(return) homePage
return
(main kit list)
New!
Receptor-based Vascular Endothelial Growth
Factor-A (VEGF-A) RELIDA
FOR THE QUANTITATIVE
DETERMINANTION OF BIOLOGICALLY ACTIVE RECOMBINANT AND NATURALLY OCCURRING
HUMAN VASCULAR ENDOTHELIAL GROWTH
FACTOR-A (VEGF-A) IN CELL CULTURE SUPERNATANTS AND COMPLEX BIOLOGICAL FLUIDS
Cat#RDI-DA066 $787.50/1kit
FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURE.
STORE KIT AT 4°C.
Order through :
Research Diagnostics Inc, Pleasant Hill Road, Concord MA 01742-3049
USA
Phone 973-584-7093 fax 973-584-0210 web : http://www.researchd.com
SAMPLE
INSERT-SEE INSERT IN EACH KIT for any batch specific information
Vascular endothelial growth factor (VEGF or VEGF-A) is a
mitogen for vascular endothelial cells derived from arteries, veins and
lymphatics, but it is devoid of consistent mitogenic activity for other cell
types. VEGF-A is also known as vascular permeability factor (VPF), based on its
ability to induce vascular leakage in the guinea-pig skin and in different
models of vascular permeability. An increase in vascular permeability is a
crucial step in angiogenesis associated with tumors and wounds. Besides the
related mitogen placenta growth factor (PlGF) new molecules of the VEGF family
were described in the last years and consequently named VEGF-B to VEGF-E. The
newly described VEGF-C can also stimulate and activate lymphatic endothelial
cells.
The human VEGF-A gene is organized in eight exons,
separated by seven introns. Alternative splicing of the eight exons leads to
the formation of at least five different molecular species of VEGF having 121,
145, 165, 189 and 206 amino acids following the cleavage of the signal
sequence. VEGF165 is the predominant molecular species produced by a
variety of normal and transformed cells. It is a basic, heparin-binding,
homodimeric glycoprotein of 45kDa. VEGF121
is also secreted from many cell types, but it is not heparin binding.
Among the mechanisms that have been proposed to participate
in the regulation of VEGF-A gene expression, oxygen tension plays a major role
both in vitro and in vivo. VEGF-A mRNA expression is rapidly induced in normal
and transformed cultured cell types by exposure to low oxygen levels.
Similarities exist in the mechanisms leading to the hypoxic regulation of
VEGF-A and erythropoietin (Epo). However, hypoxia increases the VEGF-A
production not only by transcriptional activation but also by
post-transcriptional events as increasing the VEGF-A mRNA stability.
VEGF-A binds to two different receptor tyrosine kinases
known as VEGFR-1 (Flt-1) and VEGFR-2 (KDR). Many factors are potentially involved in the
regulation of angiogenesis, but VEGF-A is a specific factor and the major key
player is involved in all varieties of physiological and pathological
angiogenesis. Therefore, the new receptor-based VEGF-A RELIDA offers a
conveniently simple and reliable method to quantify biologically active VEGF-A
in cell culture supernatants or complex biological fluids, e.g. tumour ascitis
fluids.
Receptor-based
Vascular Endothelial Growth Factor-A RELIDA
This assay was
designed to quantitate biologically active human vascular endothelial growth
factor-A (VEGF-A) in cell culture supernatants or complex biological fluids.
The type of the assay is unique based on its novel design with soluble VEGF-A
receptors as capture molecules of active ligands. For high affinity binding of
VEGF-A the soluble receptor molecules are used for solid phase binding in the
presence of biomatrix material. The assay type mimics the physiological
ligand-receptor interaction and binds e.g. VEGF165 with a Kd
of about 30pM. The assay will measure all soluble, secreted isoforms of VEGF-A
and may also detect cell-associated isoforms (VEGF145, VEGF189),
if these were solubilized by the action of proteases. The assay will recognize
only free, uncomplexed and biologically active forms of VEGF-A that are not sequestered by soluble
receptors. Sequestered, monomeric and degraded VEGF-A molecules will not be
detected. A schematic representation of
the assay system is given in Fig. 1.
Fig. 1: Schematic
representation of the assay.
