Download biomedica product listHuman VEGF ELISA Kit | BI-VEGF
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Method
Sandwich ELISA, HRP/TMB, 12×8-well detachable strips
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Sample type
Serum, EDTA plasma, citrate plasma, cell culture supernatants, urine
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Sample volume
10 µl sample / well
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Assay time
2 h / 1 h / 1h /30 min
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Sensitivity
LOD: 2.5 pg/ml; LLOQ: 15.6 pg/ml (measurable concentrations in serum AND plasma samples)
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Standard range
0 – 2000 pg/ml (0 / 31.25 / 62.5 / 125 / 250 / 500 / 1000 / 2000)
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Precision
In-between-run: (n=3): ≤ 6 % CV
Within-run (n=3): ≤ 3 % CV
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Specificity
This assay recognizes recombinant and endogenous (natural) human VEGF including all circulating VEGF isoforms (incl. VEGF165b)
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Validation Data
See validation data tab for: precision, accuracy, dilution linearity, values for healthy donors, etc
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Use
Research use only
Human VEGF ELISA Product Overview
The Biomedica human Vascular Endothelial Growth Factor (VEGF) ELISA kit is a 4.5 hour 96-well sandwich enzyme immunoassay for the quantitative determination of human VEGF in serum, plasma (EDTA, citrate) , cell culture supernatants, and urine samples.
The VEGF ELISA assay recognizes both natural and recombinant human VEGF with high sensitivity. The assay employs highly specific epitope mapped antibodies as well as eight human serum-based standards and two controls to ensure the measurement of biologically reliable data.
Human VEGF ELISA Assay Principle
The human Vascular Endothelial Growth Factor (VEGF) ELISA kit is a sandwich enzyme immunoassay that has been optimized and fully validated for the quantitative determination of human VEGF in serum, EDTA-plasma, and citrate plasma. Validation experiments have been performed according to international quality guidelines (ICH/ FDA/ EMEA). Cell culture supernatant and urine samples are compatible with this ELISA. The VEGF ELISA assay recognizes both natural and recombinant human VEGF.
The figure below explains the principle of the human VEGF sandwich ELISA:
Capture antibody: recombinant anti-human VEGF antibody
Detection antibody: polyclonal anti-human VEGF antibody, Biotin-labeled
Target antigen: human VEGF
This kit is a sandwich enzyme immunoassay for the quantitative determination of human VEGF (Vascular Endothelial Growth Factor) in human serum, plasma, cell culture supernatants, and urine samples. In a first step, assay buffer is pipetted into the wells of the microtiter strips. Thereafter, STD/sample/CTRL are pipetted into the wells, which are pre-coated with the recombinant anti-human VEGF antibody. Any soluble VEGF present in the STD/sample/CTRL binds to the pre-coated anti-VEGF antibody in the well. After incubation, a washing step is applied where all non-specific unbound material is removed. In a next step, the biotinylated anti-VEGF antibody (AB) is pipetted into the wells and reacts with the VEGF present in the sample, forming a sandwich. Next, all unbound antibody is removed during another washing step. In the next step, the conjugate (streptavidin-HRPO) is added and reacts with the biotinylated anti-VEGF antibody. After another washing step, the substrate (Tetramethylbenzidine; TMB) is pipetted into the wells. The enzyme catalysed colour change of the substrate is directly proportional to the amount of VEGF present in the sample. This colour change is detectable with a standard microtiter plate ELISA reader. A dose response curve of the absorbance (optical density, OD at 450 nm) versus standard concentration is generated, using the values obtained from the standards. The concentration of soluble VEGF in the sample is determined directly from the dose response curve.
Typical Data
This standard curve and the displayed O.D. values are for demonstration only. A standard curve should be generated for each assay run.
| Standard | VEGF pg/ml | OD | |||
| #1 | #2 | Average | CV% | ||
| STD1 | 0 | 0.098 | 0.090 | 0.094 | 6 |
| STD2 | 31.25 | 0.151 | 0.157 | 0.154 | 3 |
| STD3 | 62.5 | 0.210 | 0.218 | 0.214 | 3 |
| STD4 | 125 | 0.322 | 0.322 | 0.322 | 0 |
| STD5 | 250 | 0.557 | 0.545 | 0.551 | 2 |
| STD6 | 500 | 0.969 | 0.928 | 0.949 | 3 |
| STD7 | 1000 | 1.577 | 1.619 | 1.619 | 4 |
| STD8 | 2000 | 2.894 | 2.894 | 2.894 | 0 |
The quality control protocol supplied with the kit shows the results of the final release QC for each kit at the production date. ODs obtained by customers may differ due to various influences including a normal decrease of signal intensity throughout shelf life. However, this does not affect the validity of the results, provided an OD of 1.50 or higher is obtained for the standard with the highest concentration, and the measured control values fall into their respective target range (see labels).
VEGF ELISA Kit Components
All reagents supplied in the human VEGF ELISA kit are stable at 2-8°C until the expiry date stated on the label of each reagent. Reconstituted reagents must be stored at -25°C until the expiry date.
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Contents |
Description |
Quantity |
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PLATE |
Microtiter strips coated with recombinant VEGF antibody specific for human VEGF in strip holder packed in an aluminum bag with desiccant |
12 x 8 tests |
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WASHBUF |
20x wash buffer concentrate, transparent cap |
1 x 50 ml |
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STD |
Recombinant VEGF165 standards (0 / 31.25 / 62.5 / 125 / 250/ 500 / 1000 / 2000 pg/ml), human serum based, white caps, lyophilized |
8 vials |
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CTRL |
Control A and B, human serum based, yellow cap, lyophilized, exact concentration is stated on labels |
2 vials |
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ASYBUF |
Assay buffer, red cap, ready to use |
1 x 18 ml |
|
AB |
Polyclonal VEGF antibody specific for human VEGF, biotin-labeled, green cap, ready to use |
1 x 13 ml |
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CONJ |
Conjugate (streptavidin-HRPO), brown cap, ready to use |
1 x 13 ml |
|
SUB |
Substrate (TMB solution), blue cap, ready to use |
1 x 13 ml |
|
STOP |
Stop solution, white cap, ready to use |
1 x 7 ml |
Serum, EDTA plasma, citrate plasma, cell culture supernatants, and urine samples are suitable for use in this assay. Do not change sample type during studies. We recommend duplicate measurements for all samples, standards and controls. The sample collection and storage conditions listed are intended as general guidelines.
Serum & Plasma
Collect venous blood samples by using standardized blood collection tubes. Perform plasma or serum separation by centrifugation according to supplier’s instructions of the blood collection devices. Assay the acquired samples immediately or aliquot and store at -25°C or lower. Lipemic or haemolyzed samples may give erroneous results. Samples are stable for up to five freeze-thaw cycles. Samples should be mixed well before assaying. We recommend duplicates for all values.
Cell Culture Supernatant
Cell culture supernatants should contain at least 1% fetal bovine serum for stability of the VEGF. Remove particles by centrifugation and assay immediately or aliquot and store samples at ≤ -25 °C or lower. Avoid repeated freeze-thaw cycles.
Urine
Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particles matter, assay immediately or aliquot and store at -25°C or lower. Avoid repeated freeze-thaw cycles.
Reagent Preparation
Wash Buffer
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1. |
Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature (18-26°C) |
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2. |
Dilute the WASHBUF concentrate 1:20, e.g. 50 ml WASHBUF + 950 ml distilled or deionized water. Only use diluted WASHBUF when performing the assay. |
The diluted WASHBUF is stable up to one month at 4°C (2-8°C).
STANDARDS & CONTROLS FOR SERUM, PLASMA, CELL CULTURE SUPERNATANTS, AND URINE MEASUREMENTS
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1. |
Pipette 200 µl of distilled or deionized water into each standard (STDs) and control (CTRL) vial. The exact concentration is printed on the label of each vial. |
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2. |
Leave at room temperature (18-26°C) for 15 min. Vortex gently. |
Reconstituted STDs and CTRLs are stable -25°C or lower until expiry date stated on the label. STDs and CTRLs are stable up to five freeze-thaw cycles.
