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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                   $750.00/1kit    $562.00/kit 5+ 

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


Introduction


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.

Add streptavidine-enzyme to each well.

Incubate 1h.

Wash four times.

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

 

 

 

Fig. 2: 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                    $562.00/kit

 VEGF-A                    cat#RDI-DA066                    $625.00/kit

See also recombinant VEGFR’s at:

http://www.researchd.com/cytokines/rdicyt1.htm

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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

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