Method for cAMP production

Details Method for cAMP production Method for cAMP production

2001-07-23

2003-04-01

Gitomer, Ralph (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S029000, C435S069200

Reexamination Certificate

active

06541196

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of detecting, quantifying and purifying chemicals responsible for cellular signaling. The instant invention concerns a novel method for measuring the levels of cyclic adenosine monophosphate (cAMP) produced by cells which does not require that the cells be lysed. Specifically, the present invention provides for a method of detecting and quantifying cAMP extracellularly and for kits useful in employing these methods. Additionally, the instant invention provides for a method of isolating cAMP produced by cell culture.
2. Technical Problem Addressed by the Invention
A. Measurement for cAMP for Intact Cells
Heretofore in the field of cell signaling, in order to measure the level of cAMP produced by a cell during a cell-signaling event, it has been necessary to break open (or lyse) the cells. The instant invention provides for a method of measuring cellular cAMP levels without lysing the cells. This is advantageous because it allows for the continued growth and monitoring of the cells whose cAMP levels are being monitored.
A related problem has been the high cost of producing cAMP for experimental and other uses. cAMP is typically produced today by expensive synthetic chemical means. The instant invention also provides for a more cost-effective method of producing cAMP by doing so in a tissue culture or bio-reactor system.
SUMMARY OF THE INVENTION
The present invention provides for a method of measuring and producing cAMP in a cell culture or bioreactor system. The instant invention also provides for kits useful for measuring cAMP in these types of systems.
One embodiment of the instant invention provides for a method of measuring the cAMP concentrations produced by cells without disrupting the cell's membranes. This method comprises the following steps:
a) Providing cells growing in culture.
b) Removing the growth media from the cells.
c) Adding cAMP collection medium to the cells.
d) Incubating said cells with said collection medium.
e) Removing the collection medium and determining the cAMP concentration in the collection medium.
Another aspect of this embodiment of the invention provides for a kit for use to determine the amounts of cAMP produced by cells.
Another embodiment of the instant invention provides for a method of producing cAMP from tissue culture or a bioreactor. One aspect of this embodiment of the invention comprises the steps of:
a) Providing a culture of cells.
b) Removing the growth media from the cells.
c) Adding a cAMP collection medium.
d) Incubating said cells with said collection medium.
e) Removing the collection medium, and purifying the cAMP from the collection medium.


REFERENCES:
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Sigiyama A. An Enzymatic Fluorometric Assay for Adenosine 3′:5′-Monophosphate. Analytical Biochem 218 20-25, 1994.*
Suidan H. Simultaneous Analysis of Adenosine 3′,5′-cyclic Monophosphate . . . Anal Biochem 205(1)159-165, 1992.*
Ethier M. Mechanism of Enhanced cAMP Stimulation by Isoproterenol in Aged Human Fibroblasts. Exp Gerontology 27(3)287-300, 1992.*
Izevbigie, E.B. and Bergen, W.G. (2000)Beta-adrenergic agonist hyperplastic effect is associated with increased fibronectin gene expression and not mitogen-activated protein kinase modulation in C2C12 cells, Proc. Soc. Exp. Biol. Med. 223:302-309.
Izevbigie, E.B., Gutkind, J.S., and Ray, P.E. (2000) Angiotensin II and basic fibroblast growth factor mitogenic pathways in human fetal mesangial cells,Pediatr. Res. 47:614-621.
Izevbigie, E.B., Gutkind, J.S., and Ray, P.E. (2000) Isoproterenol inhibits fibroblast growth factor-2 growth of renal epithelial cells,Pediatr. Nephrol. 14:726-734.
Tovey, K.C., Oldham, K.G., Whelan, J.A., (1974) A simple direct assay for cyclic AMP in plasma and other biological samples using an improved competitive binding technique.Clin. Chim. Acta56:221-234.

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