Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving urea or urease
Reexamination Certificate
2000-08-01
2003-02-18
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving urea or urease
C435S007100, C435S007920, C435S004000
Reexamination Certificate
active
06521416
ABSTRACT:
Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
BACKGROUND OF THE INVENTION
With the extension of the human life span, women spend one-third of their lives beyond the reproductive years. The transition into menopause, a normal process of aging, is associated with physical risks and psychological adjustments. It is critical to improve understanding of the these changes. However, there is a lack of diagnostic tools for monitoring the temporal stages in the history of menopause despite the importance of this transition period. There is no reliable test to determine how close a woman is to menopause. Clinical decisions for treatment of perimenopausal women today are based chiefly upon subjective symptoms rather than objective diagnostic tests.
There is a lack of adequate chemical markers for defining the menopausal state since neither serum gonadotropins, estradiol, nor inhibin A or B levels are adequate for diagnosis unless daily sampling is performed for prolonged periods of time (Burger, 1996;. Burger, et al., 1995; Burger, 1994a; Burger, 1994b; Hee, et al. 1993; Metcalf, 1988 ). A number of studies of women during the periovulatory period have indicated that the currently used biochemical markers of menopause are inadequate (Burger, 1996;. Burger, et al., 1995; Burger, 1994a; Burger, 1994b; Hee, et al. 1993; Metcalf, 1988; Santoro, et al. 1996). Gonadotropin levels fluctuate from postmenopausal cncentrations back down to levels found in normal, young cycling women (Burger, 1996; Burger, et al., 995; Burger, 1994a; Metcalf, 1988; Metcalf, et al. 1982; Metcalf and Donald, 1979). What appear to be normal ovulatory cycles may follow prolonged anovulatory periods coincident with postmenopausal concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) (Burger, 1996;. Burger, et al., 1995; Burger, 1994a; Burger, 1994b; Metcalf, 1988; Metcalf, and Donald, 1979; Metcalf, et al. 1981a). Some investigators declare that all current biochemical measurements have little predictive value during the menopausal transition because of the great variations in levels of steroids and gonadotropins. (Burger, 1996;. Burger, 1994a; Burger, 1994b; Hee, et al. 1993;. Metcalf, and Donald, 1979; Metcalf, et al. 1981b; Metcalf, 1979).
Although elevations in certain serum gonadotropin levels reflecting gametogenic failure usually occur several years before a decline in estrogen and irregular cycling begins, measurement of serum gonadotropin levels, estrogen, and inhibins A and B have limited value to the practicing physician. A reliable test is essential to differentiate a premenopausal woman from a woman very early in perimenopause or the laster from one in the middle of the transition; menopausal changes could be placed in relation to the stage of menopausal transition. This would help to resolve, or example, whether treatment for osteoporosis should begin much earlier or that hormone replacement therapy should begin at a different time rather than based on symptomatic discomfit. The present invention solves these problems by providing urinary-based immunoassay methods and assay kits
Human gonadotropins undergo metabolic transformations, which result in the presence of several smaller, structurally and immunologically related forms in the urine. A major form of urinary hCG-associated immunoreactivity is an epitope on a molecule smaller than heterodimeric hCG (Birken et al., 1996; O'Connor et al., 1994; Schroeder and Halter, 1983). This molecule has been identified as an hCG beta core fragment (hCG&bgr;cf) (Birken et al., 1988; Blithe et al., 1988). In normal pregnancy, the core fragment constitutes a major mole fraction of urinary hCG excretion (Kato and Braunstein, 1988). Using polyclonal antisera raised against hCG&bgr;cf, immunoreactive beta core like activity can be detected in both postmenopausal women and in the periovulatory period of the normal menstrual cycle (Iles et al., 1992; Neven et al., 1993). However, some immunoreactivity results from cross-reactivity with the polyclonal hCG&bgr;cf antibodies. An hLH beta core fragment (hLH&bgr;cf) has been isolated from human pituitaries and a panel of monclonal antibodies has been generated (Birken et al., 1993a; Kovalevskaya et al., 1995).
The corresponding urinary fragment is inferred from mass spectral and immunochemical analysis of chromatographically separated urinary forms. Physico-chemical characteristics, primarily mass spectral and chromatographic, indicate that the pituitary and urinary forms of hLH&bgr;cf have a different structure, probably in the carbohydrate moieties. The carbohydrate moiety of the pituitary hLH&bgr;cf resembles that of pituitary hLH&bgr; rather than displaying the degraded carbohydrate chains present in urinary hCG&bgr;cf. The endogenous urinary core material is extremely stable to repeated freeze/thaw cycles and prolonged storage at 4° C. or at room temperature. HLH&bgr;cf cross-reaction with some hLH cr hLH&bgr; monoclonal antibodies may well interfere with the accurate estimation of the day of hLH surge when urinary tests are utilized. The expression of hLH&bgr;cf in the urine of both reproductive and postreproductive age women and in men, is now characterized employing assays highly specific for the pituitary form of the fragment.
Analysis or the metabolites of the gonadotropins excreted into urine can help to distinguish between healthy and abnormal physiological states. For example, the hCG &bgr; core fragment (hCG&bgr;cf) is present at high levels in the urine of normal pregnant women (Kato et al., 1988) but, also, occurs abnormally in the urine of nonpregnant patients with a variety of malignancies (O'Connor et al., 1988, Cole et al., 1988a,1988b,1990). Until now, it has not been possible to distinctly measure one of the fragments in the presence of the others. For example, the utility of the hCG&bgr;cf molecule as a marker of malignancies in postmenopausal women has been compromised by the cross-reactions of antibodies elicited to the hCG&bgr;cf with a molecule of similar structure and size (presumably the homologous fragment of hLH) excreted by normal postmenopausal women in their urine. Consequently, the high threshold measurement compromised the ability of hCG&bgr;cf to serve as a cancer marker in this important patient population. Distinguishing hLH&bgr;cf from an hCG&bgr;cf, therefore, is of great value. A preponderance of hLH&bgr;cf may indicate a normal state while a major mole fraction of the hCG fragment may be associated with malignancy (Birken et al., 1993b). The present invention provides a method to make such a distinction. Immunological analysis of the hLH&bgr;cf in normal cycling women, as compared with infertile patients, may identify a metabolic marker associated with an abnormal state (i.e. an ovulatory cycles, polycystic ovarian disease). Antibodies to the hLH&bgr;cf, isolated from pituitary extract, can also be used to measure such a molecule in urine.
Methods for specific immunometric assays which report the levels of expression of this new hLH molecular form, hHL&bgr;cf, in men and women at different stages of their reproductive history are described herein. The present invention now makes it possible to evaluate the metabolism of hLH in premenopausal, perimenopausal and postmenopausal women and in men and to distinguish between normal and abnormal physiological states.
In addition, these methods to visualize LH fragment in plasma differentiates LH fragment derived directly from pituitary from that derived by peripheral cleavage of LH. hLH&bgr;cf may circulate in plasma.
The methods described herein measure the stable m
Birken Steven
Kovalevskaya Galina I.
Maydelman Yacov
O'Connor John F.
Cooper & Dunham LLP
Deberry Regina M.
Kunz Gary L.
The Trustees of Columbia University in the City of New York
White John P.
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