Human VEGF 165b 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 VEGF 165b. 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: VEGF
VEGF (Vascular endothelial growth factor) is a potent mediator of both angiogenesis and vasculogenesis in the fetus and adult. It is required for regulating the proliferation, migration, and survival of embryonic endothelial cells and during wound healing and the female reproductive cycle in adults. Pathologically, it is involved in tumor development and tumor vascular leakage. VEGF binds to VEGF R1/Flt-1, VEGF R2/Flk-1/KDR, and Neuropilin-1. Multiple forms of VEGF are generated by alternative splicing and proteolysis.
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 VEGF 165b 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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The splicing factor YBX1 promotes the progression of osteosarcoma by upregulating VEGF165 and downregulating VEGF165b
Authors: Quan, B;Li, Z;Yang, H;Li, S;Yan, X;Wang, Y;
Heliyon
Species: Human
Sample Types: Cell Culture Supernates
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A novel anti-angiogenic radio/photo sensitizer for prostate cancer imaging and therapy: 89Zr-Pt@TiO2-SPHINX, synthesis and in vitro evaluation
Authors: V Tekin, T Aweda, O Kozgus Gul, FZ Biber Muft, J Bartels, SE Lapi, P Unak
Nuclear medicine and biology, 2021-01-09;94(0):20-31.
Species: Human
Sample Types: Cell Culture Supernates
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Plasma Ephrin-A1 level in a cohort of diabetic retinopathy patients
Authors: D Mao, Y Hu, Q Bao, K Wu, Y Zheng, Y Yang, B Lei, Y Jiang
BMC Ophthalmol, 2020-08-05;20(1):319.
Species: Human
Sample Types: Serum
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VEGF-A165b levels are reduced in breast cancer patients at primary diagnosis but increase after completion of cancer treatment
Authors: MM Karsten, MH Beck, A Rademacher, J Knabl, JU Blohmer, J Jückstock, JC Radosa, P Jank, B Rack, W Janni
Sci Rep, 2020-02-27;10(1):3635.
Species: Human
Sample Types: Whole Blood
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VEGF165b, a splice variant of VEGF-A, promotes lung tumor progression and escape from anti-angiogenic therapies through a ?1 integrin/VEGFR autocrine loop
Authors: A Boudria, C Abou Fayca, T Jia, S Gout, M Keramidas, C Didier, N Lemaître, S Manet, JL Coll, AC Toffart, D Moro-Sibil, C Albiges-Ri, V Josserand, E Faurobert, C Brambilla, E Brambilla, S Gazzeri, B Eymin
Oncogene, 2018-09-07;0(0):.
Species: Human
Sample Types: Serum
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Mechanisms regulating angiogenesis underlie seasonal control of pituitary function
Authors: J Castle-Mil, DO Bates, DJ Tortonese
Proc. Natl. Acad. Sci. U.S.A, 2017-03-07;114(12):E2514-E2523.
Species: Ovine
Sample Types: Tissue Homogenates
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The role of VEGF-A165b in trophoblast survival.
Authors: Bills V, Hamdollah-Zadeh M, Soothill P, Harper S, Bates D
BMC Pregnancy Childbirth, 2014-08-15;14(0):278.
Species: Human
Sample Types: Cell Lysates
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The relationship between endothelial progenitor cell populations and epicardial and microvascular coronary disease-a cellular, angiographic and physiologic study.
Authors: Chan, Kim H, Simpson, Philippa, Yong, Andy S, Dunn, Louise L, Chawantanpipat, Chirapan, Hsu, Chijen, Yu, Young, Keech, Anthony, Celermajer, David S, Ng, Martin K
PLoS ONE, 2014-04-15;9(4):e93980.
Species: Human
Sample Types: Plasma
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Abnormal histone methylation is responsible for increased vascular endothelial growth factor 165a secretion from airway smooth muscle cells in asthma.
Authors: Clifford RL, John AE, Brightling CE
J. Immunol., 2012-06-11;189(2):819-31.
Species: Human
Sample Types: Cell Culture Supernates
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Biomarkers for antitumor activity of bevacizumab in gastric cancer models.
Authors: Yamashita-Kashima Y, Fujimoto-Ouchi K, Yorozu K, Kurasawa M, Yanagisawa M, Yasuno H, Mori K
BMC Cancer, 2012-01-25;12(0):37.
Species: Human
Sample Types: Tissue Homogenates
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VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo.
Authors: Rennel ES, Varey AH, Churchill AJ, Wheatley ER, Stewart L, Mather S, Bates DO, Harper SJ
Br. J. Cancer, 2009-08-25;101(7):1183-93.
Species: Human
Sample Types: Cell Culture Supernates
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