The
„ready to use“ plates are precoated with a recombinant VEGF receptor (VEGFR)
and a specific proteoaminoglycan to mimic a biological matrix and enhance
VEGF-A binding (A). Recombinant human VEGF-A standard and samples are added in
the first incubation step (B). VEGFR-bound VEGF-A is detected by incubation
with a biotin-labeled, specific anti-VEGF-A antibody (C). After incubation with
a horseradish peroxidase (HRP)-labeled streptavidine (D) the assay can be
developed with TMB-substrate.
The kit includes reagents for one
96-well ELISA plate. We recommend running the standard and the samples in
duplicate.
Sensitivity: 11 pg/ml
Range of detection: 16 pg/ml to 1000 pg/ml
Intra-assay variation: 5.9 %
Inter-assay variation: 17.7 %
Kit materials
Microtiter
plate: One precoated
and saturated 96-well microtiter ELISA plate, lyophilized and sealed with foil,
removable 16-well racks
Plate
sealer/Manual: Resealable
bag containing two adhesive strips and manual
Wash buffer: 30ml
of 20x concentrate (1x Wash
buffer: PBS, 0.05% TWEEN 20, pH 7.0)
Assay buffer: 11ml of 10x
concentrate
Sample diluent: 12ml of 1x diluent
VEGF-A standard: Two vials each containing
0.6ng lyophilized VEGF-A
Biotinylated detector: One vial containing 60µl
pre-diluted rabbit IgG
Streptavidin
enzyme: One vial containing
60µl pre-diluted poly-HRP conjugated streptavidin
Color
reagent: 12ml
one-component TMB (tetramethyl-benzidine) solution
Stop solution: 12ml 0.2M Sulphuric acid (H2SO4)
WARNING:
SOME LIQUID MATERIALS CONTAIN SODIUM AZIDE OR THIMEROSAL AS PRESERVATIVE. AVOID SKIN
CONTACT. H2SO4 CAUSES SKIN IRRITATIONS.
TMB IS HIGHLY TOXIC. AVOID BREATHING IT. PROTECT EYES, FACE, HANDS AND CLOTHES
WHILE WORKING WITH ALL ELISA COMPONENTS.
Materials
required but not provided
·
Multichannel
or repeating pipettes
·
Pipettes
capable of accurately measuring 1-1000µl
·
Orbital
shaker
·
Clean
10-15ml serological tubes and Eppendorf tubes for preparation of working
dilutions
·
96-well
microtiter plate reader with 450nm and 650nm filter
·
Distilled
water
·
Computerized
data plotting or graph paper for manual plotting of data.
Manual plate
washing
Washing
and complete removal of all liquid at the end of each incubation step is very
important to obtain low background values. The following washing procedure is
recommended:
1. Remove existing fluid from
each well by flicking the plate over a sink.
2. Blot the plate on clean paper
towels.
3. Forcefully pipet 250µl diluted
wash buffer into each well.
4. Repeat steps 1-3 twice.
5. Always remove wash buffer
immediately. Do not incubate plate in wash buffer.
Preparation of reagents
Reagents supplied as
small volumes must be spinned down before opening the tubes to avoid loss of
reagents. Prepare all reagents right before usage and keep cool until
application.
1. Wash- and assay buffer: Dilute 10x assay buffer concentrate and 20x wash buffer concentrate
with destilled water (final volume 100ml (assay buffer) and 600ml (wash
buffer)).
2.
Microtiter plate: Unpack ELISA plate and remove foil
seal. Reconstitute the plate by pipetting 100µl 1x wash buffer into each well,
wait 5 min, flick and blot the plate. Use plate sealer to avoid drying of plate.
3. Standard:
Reconstitute VEGF-A by addition of 300µl assay buffer to make up 2ng/ml. Mix
well. Serial dilutions are prepared within the wells. The reconstituted
standard should not be stored for longer than 24h at 4°C.
4. Biotinylated detector: Dilute 1/100 in assay buffer (final volume 6ml).
5. Streptavidin enzyme: Dilute 1/200 in assay buffer
(final volume 12ml).
HALF OF THE SOLUTIONS
IS NEEDED FOR WORKING WITH A HALF PLATE.
USE A NEW LYOPHILIZED STANDARD PROTEIN EVERY
TIME.
WE RECOMMEND TO RUN STANDARD AND SAMPLES IN
DUPLICATE.