The standards and controls provided in the kit are suitable for all sample types.
Sample Preparation
Bring samples to room temperature and mix samples gently to ensure the samples are homogeneous. We recommend duplicate measurements for all samples.
Samples with analyte concentrations outside of the calibration range of the assay (2000 pg/ml) should be diluted with assay buffer (ASYBUF) and remeasured.
Human VEGF ELISA Assay Protocol
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Read the entire instructions for use before beginning the assay. |
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All reagents and samples must be at room temperature (18-26°C) before use in the assay. |
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Mark position for STD/CTRL/SAMPLE (standard/control/sample) on the protocol sheet. |
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Take microtiter strips out of the aluminium bag. Store unused strips with desiccant at 4°C (2-8°C) in the aluminium bag. Strips are stable until expiry date stated on the label. |
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1. |
Pipette 100 µl ASYBUF (Assay Buffer, natural cap) into each well |
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2. |
Add 10 ul of STD/SAMPLE/CTRL (Standard, Sample/Control) in duplicate into respective well. Swirl gently. |
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3. |
Cover tightly and incubate for 2 hours at room temperature (18-26°C). |
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4. |
Aspirate and wash wells 5x with 300 µl diluted WASHBUF (Wash buffer, natural cap). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel. |
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5. |
Add 100 µl AB (biotinylated anti-VEGF antibody, green cap) into each well, swirl gently. |
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6. |
Cover tightly and incubate for 1 hour at room temperature (18-26°C). |
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7. |
Aspirate and wash wells 5x with 300 µl diluted WASHBUF (Wash buffer, natural cap). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel. |
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8. |
Add 100 µl CONJ (Conjugate, amber cap) into each well, swirl gently. |
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9. |
Cover tightly and incubate for 1 hour at room temperature (18-26°C). |
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10. |
Aspirate and wash wells 5x with 300 µl diluted WASHBUF (Wash buffer, natural cap). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel.. |
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11. |
Add 100 µl SUB (Substrate, blue cap) into each well, swirl gently. |
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12. |
Incubate for 30 min at room temperature (18-26°C), in the dark. |
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13. |
Add 50 μl STOP (stop solution, white cap) into each well, swirl gently. |
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14. |
Measure absorbance immediately at 450 nm with reference 630 nm, if available. |
Calculation of Results
Construct a standard curve from the absorbance read-outs of the standards using commercially available software capable of generating a four-parameter logistic (4-PL) fit.
Alternatively, plot the standards’ concentration on the x-axis against the mean absorbance for each standard on the y-axis and draw a best fit curve through the points on the graph.
Curve fitting algorithms other than 4-PL have not been validated and will need to be evaluated by the user.
Obtain sample concentrations from the standard curve. If required, pg/ml can be converted into pmol/l by applying a conversion factor ( 1pg/ml=0.052pmol/l).
Concentrations of high-measuring samples that have been diluted during sample preparation must be multiplied by the dilution factor.
Vascular endothelial growth factor (VEGF or VEGF-A), is a growth hormone secreted by endothelial cells, fibroblasts, smooth muscle cells, platelets, macrophages, and many other cell types. It belongs to the cysteine-knot growth factor superfamily (Peach C et al. ) and has a molecular weight of about 40 kDa.
Up to date 17 different VEGF isoforms were described to be expressed from one single gene. They are produced by alternative promoter usage/initiation or alternative splicing/proteolysis after protein translation. The N-terminal region is responsible for receptor binding and conserved among all VEGF isoforms. In contrast, residues of the C-terminus differ between isoforms and determine protein length and properties: binding to co-receptor Neuropilin-1 (NRP1) or to extracellular matrix (ECM), agonist/antagonist of angiogenesis. Most isoforms result from the common transcripts: VEGF111, VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206. Additionally, a third VEGF variant (VEGFAx), that demonstrates pro- and anti-apoptotic properties, was described. Thus, vascularization is tightly controlled by the balance of various splice variants, their availability and concentration, whereas isoforms linked to the ECM constitute a reservoir of VEGF that can quickly be shed to circulating forms (Peach C et al.).
One of the most potent pro-angiogenic isoforms is VEGF165a. After secretion, 50-70% of VEGF165a is attached to the extracellular matrix (via heparin binding site), the rest is freely diffusible ( Peach C et al.). It is the most abundant isoform and enhances signaling over the VEGFR2 receptor by additionally binding to its co-receptor Neuropilin-1.
VEGF A isoforms are glycosylated, homodimeric proteins. Two anti-parallel monomers are linked by intermolecular disulfide bonds (Wiesmann C et al.), whereas eight cysteine residues form a knot-like structure at one end of each monomer (Tjwa M et al.). However, heterodimerization with PLGF has been described as well (Olsson AK et al.)
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Full Name |
VEGFA or VEGF |
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Length |
232aa canonical sequence (412-137) |
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Structure |
Homodimer, Heterodimer with PGF |
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Molecular weight |
22.3kDa (VEGF165a) , 27kDa/monomer (45.5 – 16 kDa) |
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Ligands |
VEGF R1 (FLT1), VEGF R2 (KDR), NRP-1, heparan sulfate, heparin |
| Cellular localisation |
Cell-associated or secreted |
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Post-translational modifications |
5 SH bonds (52-94, 77, 83-128, 86, 87-130), N-glycosylated (101), alternatively spliced (17 known isoforms and 15 computationally mapped) |
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Alternative names |
Vascular Endothelial Growth Factor, VEGF-A, VEGFA, VPF, MVCD1, Vascular endothelial growth factor A, vascular endothelial growth factor A121, vascular endothelial growth factor A165, Vascular permeability factor, VEGF-A, VPF |
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Entrez/NCBI ID |
Gene ID: 7422 https://www.ncbi.nlm.nih.gov/gene/7422 |
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Genecards |
VEGFA |
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OMIM |
192240 |
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Protein Structure |
Member of cysteine-knot family Anti-parallel homodimer covalently linked by 2 SH (Cys-51, Cys-60) |
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PDB / Pfam |
1FLT / VEGFA_HUMAN |
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Protein Atlas |
Protein VEGFA: https://www.proteinatlas.org/ENSG00000112715-VEGFA#gene_information Gene Name: VEGF (synonymes: VEGF, VEGF-A, VPF) |
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Sequence similarities |
VEGF family – cysteine knot superfamily |
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Expression |
Vascular endothelial cells, fibroblasts, smooth muscle cells, platelets, neutrophils, macrophages |
VEGF Function
Vascular Endothelial Growth Factor (VEGF or VEGFA) has an important role in vasculogenesis/angiogenesis stimulating cell survival, migration and proliferation of endothelial cells (Peach C et al. , Apte RS et al.). Apart from its vascular role, VEGF is also involved in skeletal bone formation and bone repair (Hu K et al.) and in the development/homeostasis of many other organs (respiratory, nervous, renal, heart, reproductive system etc.) (Tjwa M et al., Braile M et al. ) Although VEGF is essential for physiologic vascular homeostasis in diverse cells and tissues, it has been demonstrated to be important in the molecular pathogenesis of tumor growth and metastasis and in retinopathy associated with several blinding eye diseases, including age-related macular degeneration (AMD) and diabetic and hypertensive retinopathy (Ferrara N et al.)
Changes of the cellular environment (e.g. oxygen level, presence of other growth factors, cytokines, shear stress etc.) induce VEGF and VEGF receptor expression.
