Bovine IgG DuoSet ELISA
Bovine IgG DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural bovine IgG. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Available in 5 (96-well) plate pack size
- Economical alternative to complete kits
Kit Content
- Capture Antibody
- Detection Antibody
- Natural Standard
Other Reagents Required
DuoSet Ancillary Reagent Kit 3 (5 plates): (Catalog # DY009) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 3.
The components listed above may be purchased separately:
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY004), or 5% Tween 20 in PBS, 7.2-7.4, 0.2 μm filtered
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Scientific Data
Product Datasheets
Preparation and Storage
Background: IgG
R&D Systems offers a range of secondary antibodies and controls for flow cytometry, immunohistochemistry, and Western blotting. We provide species-specific secondary antibodies that are available with a variety of conjugated labels.
Our NorthernLights fluorescent secondary antibodies are bright and resistant to photobleaching. We are currently offering secondary antibodies recognizing mouse, rat, goat, sheep, and rabbit IgG as well as chicken IgY. These reagents are available with three distinct excitation and emission maxima, making them ideal for multi-color fluorescence microscopy.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 150 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block plates by adding 300 μL of Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
- Dilute the Detection Antibody to the working concentration specified on the vial label using Reagent Diluent. Prepare only as much Detection Antibody as required for each assay.
- Add 50 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well (Proceed to step 3 without washing).
- Add 100 μL of the prepared Detection Antibody to all wells containing standards, samples, and blanks. Cover with an adhesive strip, and incubate 2 hours at room temperature on a horizontal orbital microplate shaker (0.12” orbit) set at 500 ± 50 rpm.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 150 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature on the benchtop. Avoid placing the plate in direct light.
- Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Citations for Bovine IgG DuoSet ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 2
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Methods to improve efficacy of orally administered bioactive peptides using bovine colostrum as an exemplar
Authors: RJ Playford, MJ Weiser, T Marchbank
PLoS ONE, 2021-06-17;16(6):e0253422.
Species: Bovine
Sample Types: Milk
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Temporal dynamics of intradermal cytokine response to tuberculin in Mycobacterium bovis BCG-vaccinated cattle using sampling microneedles
Authors: S Steinbach, S Jalili-Fir, S Srinivasan, MB Melo, S Middleton, T Konold, M Coad, PT Hammond, DJ Irvine, M Vordermeie, V Kapur
Scientific Reports, 2021-03-29;11(1):7074.
Species: Bovine
Sample Types: Tissue Homogenates
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