Human Granzyme B DuoSet ELISA
Human Granzyme B 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 Granzyme B. 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.
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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
- Economical alternative to complete kits
Kit Content
- 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:
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 # 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)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Scientific Data
Product Datasheets
Preparation and Storage
Background: Granzyme B
Granzyme B is a member of the granzyme family of serine proteases found specifically in granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (1, 2). Granzyme B plays an essential role in granule-mediated apoptosis utilizing the substrates in this pathway, such as Caspase 3, Caspase 8 and Bid (3, 4). Recent research indicates expanded Granzyme B functionality to include extracellular roles along with its classical pro-apoptotic function. It has been found that Granzyme B is an important mediator of skin injury, repair and inflammation (4) through extracellular substrates including Laminin, VE-Cadherin, Fibronectin and the proteoglycans Aggrecan (3) and Decorin (4).
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 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.
Assay Procedure
- 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 Granzyme B 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 - 10
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Combined IgE neutralization and Bifidobacterium longum supplementation reduces the allergic response in models of food allergy
Authors: SB An, BG Yang, G Jang, DY Kim, J Kim, SM Oh, N Oh, S Lee, JY Moon, JA Kim, JH Kim, YJ Song, HW Hyun, J Kim, K Lee, D Lee, MJ Kwak, BK Kim, YK Park, CP Hong, JH Kim, HS Lim, MS Ryu, HT Jin, SW Lee, YS Chang, HS Park, YC Sung, MH Jang
Nature Communications, 2022;13(1):5669.
Species: Human
Sample Types: Cell Culture Supernates
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Genome-wide gain-of-function screening characterized lncRNA regulators for tumor immune response
Authors: Y Wang, Y Zhao, W Guo, GS Yadav, C Bhaskarla, Z Wang, X Wang, S Li, Y Wang, Y Chen, D Pattarayan, W Xie, S Li, B Lu, US Kammula, M Zhang, D Yang
Science Advances, 2022;8(49):eadd0005.
Species: Human
Sample Types: Cell Culture Supernates
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Monocyte-dependent co-stimulation of cytokine induction in human gammadelta T cells by TLR8 RNA ligands
Authors: R Serrano, C Coch, C Peters, G Hartmann, D Wesch, D Kabelitz
Scientific Reports, 2021;11(1):15231.
Species: Human
Sample Types: Cell Culture Supernates
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The Search for an Interesting Partner to Combine with PD-L1 Blockade in Mesothelioma: Focus on TIM-3 and LAG-3
Authors: E Marcq, JRM Van Audena, J De Waele, C Merlin, P Pauwels, JP van Meerbe, SA Fisher, ELJ Smits
Cancers, 2021;13(2):.
Species: Human
Sample Types: Cell Culture Supernates
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CRISPR/Cas9-mediated TGFbetaRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro
Authors: K Alishah, M Birtel, E Masoumi, L Jafarzadeh, HR Mirzaee, J Hadjati, RH Voss, M Diken, S Asad
Journal of Translational Medicine, 2021;19(1):482.
Species: Human
Sample Types: Cell Culture Supernates
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Novel Combinations of Human Immunomodulatory mAbs Lacking Cardiotoxic Effects for Therapy of TNBC
Authors: C Vetrei, M Passariell, G Froechlich, R Rapuano Le, E Sasso, N Zambrano, C De Lorenzo
Cancers, 2021;14(1):.
Species: Human
Sample Types: Cell Culture Supernates
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Influence of Indoleamine-2,3-Dioxygenase and Its Metabolite Kynurenine on gammadelta T Cell Cytotoxicity against Ductal Pancreatic Adenocarcinoma Cells
Authors: H Jonescheit, HH Oberg, D Gonnermann, M Hermes, V Sulaj, C Peters, D Kabelitz, D Wesch
Cells, 2020;9(5):.
Species: Human
Sample Types: Cell Culture Supernates
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Targetable mechanisms driving immunoevasion of persistent senescent cells link chemotherapy-resistant cancer to aging
Authors: DP Muñoz, SM Yannone, A Daemen, Y Sun, F Vakar-Lope, M Kawahara, AM Freund, F Rodier, JD Wu, PY Desprez, DH Raulet, PS Nelson, LJ van 't Vee, J Campisi, JP Coppé
JCI Insight, 2019;5(0):.
Species: Human
Sample Types: Cell Culture Supernates
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Preclinical assessment of galunisertib (LY2157299 monohydrate), a first-in-class transforming growth factor-? receptor type I inhibitor
Authors: JM Yingling, WT McMillen, L Yan, H Huang, JS Sawyer, J Graff, DK Clawson, KS Britt, BD Anderson, DW Beight, D Desaiah, MM Lahn, KA Benhadji, MJ Lallena, RB Holmgaard, X Xu, F Zhang, JR Manro, PW Iversen, CV Iyer, RA Brekken, MD Kalos, KE Driscoll
Oncotarget, 2018;9(6):6659-6677.
Species: Human
Sample Types: Cell Culture Supernates
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Folate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer
Authors: M Kim, S Pyo, CH Kang, CO Lee, HK Lee, SU Choi, CH Park
PLoS ONE, 2018;13(6):e0198347.
Species: Human
Sample Types: Cell Culture Supernates
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Soluble markers of neutrophil, T-cell and monocyte activation are associated with disease severity and parasitemia in falciparum malaria
Authors: K Otterdal, A Berg, AE Michelsen, S Patel, MG Tellevik, CG Haanshuus, B Fevang, P Aukrust, N Langeland, T Ueland
BMC Infect. Dis., 2018;18(1):670.
Species: Human
Sample Types: Plasma
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JAK2 inhibitor combined with DC-activated AFP-specific T-cells enhances antitumor function in a Fas/FasL signal-independent pathway
Onco Targets Ther, 2016;9(0):4425-33.
Species: Human
Sample Types: Cell Culture Supernates
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Reviews for Human Granzyme B DuoSet ELISA
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Detection of granzyme B produced by human BCMA-specific CAR-T cells in response to BCMA-negative and BCMA-positive cell lines.
We used this kit for the quantification of Granzyme B in human serum and plasma. Works very well and well-described protocol.