VEGF receptors VEGFR1 and VEGFR2 are tyrosine kinases on vascular/non-vascular cells that are essential for transducing VEGF signals. Homo-/heterodimerization of receptors is a prerequisite for VEGF ligand binding. Upon receptor homo-/heterodimerization, VEGF binds and leads to the phosphorylation of the intracellular receptor domains, thus activating the signal transduction cascade (Wu FTH et al.). VEGF induces pro-angiogenic signals mainly via VEGFR2: sprouting angiogenesis and endothelial cell survival. In contrast, VEGFR1 works as a decoy receptor for circulating VEGF-A and exists as soluble form as well. Furthermore, it can dimerize and inactivate other receptor monomers as it lacks the cytoplasmatic, transducing domain. VEGF has a higher affinity for its decoy receptor than for the main signaling receptor. Additionally, there are several matrix proteins in the extracellular space that contain VEGF binding sites. The glycoproteins NRP1 and NRP2 are considered as co-receptors that are non signaling (Wu FTH et al.).
VEGF-mediated pathogenic effects are primarily due to its effects on vascular permeability , and neoangiogenesis-neovascularization (Apte RS et al). A number of therapeutic approaches have thus targeted one or more isoforms of VEGF, the VEGF receptors, or signaling pathways, and some have since led to approval of drugs by regulatory authorities around the world (Ferrara amd Adamis, 2016).
Areas of interest:
- Cancer
- Metabolic disease (diabetes and diabetic kidney disease, diabetic retinopathy, obesity)
- Retinal Diseases
- Autoimmune & inflammatory disease (rheumatoid arthritis, psoriasis, psoriatic arthritis)
- Heart and cardiovascular disease
- Skeletal bone formation and bone repair
Literature:
Molecular Pharmacology of VEGF-A Isoforms: Binding and Signalling at VEGFR2
Peach C., Mignone V., Arruda M., Alcobia D., Hill S., Kilpatrick L., et. a. 2018. Int J Mol Sci.
Crystal Structure at 1.7 Å Resolution of VEGF in Complex with Domain 2 of the Flt-1 Receptor
Wiesmann C., Fuh G., Christinger HW,. Eigenbrot C., Wells JA., de Vos AM. Cell. 1997; 91(5): 695–704
VEGF and PlGF: two pleiotropic growth factors with distinct roles in development and homeostasis
Tjwa M, Luttun A, Autiero M, Carmeliet P. Cell Tissue Res. 2003; 314(1): 5–14.
VEGF receptor signalling ? in control of vascular function.
Olsson A-K, Dimberg A, Kreuger J, Claesson-Welsh L.,Nat Rev Mol Cell Biol. 2006; 7(5): 3; 59–71.
VEGF in Signaling and Disease: Beyond Discovery and Development.
Apte, Rajendra S., Daniel S. Chen, und Napoleone Ferrara, Cell. 2019; 176, 6: 1248–64.
The roles of vascular endothelial growth factor in bone repair and regeneration.
Hu K, Olsen BR.,Bone. 2016; 91:3 0–8.
VEGF-A in Cardiomyocytes and Heart Diseases.
Braile M, Marcella S, Cristinziano L, Galdiero MR, Modestino L, Ferrara AL, Varricchi G, Marone G, Loffredo S.,Int J Mol Sci. 2020; 26;21(15).
VEGF and Intraocular Neovascularization: From Discovery to Therapy.
Ferrara N.,Translational Vision Science & Technology. 2016a; 5, 2: 10.
A systems biology perspective on sVEGFR1: its biological function, pathogenic role & therapeutic use.
Wu FTH, Stefanini MO, Gabhann FM, Kontos CD, Annex BH, Popel AS.,J Cell Mol Med. 2010; 14(3): 528-52.
Ten Years of Anti-Vascular Endothelial Growth Factor Therapy.
Ferrara, N and Adamis AP., Nature Reviews Drug Discovery. 2016b: 15, 6: 385–403.
Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF.
Banks RE, Forbes MA, Kinsey SE, Stanley A, Ingham E, et al., Br J Cancer. 1998; 77:956–964.
Thrombocytes Are the Major Source for Soluble Vascular Endothelial Growth Factor in Peripheral Blood.
Gunsilius E, Petzer A, Stockhammer G, Nussbaumer W, Petra Schumacher P, Clausen J, Gast G., Oncology. 2000; 58, 2: 169–74.
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Cancer
VEGF as a potential target in lung cancer.
Daniela Frezzetti , Marianna Gallo, Monica R Maiello , Amelia D'Alessio, Claudia Esposito, Nicoletta Chicchinelli, Nicola Normanno , Antonella De Luca. Expert Opin Ther Targets- 2017: Oct;21(10):959-96.
Molecular insight of regorafenib treatment for colorectal cancer.
Arai H, Battaglin F, Wang J, Lo JH, Soni S, Zhang W, Lenz HJ. Cancer Treat Rev. 2019 Dec;81:101912.
Contribution of Angiogenesis to Inflammation and Cancer
Aguilar-Cazares D, Chavez-Dominguez R, Carlos-Reyes A, Lopez-Camarillo C, Hernadez de la Cruz ON, Lopez-Gonzalez JS. Front Oncol. 2019 Dec 12;9:1399. doi: 10.3389/fonc.2019.01399.
Bevacizumab (Avastin®) in cancer treatment: A review of 15 years of clinical experience and future outlook.
Garcia J, Hurwitz HI, Sandler AB, Miles D, Coleman RL, Deurloo R, Chinot OL. Cancer Treat Rev. 2020 Jun;86:102017.
VEGF inhibition in urothelial cancer: the past, present and future.
Ghafouri S, Burkenroad A, Pantuck M, Almomani B, Stefanoudakis D, Shen J, Drakaki A. World J Urol. 2020 May 2. doi: 10.1007/s00345-020-03213-z.
Tumor angiogenesis: causes, consequences, challenges and opportunities.
Lugano R, Ramachandran M, Dimberg A. Cell Mol Life Sci. 2020 May;77(9):1745-1770. -
Cardiovascular Disease
VEGF-A in Cardiomyocytes and Heart Diseases.
Braile M, Marcella S, Cristinziano L, Galdiero MR, Modestino L, Ferrara AL, Varricchi G, Marone G, Loffredo S. Int J Mol Sci. 2020 Jul 26;21(15):5294.
Team-Based Approach to Management of Hypertension Associated with Angiogenesis Inhibitors.
Patel S, Dushenkov A, Jungsuwadee P, Krishnaswami A, Barac A.J. Cardiovasc Transl Res. 2020 Jun;13(3):463-477.
VEGF-A plasma levels are associated with microvascular obstruction in patients with ST-segment elevation myocardial infarction.
Garcia R, Bouleti C, Sirol M, Logeart D, Monnot C, Ardidie-Robouant C, Caligiuri G, Mercadier JJ, Germain S. Int J Cardiol. 2019 Sep 15;291:19-24. -
Inflammation
Rheumatoid arthritis / Osteoarthritis
Lee YH, Bae SC. Correlation between circulating VEGF levels and disease activity in rheumatoid arthritis: a meta-analysis. Z Rheumatol. 2018 Apr;77(3):240-248.
Relationship between VEGF Gene Polymorphisms and Serum VEGF Protein Levels in Patients with Rheumatoid Arthritis.
Paradowska-Gorycka A, Pawlik A, Romanowska-Prochnicka K, Haladyj E, Malinowski D, Stypinska B, Manczak M, Olesinska M. PLoS One. 2016 Aug 11;11(8):e0160769.
Targeting VEGF and its Receptors for the Treatment of Osteoarthritis and Associated Pain.
Hamilton JL, Nagao M, Levine BR, Chen Di, Olsen BR, Im HJ. J Bone Miner Res. 2016 May; 31(5): 911–924.
Psoriasis
Novel investigational vascular endothelial growth factor (VEGF) receptor antagonists for psoriasis.
Malecic N, Young HS. Expert Opin Investig Drugs. 2016;25(4):455-62.
Psoriatric arthritis
Psoriatic Arthritis: What is Happening at the Joint?