Assay procedure
1.
Start
with the reconstituted plate. Remove the plate sealer and add 100µl sample
diluent in duplicate to the standard wells (A1/A2 to H1/H2).
2.
Add
sample diluent to the sample wells. Samples should be measured diluted 1/2 or 1/4. For 1/2 dilution add 50µl
diluent to all sample wells, for 1/4 dilution add 75µl diluent to all sample
wells (Some samples may be diluted higher, e.g. 1/8).
3.
Add
100µl of the reconstituted standard to wells H1/H2. Prepare 1/2 dilutions
within the wells by mixing and pipetting 100µl to wells G1/G2, F1/F2, ...,
B1/B2. Discard 100µl from wells B1/B2 (see Figure below). Wells A1/A2 are
background controls. Do not add standard protein to wells A1/A2. Avoid touching
the bottom of the wells with the pipette-tips.
H1/H2    
G1/G2     
F1/F2      
E1/E2     
D1/D2    
C1/C2    
B1/B2                     
A1/A2
 1000 
       500          
250          
125         
625     
0.3125    
0.16 
ng/ml           
0.0 ng/ml
Discard 100µl
100µl
VEGF-A
Standard
4.
Add
samples to the sample wells. Use 50µl of the samples for 1/2 dilution and 25µl
of the samples for 1/4 dilution.
5.
Add
50µl biotin-conjugate (diluted 1/100 in assay buffer) to each well including
the background controls (A1/A2). Seal the plate and incubate for 2h at room
temperature on an orbital-shaker.
6.
Wash
plate four times with wash-buffer. Afterwards add 100µl streptavidin-enzyme
(diluted 1/200 in assay buffer) to each well. Seal plate and incubate for 1h at
room temperature on an orbital-shaker.
7.
Wash
plate four times with wash-buffer. Aftetrwards add 100µl TMB-substrate solution
to each well. Allow the blue colour to develop for 10-30 minutes. Do not shake
plate during this incubation step. Stop the reaction by adding 50µl
stop-solution to each well. The blue colour is turned to yellow as a result of
the pH- shift.
8.
Measure
plate in a 96-well microplate reader using 450nm as measuring and 650nm as
reference wavelength.
Assay procedure summary
Prepare
samples and standard, reconstitute the plate.
Apply standard, samples
and biotinylated detector.
Incubate
2h.
Wash
four times.
Incubate
1h.
Add
TMB-Substrate to each well.
Wait
10-30min for colour development. Stop reaction and
read the plate at
450nm and 650nm as reference.
Calculation
of results
Plot the standard curve on
semi-logarithmic paper. Known concentrations of VEGF165 are
plotted on the log-scale (X-axis), the corresponding OD is plotted on the
linear scale. The standard curve should have a sigmoidal shape (Fig.2). The
concentrations of VEGF-A in unknown samples may be determined by plotting the
sample OD on the Y-axis and drawing a horizontal line that intersects with the
standard curve. A vertical line dropped from the point of intersection with the
standard curve to the X-axis intersects the X-axis at the point of the
concentration of the unknown sample. Multiply this value with the dilution
factor of the sample to get the original concentration of the undiluted sample.
Fig. 2:
Representative standard curve
Storage
and application of samples Approximate sample values (Table 1)
1.
Clear samples by centrifugation prior to storage.
2.
For short term storage
(<4 weeks) keep samples at –20°C. For long term storage (>4 weeks) keep
samples at –70°C. Avoid repeated freeze-thaw cycles.
3.
Samples containing low
amounts of VEGF165 can be concentrated by ultrafiltration (>10 kDa).
4.
We recommend desalting
of samples with a high salt content prior to measurement.
5.
Samples can be depleted
of VEGF165 by
incubation with Heparin sepharose.
References
Leung DW,
Cachianes G, Kuang WJ, Goeddel DV, Ferrara
N (1989). Vascular endothelial growth factor is a secreted angiogenic mitogen.
Science 246: 1306-1309.
Houck KA, Leung
DW, Rowland AM, Winer J, Ferrara
N (1992). Dual regulation of vascular endothelial growth factor bioavailability
by genetic and proteolytic mechanisms. J Biol Chem 267: 26031-26037.
