TNF-α, IL-1β, and IL-33 were obtained for measurement using a standardized sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (R&D system) [26 (link)]. The absorbance was read at 450 nm using an Epoch ELISA reader (Epoch, BioTek, USA).
Epoch elisa reader
Manufactured by Agilent Technologies
Sourced in United States
The EPOCH ELISA reader is a microplate reader designed for absorbance-based ELISA (Enzyme-Linked Immunosorbent Assay) experiments. It provides accurate and reliable detection of colorimetric signals generated in ELISA assays.
Automatically generated - may contain errors
Lab products found in correlation
Ap substrate, by Merck Group (6 mentions)
Anti human kappa ap, by Southern Biotech (6 mentions)
Quanti blue substrate, by InvivoGen (6 mentions)
Mst14, by R&D Systems (6 mentions)
96 well tissue culture plate, by Thermo Fisher Scientific (6 mentions)
Immulon 2hb plate, by Thermo Fisher Scientific (4 mentions)
Ifnαr2, by R&D Systems (4 mentions)
Hek blue ifna b cells, by InvivoGen (3 mentions)
Recombinant ifnα, by Novus Biologicals (3 mentions)
Bovine serum albumin (bsa), by Southern Biotech (3 mentions)
Hek blue ifnα b cells, by InvivoGen (3 mentions)
Recombinant ifna, by Novus Biologicals (3 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Cck 8 assay, by Dojindo Laboratories (2 mentions)
Each indicated peptide, by Thermo Fisher Scientific (2 mentions)
Tmb substrate, by Merck Group (2 mentions)
Ridascreen cryptosporidium, by R-Biopharm (1 mentions)
Goat anti mouse igg2a hrp, by Southern Biotech (1 mentions)
Goat anti mouse igg1 hrp, by Southern Biotech (1 mentions)
Glutathione reductase activity kit, by Enzo Life Sciences (1 mentions)
28 protocols using epoch elisa reader
1
Measurement of Inflammatory Cytokines
2
Masked Fusion Abs Binding to IFNα2 Receptor
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Example 4
In this example, it is shown that a plurality of Masked Fusion Abs of the disclosure can bind to the IFNα2 receptor. Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 ug/mL IFNαR2 (R&D Systems) overnight at 4 deg. C and blocked with 2% BSA (Fisher) for a minimum of 2 hrs. at room temperature. Then, wells were washed 3× with PBS+0.05% Tween (Sigma). Indicated Ab concentrations were overlayed overnight at 4 deg. C. Wells were then washed 3× with PBS+0.05% Tween. Bound Abs were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Absorbance changes after addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader. The results show the masked 5T4, mesothelin, CD20, and CD138 Abs bind IFNαR2 with lower affinity compared to the unmasked 5T4, mesothelin, CD20, and CD138 Fusion Abs. (See,
3
Masking Effect on IFNα Activity
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Example 21
In this example, it is shown that both Mask1 and Mask2 of the disclosure can reduce and restore IFNα activity. Briefly, HEK Blue IFNa/b cells (Invivogen) were seeded into 96-well tissue culture plates (Fisher) at a density of 1×10e4 cells/well (50 uL/well). 50 uL/well of recombinant IFNα (Novus Biologicals) or indicated Abs (5T4 or Mesothelin) were incubated with the cells at the indicated concentrations overnight at 37 deg. C. Abs cleaved with MST14 (R&D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr. At 37 deg. C. Then, 10 uL of supernatant was added to a plate containing 90 uL/well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader. The results show that the IFNα activity in masked anti-CD138-IFNα (Mask1) and masked anti-CD138-IFNα (Mask2) was reduced compared to when the mask is cleaved. (See,
4
Masked Fusion Ab Binding to IFNαR2
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Example 20
In this example, it is shown that a Masked Fusion Ab (utilizing mask2) of the disclosure can bind to the IFNα2 receptor. Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 ug/mL IFNαR2 (R&D Systems) overnight at 4 deg. C and blocked with 2% BSA (Fisher) for a minimum of 2 hrs. at room temperature. Then, wells were washed 3× with PBS+0.05% Tween (Sigma). Indicated Ab concentrations were overlayed overnight at 4 deg. C. Wells were then washed 3× with PBS+0.05% Tween. Bound Abs were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Absorbance changes after addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader. The results show that both mask1 and mask2 are able to inhibit Fusion Ab binding to IFNαR2. (See,
5
Reduction of IFNα Activity by Masked Fusion Abs
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Example 6
In this example, it is shown that a plurality of Masked Fusion Abs of the disclosure can reduce and restore IFNα activity. Briefly, HEK Blue IFNa/b cells (Invivogen) were seeded into 96-well tissue culture plates (Fisher) at a density of 1×10e4 cells/well (50 uL/well). 50 uL/well of recombinant IFNα (Novus Biologicals) or indicated Abs (5T4 or Mesothelin) were incubated with the cells at the indicated concentrations overnight at 37 deg. C. Abs cleaved with MST14 (R&D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr. At 37 deg. C. Then, 10 uL of supernatant was added to a plate containing 90 uL/well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader. The results show that the IFNα activity in masked anti-5T4-IFNα and masked anti-mesothelin-IFNα was reduced by approximately 1 to 2 logs compared to when the mask is cleaved. (See,