Belasco J, Wei N. N.Rheumatol Ther. 2019 Sep;6(3):305-315. -
Retinal Diseases
Innovative therapies for neovascular age-related macular degeneration.
Al-Khersan H, Hussain RM, Ciulla TA, Dugel PU. Expert Opin Pharmacother. 2019 Oct;20(15):1879-1891.
VEGF and Intraocular Neovascularization: From Discovery to Therapy.
Ferrara, Napoleone. Translational Vision Science & Technology 5, Nr. 2 (11. März 2016): 10.
Fundamental principles of an anti-VEGF treatment regimen: optimal application of intravitreal anti–vascular endothelial growth factor therapy of macular diseases.
Lanzetta P, Loewenstein A. The Vision Academy Steering Committee. Graefes Arch. Clin Exp Ophthalmol. 2017; 255(7): 1259–1273.
All Biomedica ELISAs are validated according to international FDA/ICH/EMEA guidelines. For more information about our validation guidelines, please refer to our quality page and published validation guidelines and literature.
- ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology.
- EMEA/CHMP/EWP/192217/2009 Guideline on bioanalytical method validation.
- Bioanalytical Method Validation, Guidance for Industry, FDA, May 2018
Calibration
The Biomedica human Vascular Endothelial Growth Factor (VEGF) ELISA kit is calibrated against a highly purified recombinant human VEGF165 protein (Ala27-Arg191; expressed in spodoptera frugiperda 21 ).
The calibrator is provided in eight lyophilized glass vials in the following concentrations: 0 / 31.25 / 62.5 / 125 / 250 / 500 / 1000 / 2000 pg/ml, and is human serum based.
CALIBRATION using WHO standard
The WHO reference reagent VEGF165/NIBSC code 02/286 (recombinant DNA, human sequence) was analysed in this human VEGF ELISA kit.
The equation below can be used to convert the sample values obtained with this kit to approximate WHO/VEGF165/NIBSC 02/286 units:
WHO/NIBSC (02/286) reference (U/ml) = 0.0006 x BI-VEGF value (pg/ml).
Human VEGF ELISA Detection Limit & Sensitivity
To determine the sensitivity of the VEGF ELISA, experiments measuring the Lower Limit of Detection (LOD) and the Lower Limit of Quantification (LLOQ) were conducted.
The LOD, also called the detection limit, is the lowest point at which a signal can be distinguished from the background signal, i.e., the signal that is measured in the absence of VEGF, with a confidence level of 99%. It is defined as the mean back-calculated concentration of standard 1 (0 pg/ml of VEGF, five independent measurements) plus three times the standard deviation of the measurements.
The LLOQ, or sensitivity of an assay, is the lowest concentration at which an analyte can be accurately quantified. The criteria for accurate quantification at the LLOQ are an analyte recovery between 75% and 125% and a coefficient of variation (CV) of less than 25%. To determine the LLOQ, standard 2, i.e., the lowest standards containing VEGF, is diluted, measured five times and its concentration back calculated. The lowest dilution, which meets both criteria, is reported as the LLOQ.
The following values were determined for the human VEGF ELISA:
|
LOD |
2.5 pg/ml |
|
LLOQ |
15.6 pg/ml |
Human VEGF ELISA Precision
The precision of an ELISA is defined as its ability to measure the same concentration consistently within the same experiments carried out by one operator (within-run precision or repeatability) and across several experiments using the same samples but conducted by several operators at different locations using different ELISA lots (in-between-run precision or reproducibility).
Within-Run Precision
Within-run precision was tested by measuring two samples of known concentrations three times within one VEGF ELISA lot by one operator.
|
ID |
n |
Mean VEGF [pg/ml] |
SD [pg/ml] |
CV (%) |
|
Sample 1 |
3 |
67.8 |
1.8 |
3 |
|
Sample 2 |
3 |
491.5 |
9.2 |
2 |
In-Between-Run Precision
Within-run precision was tested by measuring two samples of known concentrations three times within different VEGF ELISA lots by different operators.
|
ID |
n |
Mean VEGF [pg/ml] |
SD VEGF [pg/ml] |
CV (%) |
|
Sample 1 |
3 | 67.5 | 4.07 | 6 |
|
Sample 2 |
3 | 500.3 | 20.31 | 4 |
Human VEGF ELISA Accuracy
The accuracy of an ELISA is defined as the precision with which it can recover samples of known concentrations.
The recovery of the human Vascular Endothelial Growth Factor (VEGF) ELISA was measured by adding recombinant human VEGF to samples containing a known concentration of endogenous VEGF. The %recovery of the spiked concentration was calculated as the percentage of measured over the expected value.
This table shows the summary of the recovery experiments in the VEGF ELISA in different sample matrices:
|
|
|
% Recovery |
|||
|
Sample Matrix |
n |
+500 pg/ml |
+250 pg/ml |
||
|
Mean |
Range |
Mean |
Range |
||
|
Serum |
6 |
94 |
89 - 97 |
94 |
81 - 129 |
|
EDTA plasma |
6 |
102 |
94 - 114 |
92 |
81 - 104 |
|
Citrate plasma |
2 |
105 |
101- 110 |
92 |
92 - 92 |
|
+125 pg/ml |
|||||
|
Cell culture supernatant |
2 |
94 |
89 - 99 |
77 |
74 - 81 |
|
+1000 pg/ml |
|||||
|
Urine |
6 |
119 |
111 - 134 |
||
Data showing % recovery of recombinant VEGF in human serum samples:
|
Sample Matrix |
ID |
VEGF [pg/ml] |
% Recovery |
|||
|
Reference |
+500 pg/ml |
+250 pg/ml |
+500 pg/ml |
+250 pg/ml |
||
|
Serum |
S1 |
130 |
|
374 |
|
104 |
|
Serum |
S2 |
971 |
|
1173 |
|
129 |
|
Serum |
S3 |
414 |
685 |
566 |
96 |
82 |
|
Serum |
S4 |
652 |
811 |
779 |
97 |
83 |
|
Serum |
S5 |
164 |
529 |
350 |
89 |
82 |
|
Serum |
S6 |
364 |
646 |
520 |
93 |
81 |
|
Mean |
94 |
94 |
||||
Data showing % recovery of recombinant VEGF in human EDTA plasma samples:
|
Sample Matrix |
ID |
VEGF [pg/ml] |
% Recovery |
|||
|
Reference |
+500 pg/ml |
+250 pg/ml |
+500 pg/ml |
+250 pg/ml |
||
|
EDTA plasma |
E1 |
95 |
532 |
303 |
97 |
88 |
|
EDTA plasma |
E2 |
166 |
551 |
349 |
94 |
81 |
|
EDTA plasma |
E3 |
48 |
514 |
258 |
98 |
87 |
|
EDTA plasma |
E4 |
125 |
551 |
343 |
98 |
93 |
|
EDTA plasma |
E5 |
40 |
580 |
280 |
112 |
98 |
|
EDTA plasma |
E6 |
75 |
605 |
327 |
114 |
104 |
|
Mean |
102 |
92 |
||||
Data showing % recovery of recombinant VEGF in human citrate plasma samples:
|
Sample Matrix |
ID |
VEGF [pg/ml] |
% Recovery |
|||
|
Reference |
+500 pg/ml |
+250 pg/ml |
+500 pg/ml |
+250 pg/ml |
||
|
Citrate plasma |
C1 |
20 |
559 |
247 |
110 |
92 |
|
Citrate plasma |
C2 |
50 |
529 |
275 |
101 |
92 |
|
Mean |
105 |
92 |
||||
Human VEGF ELISA Dilution Linearity & Parallelism
Tests of dilution linearity and parallelism ensure that both, endogenous and recombinant samples containing VEGF, behave in a dose-dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted clinical samples spiked with known concentrations of recombinant analyte. By contrast, parallelism refers to dilution linearity in clinical samples and provides evidence that endogenous analyte behaves in the same way as the recombinant one /likewise to the recombinant analyte. Dilution linearity and parallelism are assessed for each sample type and should be within 20% of the expected concentration.
