Rat VEGF Quantikine ELISA Kit Summary

Product Summary

The Quantikine Rat VEGF Immunoassay is a 3.5 hour solid-phase ELISA designed to measure rat VEGF in cell culture supernates, serum, and plasma. It contains NS0-expressed recombinant rat VEGF and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant factor. Results obtained using natural rat VEGF showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring rat VEGF.

Preparation and Storage

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 in adults (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 formation of anti-parallel disulfide-linked dimers (4). Alternately spliced isoforms of 120, 164, and 188 amino acids (aa) have been found in rats and mice, while 121, 145, 165, 183, 189, and 206 aa isoforms have been identified in humans (2, 4). In humans, VEGF₁₆₅ appears to be the most abundant and potent isoform, followed by VEGF₁₂₁ and VEGF₁₈₉ (3, 4). The same pattern may exist in rats and mice. Isoforms other than VEGF₁₂₀ and VEGF₁₂₁ contain basic heparin-binding regions and are not freely diffusible (4). Rat VEGF164 shares 97% aa sequence identity with corresponding regions of mouse, 88% with human and bovine, 89% with porcine and canine, and 90% with feline and equine 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). The 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, 16, 17). 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 (18). 

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 (19-21). In postnatal life, 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 (22, 23). Various strategies have been employed therapeutically to antagonize VEGF-mediated tumor angiogenesis (24). Circulating VEGF levels correlate with disease activity in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus (25).

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.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.