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Product Summary
The Quantikine Human VEGF Immunoassay is a 4.5 hour solid phase ELISA designed to measure VEGF165 levels in cell culture supernates, serum, and plasma. It contains Sf 21-expressed recombinant human VEGF165 and antibodies raised against the recombinant protein. Results obtained for naturally occurring human VEGF and recombinant human VEGF121 showed linear curves that were parallel to the standard curves obtained using the Quantikine Human VEGF Immunoassay standards. These results indicate that this kit can be used to determine relative mass values for natural human VEGF.
Sample Values
Serum/Plasma - Samples from apparently healthy volunteers were evaluated for the presence of human VEGF in this assay. No medical histories were available for the donors used in this study.| Sample Type | Mean of detectable (pg/mL) | % Detectable | Range (pg/mL) |
| Serum (n=37) | 220 | 100 | 62-707 |
| EDTA plasma (n=37) | 61 | 24 | ND-115 |
| Heparin plasma (n=37) | 41 | 22 | ND-55 |
| Citrate plasma (n=37) | ___ | 0 | ND |
Cell Culture Supernates:
Human peripheral blood mononuclear cells (10 x 106 cells/mL) were cultured in RPMI supplemented with 5% fetal bovine serum, 50 μM beta -mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. The cells were cultured unstimulated or stimulated with 10 μg/mL PHA for 5 days. Aliquots of the cell culture supernates were removed, assayed for levels of human VEGF, and measured 87.1 pg/mL and 1041 pg/mL, respectively.
PC-3 human prostate cancer cells were cultured in RPMI supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. The cells were cultured unstimulated or stimulated with 60 nM of PMA for 24 hours. Aliquots of the cell culture supernates were removed, assayed for levels of human VEGF, and measured 3373 pg/mL and 3652 pg/mL, respectively.
JEG-3 human epithelial choriocarcinoma cells were cultured in MEM + NEAA supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. The cells were cultured unstimulated or stimulated with 100 ng/mL of LPS for 1 day. Aliquots of the cell culture supernates were removed, assayed for levels of human VEGF, and measured 1538 pg/mL and 1938 pg/mL, respectively.
Recovery
The recovery of human VEGF spiked to three different levels throughout the range of the assay in various matrices was evaluated.
| Sample Type | Average % Recovery | Range % |
|---|---|---|
| Cell Culture Media (n=5) | 102 | 95-111 |
| Citrate Plasma (n=5) | 100 | 88-113 |
| EDTA Plasma (n=5) | 97 | 82-113 |
| Heparin Plasma (n=5) | 93 | 82-102 |
| Serum (n=5) | 102 | 92-115 |
Linearity
To assess linearity of the assay, samples were spiked with high concentrations of human VEGF and diluted with the appropriate calibrator diluent to produce samples with values within the dynamic range of the assay.
Scientific Data
Assay Procedure
Refer to the product datasheet for the complete assay procedure.
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
- Prepare all reagents, standard dilutions, and samples as directed in the product insert.
- Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
- For Serum & Plasma Samples: Add 100 µL of Assay Diluent to each well.
For Cell Culture Supernate Samples: Add 50 µL Assay Diluent to each well. - For Serum & Plasma Samples: Add 100 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.
For Cell Culture Supernate Samples: Add 200 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours. - Aspirate each well and wash, repeating the process twice for a total of 3 washes.
- Add 200 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.
- Aspirate and wash 3 times.
- Add 200 µL Substrate Solution to each well.
- For Serum & Plasma Samples: Incubate at room temperature for 25 minutes. PROTECT FROM LIGHT. For Cell Culture Supernate Samples: Incubate at room temperature for 20 minutes.
PROTECT FROM LIGHT. - For Serum & Plasma Samples: Incubate at room temperature for 25 minutes. PROTECT FROM LIGHT. For Cell Culture Supernate Samples: Incubate at room temperature for 20 minutes.
PROTECT FROM LIGHT.
Add Assay Diluent
Add Standard, Control, or Sample
200 µL Conjugate
200 µL Substrate Solution
50 µL Stop Solution
Human VEGF Quantikine ELISA Kit Summary
Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Assay Length
4.5 hours
Sample Type & Volume Required Per Well
Cell Culture Supernates (200 uL), Serum (100 uL), EDTA Plasma (100 uL), Heparin Plasma (100 uL), Citrate Plasma (100 uL)
Sensitivity
9 pg/mL
Assay Range
15.6 - 1,000 pg/mL (Cell Culture Supernates), 31.3 - 2,000 pg/mL (Serum, EDTA Plasma, Heparin Plasma, Citrate Plasma)
Specificity
Natural and recombinant human VEGF. This assay also recognizes recombinant human VEGF165b.
