, Cambridge, MA, USA; 1:2000 dilution) was used as the capture antibody for ApoB. A biotinylated anti-ApoB antibody (Abcam, Cambridge, MA, USA; 1:2000), followed by horseradish peroxidase (HRP)-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA; 1:200) were used for detection of both MDA-LDL and ApoB. For these assays, and all other ELISAs, development was achieved by adding 3,3′,5,5′-tetramethylbenzidine (Sigma Aldrich, Poole, UK), after which the reaction was stopped with 0.5 M H2SO4. Plates were read at an optical density of 450 nm using a Synergy HT microplate reader (BioTek, VT, USA).
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4

Quantitative VEGF165 ELISA Assay

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VEGF165a capture antibody, at a concentration of 0.25  μg/ml, was incubated on high-binding 96-well plates overnight at room temperature. The plates were blocked (1% BSA in PBS) and serial dilutions of recombinant human (rh)VEGF165 standards (ranging from 500 pg/ml to 1.95 pg/ml) were added, incubated alongside sample lysates, typically 200 μg in 100 μl per well. The plate was incubated for 2 h at room temperature with shaking, washed and incubated with 100 μl/well of biotinylated goat anti-human VEGF (0.1 μg/ml; R&D systems) for 2 further hours at room temperature. After washing, 100 μl/well of Horseradish Peroxidase (HRP)-conjugated streptavidin (1:200; R&D Systems) was added and plates were left at room temperature for 20 min. The plates were washed and color change induced with substrate A and B (DY-999; R&D Systems) for 1 h under light protection. The reaction was stopped by addition of 100 μl/well of 1 M HCl and the absorbance was read immediately in an ELISA plate reader (Dynex Technologies Opsys MR system plate reader) at 450 nm with a control reading at 620 nm. A standard curve was calculated from mean absorbance values of standards enabling the estimation of VEGF concentration for each sample.
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5

Measurement of Total and Antigen-Specific IgE

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Total IgE in the serum and culture supernatant was measured using OptEIA™ Mouse IgE ELISA Set (BD Biosciences) according to the manufacturer’s instruction. The concentration of IgE was calculated using a standard curve obtained with mouse IgE of known concentrations. To measure serum levels of OVA-specific IgE, serially diluted sera was added into 96-well plates pre-coated with anti-IgE (2 μg/ml, BD Biosciences), followed by incubation with biotinylated OVA (1.25 μg/ml, Chondrex, Redmond, WA). The bound biotinylated OVA was detected with horseradish peroxidase (HRP)-conjugated streptavidin (R&D Systems) using tetramethylbenzidine as substrate (Sigma-Aldrich). The absorbance was read at 450/490 nm.
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6

HER2 Extracellular Domain ELISA Assay

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Anti-HER2 monoclonal antibodies m4D5 (provided by Genentech) and biotinylated 6E2 [16 (link)] were used as the capture and detection antibody, respectively. HER2 ECD standard was originally purchased from Oncogene Sciences. For ELISA, the capture antibody (m4D5, 5 μg/ml, 50 μL) was coated in PBS to a 96-well plate for overnight incubation at 4°C. After wash with PBST, the plate was blocked with 5% BSA for 1 h at room temperature (22°C). HER2 ECD standard and serum samples were diluted in the MBB buffer [13 (link)] and incubated for 1-h at 22°C. Diluted biotinylated detection antibody (50 μL, 1 μg/mL, with MBB) was added to each well for a 1 hr-incubation at room temperature. Streptavidin-conjugated horseradish peroxidase (HRP) (R&D systems) was used as the secondary antibody to detect the antigen-antibody complex. The plate was washed three times with PBST (0.1% Tween 20 in PBS) in-between incubations. Following six washes with PBST to remove excess detection antibodies, 50 μL of Tetramethyl Benzidine (TMB) substrate (0.1 mg/mL, 0.05 M phosphate-citrate buffer, pH 5.0) was incubated in each well at 22°C. The reaction was stopped within 15–30 min with 50 μL of 2 M H2SO4, and the data was collected at 450 nm (absorbance filter) using the SpectraFluor reader (Tecan).
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7

HER2 Extracellular Domain ELISA Assay

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Anti-HER2 monoclonal antibodies m4D5 (provided by Genentech) and biotinylated 6E2 [16 (link)] were used as the capture and detection antibody, respectively. HER2 ECD standard was originally purchased from Oncogene Sciences. For ELISA, the capture antibody (m4D5, 5 μg/ml, 50 μL) was coated in PBS to a 96-well plate for overnight incubation at 4°C. After wash with PBST, the plate was blocked with 5% BSA for 1 h at room temperature (22°C). HER2 ECD standard and serum samples were diluted in the MBB buffer [13 (link)] and incubated for 1-h at 22°C. Diluted biotinylated detection antibody (50 μL, 1 μg/mL, with MBB) was added to each well for a 1 hr-incubation at room temperature. Streptavidin-conjugated horseradish peroxidase (HRP) (R&D systems) was used as the secondary antibody to detect the antigen-antibody complex. The plate was washed three times with PBST (0.1% Tween 20 in PBS) in-between incubations. Following six washes with PBST to remove excess detection antibodies, 50 μL of Tetramethyl Benzidine (TMB) substrate (0.1 mg/mL, 0.05 M phosphate-citrate buffer, pH 5.0) was incubated in each well at 22°C. The reaction was stopped within 15–30 min with 50 μL of 2 M H2SO4, and the data was collected at 450 nm (absorbance filter) using the SpectraFluor reader (Tecan).
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