Dilution linearity was assessed by serially diluting human serum and plasma samples spiked with recombinant VEGF in assay buffer.
The figure and table below show the mean recovery and range of serially diluted recombinant VEGF in several sample matrices:
|
% Recovery of recombinant VEGF in diluted samples |
|||||||
|
Sample Matrix |
n |
1+1 |
1+3 |
1+7 |
|||
|
Mean |
Range |
Mean |
Range |
Mean |
Range | ||
|
Serum |
3 |
112 |
99 - 118 |
106 |
97- 110 |
100 |
88- 109 |
|
Plasma |
2 |
96 |
84 - 108 |
93 |
83- 102 |
85 |
81 - 92 |
|
Citrate plasma |
2 |
102 |
100 - 104 |
108 |
105- 111 |
100 |
97-103 |
|
Cell culture supernatant |
2 |
103 |
100 - 106 |
98 |
96- 99 |
105 |
102-107 |
|
Urine |
2 |
106 |
106 - 106 |
105 |
98- 113 |
- |
- |
Data showing dilution linearity of recombinant VEGF spiked into human serum and plasma samples (ref) containing endogenous VEGF.
Calculation of dilution linearity of spiked serum samples:
|
VEGF [pg/ml] |
% Recovery |
||||||||
|
Sample Matrix |
ID |
Ref +2000 pg/ml |
ref |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Serum |
S1 |
1497 |
778 |
885 |
404 |
199 |
118 |
108 |
106 |
|
Serum |
S2 |
1157 |
130 |
660 |
310 |
127 |
114 |
107 |
88 |
|
Serum |
S3 |
1329 |
292 |
770 |
357 |
176 |
116 |
107 |
106 |
|
Serum |
S4 |
1466 |
477 |
724 |
357 |
170 |
99 |
97 |
93 |
|
Serum |
S5 |
1619 |
971 |
915 |
447 |
220 |
113 |
110 |
109 |
|
Mean R[%] |
112 |
106 |
100 |
||||||
Calculation of dilution linearity of spiked EDTA plasma samples:
|
VEGF [pg/ml] |
% Recovery |
||||||||
|
Sample Matrix |
ID |
Ref +1000 pg/ml |
ref |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
EDTA plasma |
E1 |
580 |
40 |
244 |
148 |
67 |
84 |
102 |
92 |
|
EDTA plasma |
E2 |
605 |
75 |
298 |
142 |
62 |
98 |
94 |
82 |
|
EDTA plasma |
E3 |
659 |
122 |
282 |
137 |
67 |
86 |
83 |
81 |
|
EDTA plasma |
E4 |
1298 |
133 |
702 |
303 |
132 |
108 |
93 |
81 |
|
EDTA plasma |
E5 |
1265 |
57 |
653 |
297 |
139 |
103 |
94 |
88 |
|
Mean R[%] |
96 |
93 |
85 |
||||||
Calculation of dilution linearity of spiked citrate plasma samples:
|
VEGF [pg/ml] |
% Recovery |
|||||||
|
Sample Matrix |
ID |
Ref +1000 pg/ml |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Citrate plasma |
C1 |
519 |
17 |
270 |
144 |
67 |
104 |
111 |
|
Citrate plasma |
C2 |
560 |
55 |
279 |
148 |
68 |
100 |
105 |
| Mean R[%] |
102 |
108 |
100 |
|||||
Parallelism
Parallelism was assessed by serially diluting samples containing endogenous VEGF with assay buffer.
|
|
|
|
% Recovery of endogenous human VEGF in diluted samples |
|||||
|
Sample Matrix |
n |
1+1 |
1+3 |
1+7 |
||||
|
Mean |
Range |
Mean |
Range |
Mean |
Range |
|||
|
Serum |
6 |
108 |
102 - 110 |
110 |
103 - 113 |
109 |
102 - 115 |
|
|
EDTA plasma |
5 |
108 |
97 - 114 |
113 |
107 - 116 |
100 |
93 - 112 |
|
|
Citrate plasma |
2 |
103 |
99 - 108 |
91 |
80 - 102 |
90 |
85 - 96 |
|
|
Cell culture supernatant |
2 |
93 |
89 - 98 |
109 |
108 - 110 |
114 |
108 - 119 |
|
|
Urine |
6 |
85 |
70 - 94 |
n.d. |
n.d. |
n.d. |
n.d. |
|
n.d. : not determined
Data showing dilution linearity of endogenous VEGF in human serum samples:
|
VEGF [pg/ml] |
% Recovery |
|||||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Serum |
s1 |
1153 |
643 |
337 |
159 |
112 |
117 |
110 |
|
Serum |
s2 |
1002 |
521 |
282 |
144 |
104 |
113 |
115 |
|
Serum |
s3 |
460 |
249 |
123 |
59 |
108 |
107 |
102 |
|
Serum |
s4 |
845 |
465 |
227 |
114 |
110 |
108 |
108 |
|
Serum |
s5 |
591 |
322 |
167 |
85 |
109 |
113 |
115 |
|
Serum |
s6 |
559 |
285 |
144 |
73 |
102 |
103 |
104 |
|
|
Mean R[%] |
108 |
110 |
109 |
||||
Data showing dilution linearity of endogenous VEGF in human EDTA plasma samples:
|
VEGF [pg/ml] |
% Recovery |
|||||||
|
Sample Matrix |
ID |
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
EDTA plasma |
e1 |
324 |
179 |
94 |
38 |
110 |
116 |
93 |
|
EDTA plasma |
e2 |
739 |
420 |
213 |
104 |
114 |
115 |
112 |
|
EDTA plasma |
e3 |
524 |
291 |
140 |
61 |
111 |
107 |
93 |
|
EDTA plasma |
e4 |
55 |
26,4 |
11.5* |
|
97 |
|
|
|
EDTA plasma |
e5 |
983 |
532 |
274 |
123 |
108 |
111 |
100 |
|
EDTA plasma |
e6 |
324 |
179 |
94 |
38 |
110 |
116 |
93 |
|
|
Mean R[%] |
108 |
113 |
100 |
||||
Data showing recovery of endogenous VEGF in human citrate plasma samples:
|
VEGF [pg/ml] |
% Recovery |
|||||||
|
Sample Matrix |
ID |
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Citrate plasma |
c1 |
943 |
467 |
240 |
113 |
99 |
102 |
96 |
|
Citrate plasma |
c2 |
198 |
106 |
40 |
21 |
108 |
80 |
85 |
|
|
Mean R[%] |
103 |
91 |
90 |
||||
VEGF ELISA Specificity
The specificity of an ELISA is defined as its ability to exclusively recognize the analyte of interest.
The specificity of the VEGF ELISA was shown by characterizing both the capture and the detection antibodies through epitope mapping. In addition, the specificity of the ELISA was established through competition experiments, which measure the ability of the antibodies to exclusively bind VEGF.
This assay recognizes recombinant and endogenous (natural) human VEGF including all circulating VEGF isoforms (incl. VEGF165b).
The Biomedica human VEGF ELISA detects all human VEGF isoforms that are found in circulation and that are not bound to the soluble decoy receptors VEGFR1 and VEGFR2.
However, Neuropilin-1-bound VEGF forms can be quantified, if present. This is due to the binding properties of the employed antibodies. The recombinant anti-human VEGF capture antibody detects a structural epitope near the receptor binding site of the VEGF molecule and hinders the binding of soluble VEGFR1/2 receptors but does not affect the NRP1 epitope. Indeed, no interference at high NRP1 concentrations (10x higher than physiological levels) was observed.
Linear epitopes of the polyclonal anti-human VEGF detection antibody are concentrated within the N-terminal region of the VEGF protein.