6
Masking of IFNα Activity by Fusion Abs
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Example 6
In this example, it is shown that a plurality of Masked Fusion Abs of the disclosure can reduce and restore IFNα activity. Briefly, HEK Blue IFNα/b cells (Invivogen) were seeded into 96-well tissue culture plates (Fisher) at a density of 1×10e4 cells/well (50 uL/well). 50 uL/well of recombinant IFNa (Novus Biologicals) or indicated Abs (5T4 or Mesothelin) were incubated with the cells at the indicated concentrations overnight at 37 deg. C. Abs cleaved with MST14 (R&D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr. At 37 deg. C. Then, 10 uL of supernatant was added to a plate containing 90 uL/well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader. The results show that the IFNα activity in masked anti-5T4-IFNα and masked anti-mesothelin-IFNα was reduced by approximately 1 to 2 logs compared to when the mask is cleaved. (See,
7
Cisplatin Dose-Response Assay in A2780 Cells
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Cell growth and viability were measured using the CCK8 assay (Dojindo, Kumamoto, Japan). Briefly, the A2780 and A2780-cp cells were plated in 96-well plates (5000/well). After 12 h, the cells were treated with various concentrations of cisplatin (0, 10, 20, 30, 40, and 50 μg/ml) for 24 h, and 10 μl of the CCK8 reagent was subsequently added to the cells. The optical density (OD) value of the cells was then measured at 450 nm using an EPOCH ELISA reader (BioTek Instruments, Vermont, USA) according to the manufacturer’s instructions.
8
Copro-antigen ELISA for Cryptosporidium Detection
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A commercial Copro-antigen ELISA EIA kit [RIDASCREEN®Cryptosporidium (R-BIOPHARM-AG, Germany)] was used according to the manufacturer's instructions (Fig. 1 ). Briefly, 100 μL of the liquid faecal or stool samples were diluted 1:10 with dilution buffer, mixed well and incubated at room temperature for 15 min. Then, 100 μL of the supernatant from each sample was placed in a test well. Then, 100 μL of the enzyme conjugate was added and plates were incubated at room temperature for 60 min. After washing five times with diluted washing buffer, the wells were incubated with 100 μL substrate solution for 15 min before 50 μL of stop reagent were added. Optical densities were determined at 450 and 620 nm using an Epoch ELISA reader (Biotek, Bad Friedrichshall). According to the cut-off value provided by the manufacturer (extinction for the negative control + 0.15), samples were considered positive if their extinction was more than 10% above the calculated cut-off value and questionable if it was between the cut-off and the cut-off plus 10%.![]()
Comparison of three commercially available Copro-antigen tests. (A) RIDASCREEN®Cryptosporidium test [Enzyme Immunoassay (EIA)], (B) Cryptosporidium 2nd Generation [Enzyme Linked Immunosorbent Assay (ELISA)], (C) RIDA®QUICK Cryptosporidium [Immuno-chromatographic test (ICT)].
9
Recombinant Protein and Crude Extract ELISA
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ELISA plates (MaxiSorp for IgG and Immulon 4HBX for IgE) were coated with 5 μg/mL recombinant protein or 40 μg/mL crude JRC extract overnight and then blocked with 2% BSA in PBS. Serum samples were diluted 1 : 100- or 1 : 1000-fold in 1% BSA in PBS and then a 1 : 3 serial dilution was made. To detect IgE, sera were treated with Agarose-Protein G (Thermo Fisher Scientific, Rockford, IL) for 50 minutes and then 1 : 20 diluted samples were loaded to ELISA plates. Samples were detected with goat anti-mouse IgG1-HRP, goat anti-mouse IgG2a-HRP (Southern Biotech, Birmingham, AL), or rat anti-mouse-IgE-biotin (R35-118, BD Pharmingen, San Jose, CA) followed by Pierce Streptavidin-HRP (Thermo Fisher Scientific, Rockford, IL). Reaction was developed with SureBlue TMB Substrate (KPL, Gaithersburg, MD) and stopped with TMB Stop Solution (KPL, Gaithersburg, MD). Plates were read (OD450) by using Epoch ELISA reader (BioTek, Winooski, VT). Average background (PBS only) was calculated, and samples which have OD450 value more than 2 ∗ average background are considered as positive. The dilutions of such samples are determined as the endpoint titers.
10
Measuring Cisplatin Cytotoxicity in A2780 Cells
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Cell growth and viability were measured using the CCK8 assay (Dojindo, Kumamoto, Japan). Briefly, A2780 cells and three cisplatin-resistant variants were seeded into 96-well plates (5,000 cells/well). After 12 h, cells were treated with various concentrations of cisplatin (0, 0.5, 1, 2, 4, 8, 16, 32, and 64 µg/mL) for 48 h. Then, 10 µL of the CCK8 reagent were added to the cells and absorbance at 450 nm was measured by an EPOCH ELISA reader (BioTek Instruments, Vermont, USA) according to the manufacturer’s instructions.
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