Human Serpin A4/Kallistatin 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 and recombinant human Serpin A4/Kallistatin. 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.
- 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
- Economical alternative to complete kits
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.
The components listed above may be purchased separately:
Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 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)
Human Serpin A4 / Kallistatin ELISA Standard Curve
Preparation and Storage
Background: Serpin A4/Kallistatin
The human serpin superfamily consists of at least 35 members that target not only serine proteases, but also selected cysteine proteases and non-protease proteins. Serpins bind the protease active site resulting in a major conformational rearrangement that traps the enzyme in a covalent acyl-enzyme intermediate. As protease inhibitors, serpins have an array of functions including regulating blood clotting, the complement pathway, extracellular matrix remodeling, and cell motility. They are also involved in activities that extend beyond their ability to inhibit proteases. For instance, they may also regulate blood pressure, angiogenesis, or act as storage/transport proteins.
GENERAL ELISA PROTOCOL
- Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 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 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.
- Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. 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 Human Serpin A4/Kallistatin 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.
Citations: Showing 1 - 4
Filter your results:
Kallistatin protects against diabetic nephropathy in db/db mice by suppressing AGE-RAGE-induced oxidative stress.
Authors: Yiu W, Wong D, Wu H, Li R, Yam I, Chan L, Leung J, Lan H, Lai K, Tang S
Kidney Int, 2015;0(0):.
Sample Types: Cell Culture Supernates
Wnt signaling in age-related macular degeneration: human macular tissue and mouse model.
Authors: Tuo J, Wang Y, Cheng R, Li Y, Chen M, Qiu F, Qian H, Shen D, Penalva R, Xu H, Ma J, Chan C
J Transl Med, 2015;13(0):330.
Sample Types: Plasma
Elevated circulation levels of an antiangiogenic SERPIN in patients with diabetic microvascular complications impair wound healing through suppression of Wnt signaling.
Authors: McBride J, Jenkins A, Liu X, Zhang B, Lee K, Berry W, Janknecht R, Griffin C, Aston C, Lyons T, Tomasek J, Ma J
J Invest Dermatol, 2014;134(6):1725-34.
Sample Types: Serum
Antiangiogenic and antineuroinflammatory effects of kallistatin through interactions with the canonical Wnt pathway.
Authors: Liu, Xiaochen, Zhang, Bin, McBride, Jeffrey, Zhou, Kevin, Lee, Kyungwon, Zhou, Yueping, Liu, Zuguo, Ma, Jian-xin
Sample Types: Tissue Homogenates
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