As all VEGF isoforms posess conserved N-termini and both antibody epitopes fall within this region, all VEGF isoforms will be detected. This includes all circulating pro- and anti-angiogenic VEGF isoforms (incl. VEGF165b and VEGFAx).
Epitope Mapping
Antibodies were characterized by epitope mapping of linear epitopes with microarray technology and by the determination of binding kinetics with biolayer interferometry.
Capture Antibody: the peptide-specific recombinant capture antibody recognizes a structural epitope in the conserved receptor binding-site of VEGF and thus, specifically binds to all isoforms of VEGF.
Detection Antibody: Multiple linear epitopes recognized by the polyclonal detection antibody are concentrated in the first 120 amino acids of the VEGF molecule. The polyclonal detection antibody recognizes linear epitopes N-terminal of VEGF.
Microarray of detection antibody
High resolution epitope mapping of the polyclonal detection antibody on human VEGF-A.
The canonical sequence of VEGF-A (P15692-1) was printed as 15mers with an 14 amino acid overlap in duplicates on a glass chip. Green fluorescent signals on the microarray illustrate binding of the polyclonal detection antibody to VEGF and corresponds to its epitopes. Red fluorescent signals mark the position of control peptides. The polyclonal detection antibody recognizes linear epitopes N-terminal of VEGF.
Antibody epitopes on human VEGF-A molecule
3D structure of human VEGF-A dimer (V14-K107, pdb 1BJ1) with antibody epitopes. The recombinant capture antibody recognizes a structural epitope (purple) in the conserved receptor binding-site of VEGF-A (shown as dimer, dark and light grey) and thus, specifically binds to all bioactive isoforms of VEGF-A. Linear epitopes (blue) of the detection antibody are concentrated in the first 120 amino acids of the VEGF molecule.
Competition of Signal
Competition experiments were carried out by pre-incubating human samples containing endogenous VEGF with an excess of capture antibody (AB). The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample without the pre-incubation step. Mean competition in serum and plasma samples was 97%.
|
VEGF [pg/ml] |
% Competition |
|||
|
Sample Matrix |
ID |
Reference |
Reference + capture AB |
|
|
Serum |
s1 |
694 |
48 |
93 |
|
Serum |
s2 |
242 |
7 |
97 |
|
Serum |
s3 |
90 |
0 |
100 |
|
Serum |
s4 |
699 |
32 |
95 |
|
EDTA plasma |
e1 |
186 |
0 |
100 |
|
EDTA plasma |
e2 |
740 |
34 |
95 |
|
Citrate plasma |
c1 |
15 |
0 |
100 |
|
Mean |
97% |
Isomer Forms
17 described (+ 15 computationally mapped) isoforms produced by alternative promoter usage, alternative splicing and alternative initiation with distinct biological activity.
There are 5 dominant isoforms (121, 145, 165, 189, 206). Of these VEGF 165 is the most common VEGF-A isoform.
The Biomedica human VEGF ELISA detects all human circulating VEGF that are not VEGF R1/R2 receptor-bound.
Ligands
VEGF R1 (FLT1), VEGF R2 (KDR), NRP-1, heparan sulfate, heparin
Cross Reactivity
CROSS REACTIVITY with other VEGF Family Members
The low level of sequence homology between the different VEGF family members indicates that other VEGF family members e.g. VEGF-B / VEGF-C / VEGF-D, are not recognized using this ELISA.
CROSS REACTIVITY with non-human samples
This ELISA was tested in rat, mouse and porcine samples. According to our data the kit cannot be used for the detection of rat and mouse VEGF.
Porcine VEGF: The sequence homology of the capture antibody utilized in the kit (recombinant human VEGF antibody) to the porcine VEGF sequence is 100%.
The presence of endogenous VEGF signal was analyzed in pig samples; its specificity was determined with a competition experiment by pre-incubating porcine samples containing endogenous VEGF with an excess of capture antibody (AB). The concentration measured in this mixture was then compared to the reference value, obtained from the same sample without the pre-incubation step. The mean competition in porcine samples was 100% (see table below).
|
Sample matrix |
Sample ID |
OD |
c (pg/ml) |
R [%] |
||
|
ref |
comp |
ref |
comp |
|||
|
pig serum |
P1 |
0.109 |
0.078 |
12.96 |
0.0 |
100 |
|
pig plasma |
P2 |
0.135 |
0.095 |
32.43 |
0.0 |
100 |
|
|
Mean R [%] |
100 |
||||
Porcine sample values: 12 samples from healthy pigs measured with this assay showed a mean VEGF concentration of 33 pg/ml (range: 13 – 53 pg/ml).
Sample Stability
Serum, EDTA plasma, citrate plasma, cell culture supernatants, and urine samples are suitable for use in this assay. Do not change sample type during studies. We recommend duplicate measurements for all samples, standards and controls. The sample collection and storage conditions listed are intended as general guidelines.
Freeze-thaw Stability
The stability of endogenous Vascular Endothelial Growth Factor (VEGF) was tested by comparing samples that had undergone five freeze-thaw cycles (F/T).
For freeze-thaw experiments, samples were collected according to the supplier’s instruction using blood collection devices and stored at -80°C. Reference samples were freeze-thawed once. The mean recovery of sample concentration after five freeze-thaw cycles is 97%.
[/collapsible-block]
VEGF concentrations of samples after freeze-thaw (F/T) cycles:
|
VEGF [pg/ml] |
% Recovery, referred to reference |
|||||
|
Sample Matrix |
ID |
Reference |
1x F/T |
5x F/T |
1x F/T |
5x F/T |
|
Serum |
S1 |
402 |
382 |
355 |
95% |
88% |
|
Serum |
S2 |
744 |
778 |
708 |
105% |
95% |
|
EDTA plasma |
E1 |
499 |
523 |
518 |
105% |
104% |
|
EDTA plasma |
E2 |
572 |
626 |
577 |
109% |
101% |
|
|
|
|
|
Mean |
103% |
97% |
All samples should undergo a maximum of five freeze-thaw cycles.
Benchtop Stability
The benchtop stability of endogenous Vascular Endothelial Growth Factor (VEGF) was tested by comparing VEGF measurements in human samples that had been stored at different temperatures.
For the assessment of the benchtop stability, a set of human samples was aliquoted and stored at room temperature or at 4°C. Samples can be stored for at least three hours at room temperature as well as overnight at 4°C. The mean recovery of sample concentrations after overnight storage at 4°C is 102%.
VEGF concentrations of samples stored at -25°C (reference), at room temperature (RT) or overnight (o.n.) at 4°C:
|
|
|
VEGF (pg/ml) |
% Recovery, referred to reference |
|||
|
Sample matrix |
ID |
Reference |
3h RT |
oN 4°C |
3h RT |
oN 4°C |
|
Serum |
s1 |
389 | 411 |
423 |
99 | 103 |
|
Serum |
S1 |
771 | 778 | 783 | 97 | 101 |
|
EDTA plasma |
e1 |
727 | 827 | 119 | n.d. | |
|
Citrate plasma |
c1 |
175 | 187 | 85 | n.d. | |
|
Mean |
100 |
102 |
||||
n.d.: not determined
Sample Values
VEGF Values in Apparently Healthy Individuals
Serum / Plasma: VEGF was measured in samples from apparently healthy donors (no medical histories were available).
|
|
VEGF [pg/ml] |
|
|||
|
Sample Matrix |
n |
Mean |
Range |
Median |
% Detectable |
|
Serum* |
23 |
491 |
130-971 |
540 |
100% |
|
EDTA plasma |
23 |
103 |
47-149 |
111 |
100% |
|
Citrate plasma |
23 |
78 |
21-152 |
79 |
100% |
It is recommended to establish the normal range for each laboratory.
*Platelets and leukocytes can release VEGF during blood clotting which is reflected in higher serum sample concentrations compared to plasma samples levels (Banks RE et al., , Gunsilius E et al.).
