Human Activin A 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 Activin A. 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
- 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: Activin A
Activins, members of the TGF-beta superfamily, are disulfide-linked dimeric proteins originally purified from gonadal fluids as proteins that stimulated pituitary follicle stimulating hormone (FSH) release. Activin proteins have a wide range of biological activities, including mesoderm induction, neural cell differentiation, bone remodeling, hematopoiesis and roles in reproductive physiology. Activin isoforms and other members of the TGF-beta superfamily exert their biological effects by binding to heteromeric complexes of a type I and a type II serine-threonine kinase receptor, both of which are essential for signal transduction.
Activins are homodimers or heterodimers of the various beta subunit isoforms, while inhibins are heterodimers of a unique alpha subunit and one of the various beta subunits. Five beta subunits (mammalian beta A, beta B, beta C, beta E and Xenopus beta D) have been cloned to date. The activin/inhibin nomenclature reflects the subunit composition of the proteins: Activin A (beta A - beta A), Activin B (beta B - beta B), Activin AB (beta A - beta B), Inhibin A (alpha - beta A) and Inhibin B (alpha - beta B).
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 Activin A 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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Pro-Inflammatory Biomarkers Combined with Body Composition Display a Strong Association with Knee Osteoarthritis in a Community-Based Study
Authors: Tarabeih, N;Kalinkovich, A;Shalata, A;Higla, O;Livshits, G;
Biomolecules
Species: Human
Sample Types: Plasma
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Activin A Limits VEGF-Induced Permeability via VE-PTP
Authors: Baccouche, B;Lietuvninkas, L;Kazlauskas, A;
International journal of molecular sciences
Species: Human
Sample Types: Cell Culture Supernates
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A SNAI2-PEAK1-INHBA stromal axis drives progression and lapatinib resistance in HER2-positive breast cancer by supporting subpopulations of tumor cells positive for antiapoptotic and stress signaling markers
Authors: S Hamalian, R Güth, F Runa, F Sanchez, E Vickers, M Agajanian, J Molnar, T Nguyen, J Gamez, JD Humphries, A Nayak, MJ Humphries, J Tchou, IK Zervantona, JA Kelber
Oncogene, 2021-07-08;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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Potential novel biomarkers for chronic lung allograft dysfunction and azithromycin responsive allograft dysfunction
Authors: C Veraar, J Kliman, A Benazzo, F Oberndorfe, M Laggner, P Hacker, T Raunegger, S Janik, P Jaksch, W Klepetko, HJ Ankersmit, B Moser
Scientific Reports, 2021-03-24;11(1):6799.
Species: Human
Sample Types: Serum
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Exploring the potential effect of paricalcitol on markers of inflammation in de novo renal transplant recipients
Authors: HK Pihlstrøm, T Ueland, AE Michelsen, P Aukrust, F Gatti, C Hammarströ, M Kasprzycka, J Wang, G Haraldsen, G Mjøen, DO Dahle, K Midtvedt, IA Eide, A Hartmann, H Holdaas
PLoS ONE, 2020-12-16;15(12):e0243759.
Species: Human
Sample Types: Plasma
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Clinical Response to the CD95-Ligand Inhibitor Asunercept Is Defined by a Pro-Inflammatory Serum Cytokine Profile
Authors: A Radujkovic, T Boch, F Nolte, D Nowak, C Kunz, A Gieffers, C Müller-Tid, P Dreger, WK Hofmann, T Luft
Cancers, 2020-12-08;12(12):.
Species: Human
Sample Types: Serum
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Follistatin impacts Tumor Angiogenesis and Outcome in Thymic Epithelial Tumors
Authors: S Janik, C Bekos, P Hacker, T Raunegger, AI Schiefer, L Müllauer, C Veraar, B Dome, W Klepetko, HJ Ankersmit, B Moser
Sci Rep, 2019-11-22;9(1):17359.
Species: Human
Sample Types: Serum
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Activin A-mediated epithelial de-differentiation contributes to injury repair in an in vitro gastrointestinal reflux model
Authors: C Roudebush, A Catala-Val, T Andl, GF Le Bras, CD Andl
Cytokine, 2019-07-29;123(0):154782.
Species: Human
Sample Types: Cell Culture Supernates
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Evaluation of a Miniaturized Biologically Vascularized Scaffold in vitro and in vivo
Authors: S Kress, J Baur, C Otto, N Burkard, J Braspennin, H Walles, J Nickel, M Metzger
Sci Rep, 2018-03-16;8(1):4719.
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Stem cells from human apical papilla decrease neuro-inflammation and stimulate oligodendrocyte progenitor differentiation via activin-A secretion
Authors: P De Berdt, P Bottemanne, J Bianco, M Alhouayek, A Diogenes, A Llyod, J Gerardo-Na, GA Brook, V Miron, GG Muccioli, AD Rieux
Cell. Mol. Life Sci., 2018-02-07;0(0):.
Species: Human/Rat, Rat
Sample Types: Cell Culture Supernates
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Activin A and follistatin in patients with asthma. Does severity make the difference?
Authors: Stelios Loukides
Respirology, 2016-11-02;0(0):.
Species: Human
Sample Types: BALF, Sputum
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Activin A as a mediator of NK-dendritic cell functional interactions.
Authors: Seeger P, Bosisio D, Parolini S, Badolato R, Gismondi A, Santoni A, Sozzani S
J Immunol, 2014-01-06;192(3):1241-8.
Species: Human
Sample Types: Cell Culture Supernates
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Adiponectin upregulates monocytic activin A but systemic levels are not altered in obesity or type 2 diabetes.
Authors: Weigert J, Neumeier M, Wanninger J, Schober F, Sporrer D, Weber M, Schramm A, Wurm S, Stogbauer F, Filarsky M, Schaffler A, Aslanidis C, Scholmerich J, Buechler C
Cytokine, 2009-01-06;45(2):86-91.
Species: Human
Sample Types: Plasma
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Innate immune responses to TREM-1 activation: overlap, divergence, and positive and negative cross-talk with bacterial lipopolysaccharide.
Authors: Dower K, Ellis DK, Saraf K, Jelinsky SA, Lin LL
J. Immunol., 2008-03-01;180(5):3520-34.
Species: Human
Sample Types: Cell Culture Supernates
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Activin A is an anticatabolic autocrine cytokine in articular cartilage whose production is controlled by fibroblast growth factor 2 and NF-kappaB.
Authors: Alexander S, Watt F, Sawaji Y, Hermansson M, Saklatvala J
Arthritis Rheum., 2007-11-01;56(11):3715-25.
Species: Porcine
Sample Types: Cell Culture Supernates
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The activin A-follistatin system: potent regulator of human extracellular matrix mineralization.
Authors: Eijken M, Swagemakers S, Koedam M, Steenbergen C, Derkx P, Uitterlinden AG, van der Spek PJ, Visser JA, de Jong FH, Pols HA, van Leeuwen JP
FASEB J., 2007-04-20;21(11):2949-60.
Species: Human
Sample Types: Cell Culture Supernates
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Performed ideally the very first time. We used undiluted samples of medium conditioned for 48h. Carefully read the protocol - this one calls for 1M Urea. Color reaction developed in exactly 20min. Our samples measured within the standard curve range suggested (8000-250pg/ml).
Further testing would be required for cross-reactivity with horse serum
