Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2002-02-07
2004-09-07
Falk, Anne-Marie (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S029000, C435S069700, C435S090000, C435S194000, C435S325000
Reexamination Certificate
active
06787323
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to detection of vascular endothelial growth factor (VEGF) in a sample. Specifically, the present invention relates to a VEGF responsive cell-based assay for use in the measurement of the biological activity of VEGF.
BACKGROUND OF THE INVENTION
A number of biological properties have been described for VEGF, including the promotion of angiogenesis. Other properties include endothelial cell migration, endothelial cell proliferation, in vitro capillary tube formation, inhibition of endothelial cell apoptosis, and increased in vivo vascular permeability producing edema.
Because VEGF appears to have a number of significant biological properties, assays for the detection of VEGF and its properties have become increasingly important.
One in vitro bioassay which has been developed is based on the ability of human umbilical vein endothelial cells (HUVEC) to migrate in response to VEGF. This bioassay includes the steps of first virally transfecting Rat-2 cells with the consequent production of VEGF protein, and then testing the produced protein extract for the ability to stimulate HUVEC migration. While this assay can be utilized to demonstrate endothelial cell migration stimulated by VEGF protein in the conditioned media of cells, this assay includes a number of time-consuming steps, which cannot easily be automated.
Recently, it was proposed that VEGF could be a marker of cardiovascular disease risk in patients with hypertension. The measurement of a patient's VEGF and/or FLT-2 levels may be an indicator of the effectiveness of a hypertensive therapy.
Currently, with the seemingly greater importance of VEGF, it would be both advantageous and desirable to have a VEGF responsive assay that is less complex than previous assays, that can be automated, and that is also directly linked to a reporter protein tied to the VEGF receptor (VEGF-R)/FLK-1 signal transduction pathway.
SUMMARY OF THE INVENTION
The present invention provides a method for determining VEGF activity in a sample.
The present invention also relates to a secondary screen which is useful for identifying compounds that modulate VEGF receptors.
The present invention also provides a primary screening mechanism for screening compounds as inhibitors of VEGF function.
The present invention also provides a stable cell line for use in determining VEGF bioactivity and for use in screening compounds which modulate VEGF function or VEGF receptor function.
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Batley Brian Lee
Dahring Tawny Kay
Karathanasis Sotirios K.
Panek Robert Lee
Tian Ye Edward
Falk Anne-Marie
Jacobs Seth H.
Pfizer Inc
Richardson Peter C.
Sullivan Daniel M.
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