Cross-reactivity
Cross-reactivity observed with 1 or more available related molecules.Cross-species reactivity observed with 1 or more species tested.
Interference
Interference observed with 1 or more available related molecules.
背景
别名
MVCD1,VAS,vascular endothelial growth factor A,Vascular permeability factor,Vasculotropin,VEGF,VEGFA,VEGF-A,VEGFMGC70609,VPF,VPFvascular endothelial growth factor,Vascular Endothelial Growth Factor
背景
Background: VEGF
Vascular endothelial growth factor (VEGF or VEGF-A), also known as vascular permeability factor (VPF), is a potent mediator of both angiogenesis and vasculogenesis in the fetus and adult (1-3). It is a member of the PDGF family that is characterized by the presence of eight conserved cysteine residues in a cystine knot structure and the formation of antiparallel disulfide-linked dimers (4). Humans express alternately spliced isoforms of 121, 145, 165, 183, 189, and 206 amino acids (aa) in length (4). VEGF165 appears to be the most abundant and potent isoform, followed by VEGF121 and VEGF189 (3, 4). Isoforms other than VEGF121 contain basic heparin-binding regions and are not freely diffusible (4). Human VEGF165 shares 88% aa sequence identity with corresponding regions of mouse and rat VEGF. VEGF is expressed in multiple cells and tissues including skeletal and cardiac muscle (5, 6), hepatocytes (7), osteoblasts (8), neutrophils (9), macrophages (10), keratinocytes (11), brown adipose tissue (12), CD34+ stem cells (13), endothelial cells (14), fibroblasts, and vascular smooth muscle cells (15). VEGF expression is induced by hypoxia and cytokines such as IL-1, IL-6, IL-8, oncostatin M, and TNF-alpha (3, 4, 9, 16). VEGF isoforms are differentially expressed during development and in the adult (3).
VEGF dimers bind to two related receptor tyrosine kinases, VEGF R1 (also called Flt-1) and VEGF R2 (Flk-1/KDR), and induce their homodimerization and autophosphorylation (3, 4, 7, 17, 18). These receptors have seven extracellular immunoglobulin-like domains and an intracellular split tyrosine kinase domain. They are expressed on vascular endothelial cells and a range of non-endothelial cells. Although VEGF affinity is highest for binding to VEGF R1, VEGF R2 appears to be the primary mediator of VEGF angiogenic activity (3, 4). VEGF165 also binds the semaphorin receptor, neuropilin-1, which promotes complex formation with VEGF R2 (19).
VEGF is best known for its role in vasculogenesis. During embryogenesis, VEGF regulates the proliferation, migration, and survival of endothelial cells (3, 4), thus regulating blood vessel density and size, but playing no role in determining vascular patterns. VEGF promotes bone formation through osteoblast and chondroblast recruitment and is also a monocyte chemoattractant (20-22). After birth, VEGF maintains endothelial cell integrity and is a potent mitogen for micro- and macro-vascular endothelial cells. In adults, VEGF functions mainly in wound healing and the female reproductive cycle (3). In diseased tissues, VEGF promotes vascular permeability. It is thus thought to contribute to tumor metastasis by promoting both extravasation and tumor angiogenesis (23, 24). Various strategies have been employed therapeutically to antagonize VEGF-mediated tumor angiogenesis (25). Circulating VEGF levels correlate with disease activity in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus (26).
Long Name:
Vascular Endothelial Growth Factor
Entrez Gene IDs:
7422 (Human); 22339 (Mouse); 83785 (Rat); 281572 (Bovine); 403802 (Canine); 493845 (Feline); 30682 (Zebrafish)
Alternate Names:
MVCD1; VAS; vascular endothelial growth factor A; Vascular permeability factor; Vasculotropin; VEGF; VEGFA; VEGF-A; VEGFMGC70609; VPF; VPFvascular endothelial growth factor
研究领域
制备和贮存
保存方式
Preparation and Storage
Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
参考图片
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危险品化学品经营许可证(不带存储) 许可证编号：沪(杨)应急管危经许[2022]202944(QY) 
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