Park JE, Keller
HA, Ferrara N
(1993). The vascular endothelial growth factor (VEGF) isoforms: differential
deposition into the subepithelial extracellular matrix and bioactivity of extracellular
matrix bound VEGF. Mol Biol Cell 4: 1317-1326.
Taniguchi T,
Toi M, Inada K, Imazawa T, Yamamoto Y, Tominaga T (1995). Serum
concentrations of hepatocyte growth factor in breast cancer patients. Clin Cancer Res 1: 1031-1034.
Toi M, Inada
K, Hoshina S, Suzuki H, Kondo S, Tominaga T (1995). VEGF
and platelet-derived endothelial cell growth factor are frequently coexpressed
in highly vascularized human breast cancer. Clin
Cancer Res 1: 961-964.
Toi M, Kondo
S, Suzuki H, Yamamoto Y, Inada K, Imazawa T, Taniguchi T, Tominaga T (1996). Quantitative
analysis of VEGF in breast cancer. Cancer 77: 1101-1106.
Toi M,
Taniguchi T, Yamamoto Y, Kurisaki T, Suzuki H, Tominaga T (1996). Clinical
significance of the determination of angiogenic factors. Eur J
Cancer 32A: 2513-2519.
Yamamoto Y,
Toi M, Kondo S, Matsumoto T, Suzuki H, Kitamura M, Tsuruta K, Taniguchi T,
Okamoto A, Mori T, Yoshida M, Ikeda T, Tominaga T (1996). Concentrations
of VEGF in the sera of normal control and cancer patients. Clin Cancer Res 2: 821-826.
Ferrara N,
Davis-Smyth T (1997). The biology of vascular endothelial growth
factor. Endocr Rev 18: 4-25.
Gasparini G,
Toi M, Gion M, Verderio P, Dittadi R, Hanatani M, Matsubara I, Vinante O,
Bonoldi E, Boracchi P, Gatti C, Suzuki H, Tominaga T (1997). Prognostic
significance of VEGF in node-negative breast carcinoma. J Natl Cancer Inst 89:
139-147.
Kitamura M, Toi
M, Arai K, Iwasaki Y, Suzuki H, Matsuo K (1998). Concentrations of VEGF in the
sera of gastric cancer patients. Oncol Rep 5: 1419-1424.
Röckl W, Hecht
D, Sztajer H, Waltenberger J, Yayon A, Weich HA (1998). Differential binding
characteristics and cellular inhibition by soluble VEGF receptors-1 and –2. Exp
Cell Res 241: 161-171.
Gasparini G,
Toi M, Miceli R, Vermeulen PB, Dittadi R, Biganzoli E, Morabito A, Fanelli M,
Gatti C, Suzuki H, Tominaga T, Dirix LY, Gion M (1999). Clinical
relevance of VEGF and thymidine phosphorylase in patients with node-positive
breast cancer treatred with either adjuvant chemotherapy or hormone therapy. Cancer J Sci Am 5: 101-11.
Toi M, Gion
M, Saji H, Asano M, Dittadi R, gilberti S, Locopo N, Gasparini G (1999). Endogenous
IL-12: relationship with angiogenic factors, hormone receptors and nodal status
in human breast carcinoma. Int J Oncol 15: 1169-1175.
Ueno T, Toi
M, Tominaga T (1999). Circulating soluble Fas concentration in breast
cancer patients. Clin Cancer Res 5: 3529-33.
Vascular growth
factors and angiogenesis (1999). Claesson-Welsh (Ed.) Springer-Verlag Berlin,
Heidelberg.
Von Tiedemann B. et al. (1999) [submitted]?????
For
Research Use Only
See other VEGF products
including kit for:
VEGFR1 (total) cat#RDI-DA064 $724.50/kit
VEGF-A cat#RDI-DA066 $625.00/kit
See
also recombinant VEGFR’s at:
http://www.researchd.com/cytokines/rdicyt1.htm
RDI
Division of Fitzgerald Industries Intl
34 Junction Square Drive
Concord MA
01742-3049
USA USA
Phone
(978) 371-6446 or (800) 370-2222
Fax
(978) 371-2266
Email: antibodies@fitzgerald-fii.com
Web: http://www.researchd.com
(return) homePage
return
(main kit list)
terms
