rev: December 30, 2003
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Sample Kit Protocol: (see
actual insert with kit for batch specific information):
see also VEGF-A Elisa Kit
SOLUBLE
VEGF-RECEPTOR 1 ELISA Kit
CAT# RDI-DA064 (BMS268) now mfg by Bender Med Systems, Austria
old#RDI-M0100clb) 96
wells PRICE: $625.00 $562.00/kit 10+
-For the Quantitative
Determination of Recombinant and Natrual Occurring Human Soluble Vascular
Endothelial Growth Factor Receptor-1 (s-VEGFR-1) in cell culture supernatants
and complex biological fluids
For Research Use Only-Not
for use in diagnostics Procedures
RELIATech®
Second
Generation!
Improved Sensitivity!
Total Soluble Vascular Endothelial Growth Factor Receptor-1
(sVEGFR-1total) ELISA
|
|
|
|
|
Anti-Angiogenesis Test Kit
|
FOR THE QUANTITATIVE
DETERMINANTION OF RECOMBINANT AND NATURAL OCCURRING, HUMAN TOTAL SOLUBLE
VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR-1 (sVEGFR-1total) IN
CELL CULTURE SUPER-NATANTS AND COMPLEX BIOLOGICAL FLUIDS
FOR
RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURE.
STORE AT 4°
Introduction
Soluble
VEGFR-1 (sFLT-1) is a naturally occurring endogenous form of the VEGFR-1 and
was originally found in the supernatant
of human vascular endothelial cells. It is generated by differential splicing
of the flt-1 gene. In vitro sVEGFR-1 is used to inhibit
VEGF-A mediated signals in endothelial cells, and in vivo it can be used to block physiological angiogenesis in
several organs, e.g. in the ovary or in bones. Tumor cells transfected with the
flt-1 gene are growth restricted in vivo, because of the limitation in
developing tumor blood vessels via VEGF-A signalling. Very recent studies have
shown that this molecule is present endogenously at ng/ml concentrations in
biological fluids of normal human subjects or in the conditioned media of flt-1 positive cell types. The measurement of sVEGFR-1 in a variety of
clinical conditions may open up new insights in health and disease.
We are
pleased to announce that through a collaboration with several laboratories we
were able to develop the first simple, accurate immunoassay for the detection
of sVEGFR-1 in research samples and complex biological fluids such as cell
culture supernatants, amniotic fluids and cystic brain fluids. The assay allows
the detection of the total amount of sVEGFR-1 (free and complexed with
ligands). The product has been developed using state-of-the-art methods for
sandwich-ELISA developments and using specific, high affinity binding proteins
for sVEGFR-1. This new assay has been
validated under various conditions and is quantitatively calibrated to a
verified recombinant molecular standard.
Characterization
of angiogenic activity, such as embryonic development, placental
vasculari-zation, cancer and wound healing is measured by comparing the ratio
of angiogenic stimulators (e.g. FGF-2, FGF-1, VEGF-A, Ang-1) to angiogenic
inhibitors (e.g. sFLT-1, angiostatin, endostatin, thrombospondin). Several independently
published data of both normal and pathogenic subjects have confirmed
endogenous levels of VEGF-A and
bFGF in pg/ml to ng/ml ranges as measured by commercially
available sandwich ELISA kits. These factors have been thought to work
unopposed to cause blood vessel formation. The finding that sVEGFR-1, a strong
VEGF-A antagonist, is present in normal subjects suggests a finely tuned
balance of signal transduction, the workings of which can now be explored.
Together with other similar assay systems, positive and negative angiogenic
regulators can now be explored in many different physiological and pathological
settings using human cell culture
supernatants and biological fluids.
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
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.
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 and dilutions
Reagents
supplied as small volumes must be spinned down before opening the tubes to
avoid loss of reagents. Warm up buffers to room temperature before use.
Wash- and assay buffer: Dilute 10x assay buffer
concentrate and 20x wash buffer concentrate with destilled water.
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 sVEGFR-1 by
addition of 300µl assay buffer to make up 20ng/ml. Mix well. Serial dilutions
are prepared within the wells. The reconstituted standard should not be stored
for longer than 24h at 4°C.
Biotinylated detector: Dilute 1/100 in assay buffer (final volume
6ml).
Streptavidin enzyme: Dilute 1/100 in assay buffer (final volume 12ml).
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).
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
  10              
5            
2.5          
1.25       
0.625     
0.3125    
0.16 
ng/ml           
0.0 ng/ml
Discard
100µl
100µl
sVEGFR-1
Standard
Add
samples to the sample wells. Use 50µl of the samples for 1/2 dilution and 25µl
of the samples for 1/4 dilution.
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 (Incubate while mixing on a plate shaker/oribital shaker).
Wash plate four times.
Add 100µl streptavidin-enzyme
(diluted 1/100) in assay buffer) to each well. Seal plate and incubate for 1h
at room temperature.(Incubate
while mixing on a plate shaker/oribital shaker).
8. Wash plate four times.
9. 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.
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 plate.
Apply
standard, samples and biotinylated detector.
Incubate 2h