Vascular Endothelial Growth Factor (VEGF) Values in Disease Panels
In addition to samples of apparently healthy donors, panels of samples from patients with heart disease (cardiology panel), kidney diseases, as well as a panel of unselected hospital patients were tested.
VEGF values measured in apparently healthy subjects and patients with heart disease:
VEGF values measured in apparently healthy subjects and patients with kidney disease:
VEGF values measured in apparently healthy subjects and in an unselected hospital panel:
Summary of the results:
|
|
VEGF [pg/ml] |
|||
|
Samples |
n |
Mean |
Range |
Median |
|
Controls -Serum |
17 |
181 |
17-609 |
105 |
|
HD NHYA 3. 4.- Serum |
22 |
268 |
82-881 |
210 |
|
Apparently healthy EDTA plasma |
22 |
66 |
29-164 |
59 |
|
Nephro Panel EDTA plasma |
32 |
287 |
63-1110 |
212 |
|
Apparently healthy EDTA plasma |
51 |
88 |
29-182 |
77 |
|
Unselected hospital panel EDTA plasma |
18 |
628 |
45-1878 |
486 |
|
Apparently healthy Citrate plasma |
33 |
66 |
21-152 |
62 |
|
Unselected hospital panel Citrate plasma |
12 |
302 |
37-1054 |
302 |
Matrix Comparison
To assess whether all tested matrices behave the same way Vascular Endothelial Growth Factor (VEGF) was measured in serum, EDTA, and citrate plasma samples prepared from 23 apparently healthy donors.
Each individual donated blood in all tested sample matrices.
|
|
VEGF [pg/ml] |
|
|||
|
Sample Matrix |
n |
Mean |
Range |
Median |
% Detectable |
|
Serum |
23 |
491 |
130-971 |
540 |
100% |
|
EDTA plasma |
23 |
103 |
47-149 |
111 |
100% |
|
Citrate plasma |
23 |
78 |
21-152 |
79 |
100% |
Measurment of human VEGF in Urine samples - Performance check:
Urine
Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particles, assay immediately or aliquot and store at -25°C or lower.
|
Sample Matrix Urine* |
VEGF [pg/ml] |
|
Donor 1. Apparently healthy |
171 |
|
Donor 2. Apparently healthy |
267 |
|
Donor 3. Apparently healthy |
158 |
|
Donor 4. Kidney disease |
372 |
|
Donor 5. Kidney disease |
265 |
|
Donor 6. Kidney disease |
312 |
|
Donor 7. Kidney disease |
343 |
Urine samples were not normalized to creatinine values.
Acccuracy
Data showing % recovery of recombinant VEGF in human urine samples:
|
Sample Matrix |
ID |
VEGF [pg/ml] |
||
|
Reference |
+1000 pg/ml |
% Recovery |
||
|
Urine |
U1 |
372 |
1355 |
117% |
|
Urine |
U2 |
265 |
1272 |
114% |
|
Urine |
U3 |
312 |
1348 |
119% |
|
Urine |
U4 |
343 |
1359 |
119% |
|
Urine |
U5 |
171 |
1425 |
134% |
|
Urine |
U6 |
267 |
1246 |
111% |
|
Mean R [%] |
119% |
|||
Dilution linearity, parallelism
Data showing dilution linearity of recombinant VEGF in human urine samples:
|
Sample ID |
VEGF [pg/ml] |
R [%] |
|||
|
ref |
1+1 |
1+3 |
1+1 |
1+3 |
|
|
U1 |
1219 |
646 |
298 |
106% |
98% |
|
U2 |
1129 |
596 |
319 |
106% |
113% |
|
Mean R [%] |
106% |
105% |
|||
Data showing dilution linearity of endogenous VEGF in human urine samples:
|
Sample ID |
VEGF [pg/ml] |
R [%] |
|
|
0 |
1+1 |
||
|
U1 |
372 |
175 |
94% |
|
U2 |
265 |
116 |
87% |
|
U3 |
312 |
109 |
70% |
|
U4 |
343 |
141 |
82% |
|
U5 |
171 |
78 |
91% |
|
U6 |
267 |
112 |
84% |
|
Mean R [%] |
85% |
||
Competition experiments were carried out by pre-incubating human urine samples containing endogenous VEGF with an excess of capture antibody (AB).
The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample without the pre-incubation step.
Mean competition in serum and plasma samples was 99%.
Competition of endogenous signal:
|
Sample ID |
VEGF [pg/ml] |
R [%] |
|
|
0 |
+AB |
||
|
U1 |
372 |
0 |
100% |
|
U2 |
265 |
0 |
100% |
|
U3 |
312 |
13.6 |
96% |
|
U4 |
343 |
3.9 |
99% |
|
U5 |
171 |
0 |
100% |
|
U6 |
267 |
0 |
100% |
|
Mean R [%] |
99% |
||
Measurment of human VEGF in Cell Culture Supernatants - Performance check:
Cell Culture Supernatants (CCS):
Two human breast cancer cell lines MDA-MB-231, MCF-7 and a human macrophage cell line 4TL9.R were cultured in DMEM/Ham ´ s F12 and RPMI, respectively and supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin. Cells were grown in a humidified atmosphere of 95 % air and 5 % CO2 for 48 hours. Aliquots of the cell culture supernatants were removed, centrifuged to remove particles, and assayed for levels of human VEGF.
|
Sample Matrix CCS |
VEGF [pg/ml] |
|
CCS - MDA-MB-231 |
150 |
|
CCS - MCF-7 |
699 |
|
CCS - 4TL9.R |
788 |
|
DM-F-12 (with supplements) |
0 |
|
RPMI (with supplements) |
0 |
Accuracy
Recombinant VEGF was spiked into samples by using STD7 (1000pg/ml). Two concentration levels were generated. 500pg/ml spike is a sample:STD7 volume ratio of 1+1.
125pg/ml spike is a sample:STD7 volume ratio of 7+1.
Samples were assayed in duplicates.
|
CCS |
ID |
VEGF (pg/ml) |
R [%] |
|||
|
0 |
500 |
125 |
500 |
125 |
||
|
MDA-MB-231 |
CCS#1 |
150 |
570 |
232 |
99% |
81% |
|
MCF-7 |
CCS#2 |
699 |
793 |
703 |
89% |
74% |
|
|
Mean R [%] |
94% |
77% |
|||
Dilution linearity
Dilution linearity of recombinant analyte of cell culture supernatants.
Samples were spiked with STD 7 (1000pg/ml, +500pg/ml) and diluted with dilution medium (ASYBUF).
| CCS |
ID |
VEGF (pg/ml) | % recovery | |||||
| ref spike |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
||
| MDA-MB- 231 |
CCS#1 |
570 |
286 |
137 |
73 |
100% |
96% |
102% |
|
MCF- 7 |
CCS#2 |
793 |
421 |
196 |
108 |
106% |
99% |
109% |
|
Mean R [%] |
103% |
98% |
105% |
|||||
Dilution linearity of endogenous analyte was tested in conditioned media (48h) of MCF7 and MDA 231 cell lines. Dilution medium is assay buffer .
|
CCS |
ID |
VEGF (pg/ml) |
% recovery |
|||||
|
ref |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
||
|
MDA-MB- 231 |
CCS#1 |
150 |
66 |
41 |
22 |
89% |
110% |
119% |
|
MCF-7 |
CCS#2 |
699 |
344 |
189 |
94 |
98% |
108% |
108% |
|
|
Mean R [%] |
93% |
109% |
114% |
||||
Competition of VEGF in cell culture supernatants
Competition experiments were carried out by pre-incubating human cell culture supernatnant samples containing endogenous VEGF with an excess of capture antibody (AB). The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample without the pre-incubation step. Mean competition in serum and plasma samples was 93%.
