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).
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.
Automatically generated - may contain errors
Lab products found in correlation
Synergy ht microplate reader, by Agilent Technologies (3 mentions)
Biotinylated anti apob antibody, by Abcam (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (2 mentions)
Polyclonal goat anti human anti apob, by Abcam (1 mentions)
Dy999, by R&D Systems (1 mentions)
Biotinylated goat anti human vegf, by R&D Systems (1 mentions)
Opsys mr system plate reader, by Dynex (1 mentions)
Tetramethylbenzidine, by Merck Group (1 mentions)
Opteia mouse ige elisa set, by BD (1 mentions)
Anti ige, by BD (1 mentions)
7 protocols using horseradish peroxidase hrp conjugated streptavidin
1
Sandwich ELISA for MDA-LDL Detection
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).
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).
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.
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.
6
HER2 Extracellular Domain ELISA Assay
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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