Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-12-05
2001-04-24
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S007600, C435S008000, C435S021000, C435S325000
Reexamination Certificate
active
06221617
ABSTRACT:
TECHNICAL FIELD
This invention relates to assays for measuring the biological activity of an extracellular signal. More specifically, this invention relates to a bioassay for growth hormone releasing hormone (GHRH).
BACKGROUND OF THE INVENTION
Growth hormone releasing hormone (GHRH) is a peptide secreted by the hypothalamus. Normally, GHRH stimulates pituitary growth hormone release. The presence and activity of GHRH is therefore very important, particularly in normal growth and childhood development.
GHRH is also useful for testing pituitary function. Doses of GHRH can be used to stimulate pituitary secretion of growth hormone. If GHRH does not stimulate the pituitary to secrete growth hormone, this tends to indicate that the pituitary gland is not functioning properly.
GHRH can also be used to treat postmenopausal osteoporosis. Administration of GHRH can result in a reversal in bone mass loss in postmenopausal patients.
Because GHRH has many medical applications, it is highly desirable to be able to detect the presence or absence of GHRH in a sample or to qualitatively or quantitatively measure the activity of GHRH in a convenient, reliable manner.
Primary cultures of rat pituitary cells are typically used in a radioimmunoassay to asses the biological activity of GHRH. However, this radioimmunoassay is costly, time consuming and requires animals and radioactive materials.
A bioassay that would reduce the amount of time needed to detect GHRH in a sample would therefore be desirable. Additionally, it would be preferable if the bioassay did not require live animals, radioactive materials or specific antibodies.
SUMMARY OF THE INVENTION
This invention is directed to an in vitro bioassay method for detecting growth hormone releasing hormone (GHRH) in a sample. The method includes exposing a recombinant assay cell responsive to GHRH to a sample and measuring expression of a reporter gene whose expression is correlated to the concentration of GHRH in the sample.
A second aspect of the invention is directed to a recombinant assay cell useful for detecting GHRH in a sample. A suitable recombinant assay cell expresses a protein that is responsive to GHRH, e.g., a GHRH receptor which, upon activation by GHRH, causes the production of cAMP. The recombinant assay cell also expresses a reporter gene operatively connected to a cAMP-responsive promoter.
In the assay method of the invention, GHRH present in a sample activates the GHRH responsive protein, which then causes the production of cAMP. cAMP induces the expression of the reporter gene, and a signal generated by the reporter protein is quantitatively correlated with the concentration of GHRH in the sample.
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Grotjan H. Edward
Heinrich Julie
Wagner Fred W.
Xia Yuannan
Foley & Lardner
Kemmerer Elizabeth
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