Calculated according to calibration curve prepared from ELISA s standards
|
CCS |
ID |
VEGF (pg/ml) |
R [%] |
|
|
0 |
+ CAB |
500 |
||
|
MDA-MB- 231 |
CCS#1 |
150 |
15 |
90% |
|
MCF- 7 |
CCS#2 |
699 |
23 |
97% |
|
|
Mean R [%] |
93% |
||
Comparison with other human VEGF ELISA assays
Assay characteristics of two different human VEGF ELISA assays
|
|
Biomedica |
Another Manufacturer |
|
Method |
Sandwich ELISA, Streptavidin-HRPO/TMB, 12x8-well detachable strips |
Sandwich ELISA, HRPO/TMB, 12x8-well detachable strips |
|
Sample type |
Serum, EDTA plasma, citrate plasma, cell culture supernatants, urine |
Serum, plasma, cell culture supernates |
|
Sample volume |
10 µl sample / well |
100 µl sample (serum/plasma), 200 µl (cell culture) / well |
|
Assay time |
2 h / 1 h / 1 h / 30 min |
2 h / 2 h / 30 min |
|
Assay range |
0 – 2000 pg/ml (0 / 31.25 / 62.5/ 125 / 250/ 500 / 1000 / 2000) |
0 – 2000 pg/ml |
|
Sensitivity |
LOD: 2.5 pg/ml; LLOQ: 15.6 pg/ml (measurable concentrations in serum AND plasma samples!) |
MDD: 5 pg/ml |
|
Specificity |
Assay recognizes recombinant and endogenous (natural) human VEGF including all circulating VEGF isoforms (incl. VEGF165b). |
Assay recognizes natural and recombinant human VEGF. This assay also recognizes recombinant human VEGF165b |
|
Antibodies |
Epitope-mapped antibodies: Capture antibody: recombinant VEGF antibody specific for human VEGF Detection antibody: Polyclonal VEGF antibody specific for human VEGF, streptavidin-HRPO -labeled |
Capture antibody: monoclonal antibody specific for human VEGF Detection antibody: polyclonal VEGF antibody specific for human VEGF, HRPO-labeled |
|
Standard Matrix |
Serum based matrix containing recombinant VEGF165 8 ready to use standards, lyophilized |
Protein based matrix containing recombinant VEGF165 1 stock standard, lyophilized |
|
Values of apparently healthy samples |
Serum median (n=23): 491 pg/ml 100 % detectable EDTA-plasma mean (n=23): 103 pg/ml 100 % detectable |
Serum median (n=37): 220 pg/ml 100 % detectable EDTA-plasma mean (n=37): 61 pg/ml 24 % detectable |
|
Controls |
2 controls (high and low) included |
not included |
|
Validation |
According to FDA/ICH/EMEA guidelines |
not indicated |
|
Use |
RUO |
RUO |
Comparison of human sample concentrations measured with different human VEGF ELISA Assays
The Biomedica VEGF ELISA (Cat. No. BI-VEGF) was compared with an ELISA kit from another manufacturer. The same panel of samples, consisting of 46 samples (healthy and diseased), was tested.
Correlation of VEGF values measured with two human VEGF ELISA assays - Biomedica versus another manufacturer in 46 serum and plasma samples (healthy + diseased)
|
Pearson coefficient |
0.979 |
|
P value |
0.00001 |
|
P value summary |
**** |
Conclusion - Comparison between two assays
- Apparently healthy EDTA plasma samples (n=12) with the Biomedica ELISA are approximately 2-2.5 x higher than in other assay.
- Apparently healthy serum samples (n=8) with the Biomedica ELISA are approximately 1.5 x higher than in other assay.
- Excellent correlation for human serum and plasma samples (healthy and diseased) : Pearson correlation coefficient R = 0.979, p < 0.00001
- Cell culture supernatants (n=2): Biomedica and other assay show nearly identical pg/ml concentrations (read-out Biomedica standard curve with standard curve-cell culture from the other manufacturer- following instuctions for use)
- Concentrations obtained in both kits were converted to approximate NIBSC/WHO 02/286 units. Their comparison shows that Biomedica human VEGF values (U/ml) are approximately 0.5 fold higher for serum samples and 0.7 - 0.8 higher fold for EDTA plasma samples- when compared with other assay.
Table showing human VEGF concentrations measured with the Biomedica human VEGF ELISA and a human VEGF ELISA assay from another manufacturer:
|
ALL SAMPLES |
|||
|
n = 46 |
|||
|
Sample ID |
Biomedica (BI-VEGF) |
Other
|
|
|
Cohorts: |
|
VEGF pg/ml |
VEGF pg/ml |
|
Apparently healthy (AH) samples
|
AH s1 |
368 |
333 |
|
AH s2 |
723 |
413 |
|
|
AH s3 |
236 |
185 |
|
|
AH s4 |
836 |
541 |
|
|
AH s5 |
360 |
297 |
|
|
AH s6 |
265 |
230 |
|
|
AH s7 |
263 |
190 |
|
|
AH s8 |
371 |
331 |
|
|
AH ep9 |
83 |
37 |
|
|
AH ep10 |
84 |
35 |
|
|
AH ep11 |
76 |
33 |
|
|
AH ep12 |
74 |
36 |
|
|
AH ep13 |
48 |
19 |
|
|
AH ep14 |
97 |
45 |
|
|
AH ep15 |
98 |
36 |
|
|
AH ep16 |
74 |
41 |
|
|
AH ep17 |
42 |
13 |
|
|
AH ep18 |
126 |
58 |
|
|
AH ep19 |
34 |
14 |
|
|
AH ep20 |
180 |
71 |
|
|
AH cp21 |
42 |
17 |
|
|
AH cp22 |
43 |
14 |
|
|
AH cp23 |
34 |
9 |
|
|
AH cp24 |
35 |
15 |
|
|
Unspecific hospital panel (UHP) samples
|
UHP ep25 |
466 |
229 |
|
UHP ep26 |
291 |
147 |
|
|
UHP ep27 |
700 |
336 |
|
|
UHP ep28 |
411 |
185 |
|
|
UHP ep29 |
417 |
171 |
|
|
UHP ep30 |
181 |
86 |
|
|
UHP ep31 |
1737 |
977 |
|
|
UHP cp32 |
319 |
173 |
|
|
UHP cp33 |
381 |
197 |
|
|
UHP cp34 |
259 |
115 |
|
|
UHP s35 |
441 |
294 |
|
|
UHP s36 |
1368 |
760 |
|
|
UHP s37 |
1862 |
1007 |
|
|
UHP s38 |
208 |
44 |
|
|
Nephrology (N) samples
|
N ep39 |
107 |
32 |
|
N ep40 |
311 |
115 |
|
|
N ep41 |
323 |
123 |
|
|
N ep42 |
154 |
53 |
|
|
N ep43 |
396 |
132 |
|
|
N ep44 |
282 |
108 |
|
|
N ep45 |
266 |
105 |
|
|
N ep46 |
522 |
252 |
|
|
|
Pearson |
0.979 |
|
|
|
p value |
< 0.00001 |
|
s: serum
ep: EDTA plasma
cp: citrate plasma
Comparison of cell culture supernatant sample concentrations in human breast cancer cell lines measured with different human VEGF ELISA Assays
The Biomedica VEGF ELISA (Cat. No. BI-VEGF) was compared with an ELISA kit from another manufacturer.
Table showing human VEGF concentrations measured with the Biomedica human VEGF ELISA and a humanVEGF ELISA assay from another manufacturer:
|
Cell Culture Supernatatants (CCS) |
||
|
n = 2 |
||
|
Sample ID |
Biomedica VEGF ELISA (# BI-VEGF) |
Other VEGF ELISA
|
|
|
VEGF pg/ml |
VEGF pg/ml |
|
CCS - MDA-231 |
121.5 |
101.8 |
|
CCS - MCF-7 |
546.2 |
624.4 |
