Research Use Only

VEGF₁₆₅ ELISA

Intended Use
The VEGF₁₆₅ ELISA is an enzyme-linked immunoassay for the quantitation of the 165 amino acid length human vascular endothelial growth factor (VEGF₁₆₅) in human serum and plasma. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Background
Vascular endothelial growth factor (VEGF), a member of the PLGF/VEGF family, is a mitogen specific to the growth and maintenance of endothelial cells found in the blood vasculature and lymphatic system. There are five known variants of the VEGF ligand, named VEGF-A, -B, -C, -D, and –E [1]. All members regulate some aspect of angiogenesis. VEGF-A exists as five distinct circulating and cell-associated soluble forms, designated 121, 145, 165, 189, and 206, according to the number of amino acids present. VEGF₁₆₅ has been shown to be the predominant form found in circulation as well as the most mitogenic compared to its soluble counterpart, VEGF121 (which exhibits 50–100 fold less activity) [2,3].

Numerous studies with serum or plasma have shown an elevated expression of VEGF in a wide variety of cancers and diseases [4]. Jacobsen, et al., found that serum VEGF was a reliable predictor of survival in patients with renal cell carcinoma [5]. Lissoni, et al., saw that mean serum VEGF levels were higher in metastatic patients than in controls or nonmetastatic patients when examining a cohort of different cancer types [6]. Yamaguchi, et al., studied the relation of histological grade to VEGF expression in hepatocellular carcinoma (HCC) and found a direct correlation. They also found that various cytokines and growth factors could act to enhance VEGF expression in hepatocellular carcinoma, such as tumor necrosis factor-alpha and IFN-alpha [7]. Recently published studies, such as DeVita, et al., show that monitoring pre- and postoperative serum VEGF levels could be a useful predictor of outcome in patients with colon carcinoma who undergo surgery. They found that postoperative VEGF serum levels dropped significantly after surgery. Regression analysis selected preoperative serum VEGF levels as the variable that was significantly associated with the prediction of both disease-specific survival and disease-free survival [8].

Studies have also shown that monitoring VEGF₁₆₅ levels is of importance with respect to clinical course. Various studies suggest that determining a course of therapy for breast cancer patients based on an aggressive phenotype exhibited by HER-2/neu overexpression in association with VEGF expression would be beneficial. Based on current literature, Pegram, et al., hypothesized that new therapeutic molecules targeting VEGF may have unique activity against HER-2-overexpressing breast cancers [9]. Konecny, et al., found that the positive association between HER-2/neu and VEGF expression supports the use of combination therapies directed against both HER-2/neu and VEGF for the treatment of breast cancers that overexpress HER-2/neu [10].

Recently, VEGF₁₆₅ has been implicated in motor neuron degeneration and its effects are important in certain neurodegenerative disorders such as ALS. Oosthuyse, et al., reported that deletion of the hypoxia-response element in the VEGF promoter reduced hypoxic VEGF expression in the spinal cord and caused adult-onset progressive motor neuron degeneration. In addition, they found that VEGF₁₆₅ promoted the survival of motor neurons during hypoxia through binding to VEGFR-2 and neuropilin-1 [11]. VEGF also has a major role in the pathogenesis of many diseases, including hypervascularized tumors, rheumatoid arthritis, cutaneous diseases, and proliferative retinopathies [12]. The molecular and biological effects of each isoform of VEGF are still not completely known, therefore the ability to examine each isoform separately will help to elucidate the full implications of this pathway.

The VEGF₁₆₅ ELISA is designed to provide the investigator with a convenient, accurate, and reproducible method to determine VEGF₁₆₅ levels in human serum and plasma. Clinical research using the VEGF₁₆₅ ELISA may help to better define the role of VEGF₁₆₅ in cancer and other diseases.

Assay Principle
The VEGF₁₆₅ ELISA is a sandwich type immunoassay that uses a mouse monoclonal Capture Antibody and a biotinylated mouse monoclonal antibody as detector. The Capture Antibody recognizes a protein domain on the VEGF₁₆₅-specific isoform and does not cross-react with the VEGF121-specific isoform. The Capture Antibody has been immobilized on the interior surface of the microtiter plate wells. To perform the test, an appropriate volume of specimen is incubated in the wells to allow binding of the antigen by the Capture Antibody. The immobilized antigen is then exposed to the biotinylated Detector Antibody. Addition of streptavidin alkaline-phosphatase-labeled Conjugate to the wells leads to the availability of enzyme to act upon the Substrate. Addition of the Substrate allows the catalysis of a chromogen into a colored product, the intensity of which is proportional to the amount of VEGF₁₆₅ that is bound to the plate.

Standards are provided in the kit that allow accurate, quantitative determinations of VEGF₁₆₅ in suitable samples. Using a microtiter plate reader, one can measure simultaneously the absorbance of the colored product in the Standards and sample wells. Correlating the absorbance values of samples with the Standards allows the investigator to determine the levels of VEGF₁₆₅ in a sample. Samples are then assigned a quantitative value of VEGF₁₆₅ in picograms per mL (pg/mL) of serum and plasma.

Selected References

1. Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. Journal of Cell Science 2001;114(Pt 5):853-865.
2. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nature Medicine 2003;9(6):669-676.
3. Ferrara N. Molecular and biological properties of vascular endothelial growth factor. Journal of Molecular Medicine 1999;77(7):527-543.
4. Osella-Abate S, Quaglino P, Savoia P, et al. VEGF-165 serum levels and tyrosinase expression in melanoma patients: Correlation with the clinical course. Melanoma Res 2002;12(4):325-334.
5. Jacobsen J, Grankvist K, Rasmuson T, et al. Prognostic importance of serum vascular endothelial growth factor in relation to platelet and leukocyte counts in human renal cell carcinoma. Eur J Cancer Prev 2002;11(3):245-252.
6. Lissoni P, Malugani F, Bonfanti A, et al. Abnormally enhanced blood concentrations of vascular endothelial growth factor (VEGF) in metastatic cancer patients and their relation to circulating dendritic cells, IL-12 and Endothelin-1. J Biol Regul Homeost Agents 2001;15(2):140-144.
7. Yamaguchi R, Yano H, Iemura A, et al. Expression of vascular endothelial growth factor in human hepatocellular carcinoma. Hepatology 1998;28(1):68-77.
8. DeVita F, Orditura M, Lieto E, et al. Elevated perioperative serum vascular endothelial growth factor levels in patients with colon carcinoma. Cancer 2004;100(2):270-278.
9. Pegram MD, Reese DM. Combined biological therapy of breast cancer using monoclonal antibodies directed against HER-2/neu protein and vascular endothelial growth factor. Semin Oncol 2002;29(3 Suppl 11):29-37.
10. Konecny GE, Meng G, Untch M, et al. Association between HER-2/neu and vascular endothelial growth factor expression predicts clinical outcome in primary breast cancer patients. Clinical Cancer Res 2004; 10(5):1706-1716.
11. Oosthuyse B, Moons L, Storkebaum E, et al. Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet 2001;28(2):131-138.
12. Amoroso A, Del Porto F, Di Monaco C, et al. Vascular endothelial growth factor: A key mediator of neoangiogenesis. A review. Eur Rev Med Pharmacol Sci 1997;1(1-3):17-25.