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HepAnalyze  Heparanase ELISA

Catalog Number: INS-26-4-0000-20

PURPOSE
HepAnalyze is an enzyme immunoassay (enzyme-linked
immunosorbent assay'' ELISA) kit for the quantitative
measurement of human Heparanase in samples of body fluids''
such as plasma and urine and in cell extracts. HepAnalyzeTM is
for in-vitro research use only and is not intended for human or
animal diagnostics or therapeutic uses of any sort.

BACKGROUND
Heparanase is an endo-β-D-glucuronidase'' which degrades heparan
sulfate (HS) side chains of heparan sulfate proteoglycans (HSPGs)
in the extracellular matrix (ECM). Heparanase plays an important
role in ECM degradation'' facilitating the migration and
extravasation of tumor cells and inflammatory leukocytes (1''2''3).
Upon degradation'' Heparanase releases growth factors and
cytokines that stimulate cell proliferation and chemotaxis (4''5).
Heparanase is a glycosylated heterodimer comprised of a 50 kDa
subunit'' harboring the active site and an 8 kDa subunit. It is
produced as a latent 65 kDa precursor [H65] and proteolytically
processed to its active form [H58] (1''6). Heparanase is highly
expressed in myeloid leukocytes (i.e. neutrophils)'' in platelets and
in human placenta. Human Heparanase was found to be
upregulated in various types of primary tumors'' correlating in
some cases with increased tumor invasiveness and vascularity and
with poor prospective survival (7''8). Recent data suggest a role for
Heparanase in several proteinuric diseases and an increased
glomerular Heparanase expression is associated with loss of HS in
the glomerular basement membrane (9) leading to albuminuria
(10). Heparanase has also been reported to have a role in
inflammation and autoimmune diseases (11''12). In addition''
independent of its enzymatic activity'' Heparanase has roles in cell
adhesion and migration and enhancing signaling cascades (13''14).
REFERENCES
1. Vlodavsky'' I et al.'' Nat. Med. 5 (1999) 793–802
2. Vlodavsky'' I and Friedmann'' Y'' J. Clin. Invest. 108 (2001) 341–347
3. Parish CR et al.'' Biochim. Biophys. Acta 1471 (2001) M99-M108
4. Vlodavsky'' I et al.'' Cancer Metastasis Rev. 9 (1990) 203-226
5. Bashkin'' P et al.'' Biochemistry 28 (1989) 1737-1743.
6. Fairbanks'' MB et al.'' J. Biol. Chem. 274 (1999) 29587– 29590
7. Koliopanos'' A et al.'' Cancer Res. 61 (2001) 4655–4659
8. Gohji'' K et al.'' Int. J. Cancer 95 (2001) 295–301
9. van den Hoven'' MJ et al.'' Kidney Int. 72 (2007) 543-548
10. Maxhimer'' JB et al.'' Diabetes 54 (2005) 2172-2178
11. Parish'' CR'' Nat. Rev. Immunol. 6 (2006) 633-643
12. Li'' JP and Vlodavsky'' I'' Thromb Haemost. 102 (2009) 823-828
13. Fux'' L et al.'' Trends. Biochem. Sci. 34 (2009) 511-519
14. Goldshmidt'' O et al.'' FASEB J. 17 (2003) 1015-1025

ASSAY PRINCIPLE
The HepAnalyzeTM Heparanase immunoassay is a quantitative
solid phase ELISA designed to measure human pro-Heparanase
[H65] and the active Heparanase [H58] levels in samples of body
fluids and cell extracts. The wells of the microtiter plate are precoated
with monoclonal antibodies directed against Heparanase.
Standard and samples containing Heparanase are incubated in the
coated wells and following washing to remove unbound
substances'' a monoclonal anti-Heparanase antibody conjugated to
horseradish peroxidase is added to the wells. After incubation'' the
unbound conjugate is washed off. The amount of bound
peroxidase is proportional to the amount of Heparanase in the
samples. Having added the substrate solution'' the intensity of the
color developed is proportional to the concentration of
Heparanase in the samples.
SENSITIVITY
HepAnalyzeTM Heparanase ELISA Kit is able to detect pro-
Heparanase [H65] and active Heparanase [H58] at a minimum of
55 pg/ml in assayed samples.
RANGE
HepAnalyzeTM Heparanase ELISA Kit has a dynamic range of
55-7000 pg/ml.
REAGENTS PROVIDED WITH THE KIT
1. Microtiter plate (96-well)'' breakable to individual 8-well
strips coated with a monoclonal antibody specific to
Heparanase
2. Heparanase Standard
3. Assay Diluent. Ready for use
4. Wash Buffer
5. Antibody conjugate: anti-Heparanase monoclonal antibody
conjugated to horseradish peroxidase Substrate solution.
Ready for use
6. Stop solution. Ready for use
7. Plate sealing tape
EQUIPMENT REQUIRED (NOT SUPPLIED WITH KIT)
1. Manifold dispenser or plate washer
2. Polypropylene tubes or U-bottom 96-well microplate
3. Orbital shaker
4. Pipettes and pipette tips
5. Distilled water
6. Microplate reader for 96-well microplates'' capable of
measuring absorbance at 450nm
STORAGE
Upon arrival'' store Assay Diluent at -20°C. Other kit components
should be stored at 2-8°C. Under these conditions HepAnalyzeTM
Heparanase ELISA Kit is stable for 3 months.