rev:  16.05.2008

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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: $724.50

-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