Abstract
Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays an important role in physiological and tumor angiogenesis. The human VEGF gene has eight exons. Different VEGF isoforms are expressed via alternative RNA splicing and VEGF121 and VEGF165 are the major isoforms present in human tissues. The exact roles of these different VEGF isoforms are not totally clear. Assays to detect specific VEGF isoforms in biological samples are needed to understand the biological functions of these different VEGF isoforms and to better assess their potential use as predicative biomarkers for anti-angiogenic therapy. Because monoclonal antibodies specific to different VEGF isoforms are lacking, we used antibodies directed to different epitopes on VEGF165 in a set of three enzyme-linked immunosorbent assays (ELISAs) to assess the amount of VEGF121 and VEGF165 as well as VEGF110, which can be generated by plasmin cleavage in vivo. The first ELISA detects VEGF165. The second ELISA detects both VEGF121 and VEGF165. The third ELISA detects VEGF165, VEGF121, and VEGF110. The concentrations of VEGF121 can be assessed from the difference in VEGF concentrations measured by the second and the first ELISAs; the concentrations of VEGF110 can be assessed from the difference in VEGF concentrations measured by the third and the second ELISAs. The same assay strategy may be used to assess the amount of other VEGF isoforms if antibodies directed against the desired amino acids in those isoforms can be obtained.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ferrara N (2004) Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 25:581–611
Takahashi H, Shibuya M (2005) The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clin Sci 109:227–241
Longo R, Gasparini G (2007) Challenges for patient selection with VEGF inhibitors. Cancer Chemother Pharmacol 60:151–170
Linderholm BK, Lindahl T, Holmberg L et al (2001) The expression of vascular endothelial growth factor correlates with mutant p53 and poor prognosis in human breast cancer. Cancer Res 61:2256–2260
Escudier B, Eisen T, Stadler WM et al (2009) Sorafenib for treatment of renal cell carcinoma: final efficacy and safety results of the phase III treatment approaches in renal cancer global evaluation trial. J Clin Oncol 27:3312–3318
Dowlati A, Gray R, Sandler AB et al (2008) Cell adhesion molecules, vascular endothelial growth factor, and basic fibroblast growth factor in patients with non-small cell lung cancer treated with chemotherapy with or without bevacizumab – an Eastern Cooperative Oncology Group Study. Clin Cancer Res 14:1407–1412
Ferrara N, Hillan KJ, Gerber HP et al (2004) Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 3:391–400
Hurwitz H, Fehrenbacher L, Novotny W et al (2004) Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350:2335–2342
Sandler A, Gray R, Perry MC et al (2006) Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 355:2542–2550
Escudier B, Pluzanska A, Koralewski P et al (2007) Bevacizumab plus interferon alfa-2a for treatment of metastatic renal cell carcinoma: a randomised, double-blind phase III trial. Lancet 370:2103–2111
Houck KA, Ferrara N, Winer J et al (1991) The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 5:1806–1814
Tischer E, Mitchell R, Hartman T et al (1991) The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 266:11947–11954
Fujimoto J, Sakaguchi H, Hirose R et al (1999) Expression of vascular endothelial growth factor (VEGF) and its mRNA in uterine cervical cancers. Br J Cancer 80:827–833
Robinson CJ, Stringer SE (2001) The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci 114:853–865
Stimpfl M, Tong D, Fasching B et al (2002) Vascular endothelial growth factor splice variants and their prognostic value in breast and ovarian cancer. Clin Cancer Res 8:2253–2259
Bates DO, Cui TG, Doughty JM et al (2002) VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, is down-regulated in renal cell carcinoma. Cancer Res 62:4123–4131
Woolard J, Wang WY, Bevan HS et al (2004) VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression. Cancer Res 64:7822–7835
Harper SJ, Bates DO (2008) VEGF-A splicing: the key to anti-angiogenic therapeutics? Nat Rev Cancer 8:880–887
Catena R, Larzabal L, Larrayoz M et al (2010) VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A. Mol Cancer 9:320
Keyt BA, Nguyen HV, Berleau LT et al (1996) Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis. J Biol Chem 271:5638–5646
Houck KA, Leung DW, Rowland AM et al (1992) Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem 267:26031–26037
Plouet J, Moro F, Bertagnolli S et al (1997) Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic effect. J Biol Chem 272:13390–13396
Keyt BA, Berleau LT, Nguyen HV et al (1996) The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem 271:7788–7795
Yuan A, Yu CJ, Kuo SH et al (2001) Vascular endothelial growth factor 189 mRNA isoform expression specifically correlates with tumor angiogenesis, patient survival, and postoperative relapse in non-small-cell lung cancer. J Clin Oncol 19:432–441
Bando H, Weich HA, Brokelmann M et al (2005) Association between intratumoral free and total VEGF, soluble VEGFR-1, VEGFR-2 and prognosis in breast cancer. Br J Cancer 92:553–561
Jayson GC, Hicklin DJ, Ellis LM (2012) Antiangiogenic therapy—evolving view based on clinical trial results. Nat Rev Clin Oncol 9:297–303
Van Cutsem E, de Haas S, Kang YK et al (2012) Bevacizumab in combination with chemotherapy as first-line therapy in advanced gastric cancer: a biomarker evaluation from the AVAGAST randomized phase III trial. J Clin Oncol 30:2119–2127
Gutierrez J, Konecny GE, Hong K et al (2008) A new ELISA for use in a 3-ELISA system to assess concentrations of VEGF splice variants and VEGF(110) in ovarian cancer tumors. Clin Chem 54(3):597–601
Rodriguez CR, Fei DT, Keyt B et al (1998) A sensitive fluorometric enzyme-linked immunosorbent assay that measures vascular endothelial growth factor165 in human plasma. J Immunol Methods 219:45–55
Shifren JL, Tseng JF, Zaloudek CJ et al (1996) Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 81:3112–3118
Bjerner J, Olsen KH, Bormer OP et al (2005) Human heterophilic antibodies display specificity for murine IgG subclasses. Clin Biochem 38:465–472
Zimmermann R, Koenig J, Zingsem J et al (2005) Effect of specimen anticoagulation on the measurement of circulating platelet-derived growth factors. Clin Chem 51:2365–2368
Muller YA, Li B, Christinger HW et al (1997) Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site. Proc Natl Acad Sci 94:7192–7197
Breier G, Albrecht U, Sterrer S et al (1992) Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development 114:521–532
Gerber HP, Wu X, Yu L et al (2007) Mice expressing a humanized form of VEGF-A may provide insights into the safety and efficacy of anti-VEGF antibodies. Proc Natl Acad Sci 104:3478–3483
Mineur P, Colige AC, Deroanne CF et al (2007) Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents. J Cell Biol 179:1261–1273
Acknowledgements
We thank the Protein Chemistry department for VEGF110, the Antibody Engineering department for the mouse monoclonal antibodies directed against VEGF, and Genentech reviewers for critically reviewing this manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Vernes, JM., Meng, Y.G. (2015). Detection and Quantification of VEGF Isoforms by ELISA. In: Fiedler, L. (eds) VEGF Signaling. Methods in Molecular Biology, vol 1332. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2917-7_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2917-7_2
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2916-0
Online ISBN: 978-1-4939-2917-7
eBook Packages: Springer Protocols