Methods and compositions for assaying analytes

Details Methods and compositions for assaying analytes Methods and compositions for assaying analytes

1999-07-06

2002-04-23

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S195000, C435S252300, C435S320100, C435S455000, C435S091500, C435S023000

Reexamination Certificate

active

06376210

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to compositions and methods for assaying analytes, preferably, small molecule analytes. More particularly, assay methods that employ, in place of antibodies, modified enzymes that retain binding affinity or have enhanced binding affinity, but that have attenuated catalytic activity, are provided. The modified enzymes are also provided.
BACKGROUND OF THE INVENTION
Methods for assaying analytes have wide applications. Many analytes including small molecule analytes are essential components and/or participants of biological systems and processes. Methods for assaying these analytes can be used in monitoring the biological systems/processes, or prognosis or diagnosis of diseases or disorders caused by deficiencies and/or imbalances of the analytes. For instances, homocysteine (Hcy), a thiolated amino acid; folic acid, an organic acid; and cholesterol, a lipid are all important prognostic and diagnostic markers for a wide range of cardiovascular diseases. Vitamins are important prognostic and diagnostic markers for various vitamin deficient diseases or disorders. Glucose, a monosaccharide, is a diagnostic marker for numerous glycemic conditions such as diabetic mellitus. Ethanol, an alcohol, is important in monitoring liquor consumption and potential liver damages. Bile acids or bile salts are important prognostic and diagnostic markers for certain cancers such as colon cancer. Monitoring uric acid is important because abnormally high concentration of uric acid is the diagnostic marker and cause of hyperuricemia leading to gout, which is very painful and can damage the kidney. In addition to these prognostic and diagnostic uses, methods for assaying analytes have applications in other agricultural, industrial or environmental protection processes where determining the presence, location and amount of the analytes is critical.
Assays for Homocysteine
Homocysteine (Hcy) is a thiol-containing amino acid formed from methionine during S-adenosylmethionine-dependent transmethylation reactions. Intracellular Hcy is remethylated to methionine, or is irreversibly catabolized in a series of reactions to form cysteine. Intracellular Hcy is exported into extracellular fluids such as blood and urine, and circulates mostly in oxidized form, and mainly bound to plasma protein (Refsum et al.,
Annu. Rev. Medicine
, 49:31-62 (1998)). The amount of Hcy in plasma and urine reflects the balance between Hcy production and utilization. This balance may be perturbed by clinical states characterized by genetic disorders of enzymes involved in Hcy transsulfuration and remethylation (e.g., cystathionine &bgr;-synthase and N
5,10
-methylenetetrahydrofolate reductase or dietary deficiency of vitamins (e.g., vitamin B
6
, B
12
and folate) involved in Hcy metabolism (Baual, et al.,
Cleveland Clinic Journal of Medicine
, 64:543-549 (1997)). In addition, plasma Hcy levels may also be perturbed by some medications such as anti-folate drugs (e.g., methotrexate) used for treatments of cancer or arthritis (Foody, et al.,
Clinician Reviews
, 8:203-210 (1998)).
Severe cases of homocysteinemia are caused by homozygous defects in genes encoding for enzymes involved in Hcy metabolisms. In such cases, a defect in an enzyme involved in either Hcy remethylation or transsulfuration leads to as much as 50-fold elevations of Hcy in the blood and urine. The classic form of such a disorder, congenital homocystemia (Hcymia), is caused by homozygous defects in the gene encoding cystathionine &bgr;-synthase (CBS). These individuals suffer from thromboembolic complications at an early age, which result in stroke, myocardial infarction, renovascular hypertension, intermittent claudication, mesenteric ischemic, and pulmonary embolism. Such patients may also exhibit mental retardation and other abnormalities resembling ectopia lensis and skeletal deformities (Perry T., Homocysteine: Selected aspects in Nyham W. L. ed.
Hertable disorders of amino acid metabolism
. New York, John Wiley & Sons, pp. 419-451 (1974)). It is also known that elevated Hcy levels in pregnant women is related to birth defects of children with neurotube closures (Scott et al., “The etiology of neural tube defects” in Graham, I., Refsum, H., Rosenberg, I. H., and Ureland P. M. ed. “
Homocysteine metabolism: from basic science to clinical medicine
” Kluwer Academic Publishers, Boston, pp. 133-136 (1995)). Thus, the diagnostic utility of Hcy determinations has been well documented in these clinical conditions.
It has been demonstrated that even mild or moderately elevated levels of Hcy also increase the risk of atherosclerosis of the coronary, cerebral and peripheral arteries and cardiovascular disease (Boushey, et al.,
JAMA
, 274:1049-1057 (1995)). The prevalence of Hcymia was shown to be 42%, 28%, and 30% among patients with cerebral vascular disease, peripheral vascular disease and cardiovascular disease, respectively (Moghadasian, et al.,
Arch. Intern. Med
., 157:2299-2307 (1997)). A meta-analysis of 27 clinical studies calculated that each increase of 5 &mgr;M in Hcy level increases the risk for coronary artery disease by 60% in men and by 80% in women, which is equivalent to an increase of 20 mg·dl
−1
(0.5 mmol·dl
−1
) in plasma cholesterol, suggesting that Hcy, as a risk factor, is as strong as cholesterol in general population. Results from these clinical studies concluded that hyperhomocysteinemia is an emerging new independent risk factor for cardiovascular disease, and may be accountable for half of all cardiovascular patients who do not have any of the established cardiovascular risk factors (e.g., hypertension, hypercholesterolemia, cigarette smoking, diabetes mellitus, marked obesity and physical activity).
Mild homocysteinemia is mainly caused by heterozygosity of enzyme defects. A common polymorphism in the gene for methylenetetrahydrofolate reductase appears to influence the sensitivity of homocysteine levels to folic acid deficiency (Boers, et al.,
J. Inher. Metab. Dis
., 20:301-306 (1997)). Moreover, plasma homocysteine levels are also significantly increased in heart and renal transplant patients (Ueland, et al.,
J. Lab. Clin. Med
., 114:473-501 (1989)), Alzheimer patients (Jacobsen, et al.,
Clin. Chem
., 44:2238-2239 (1998)), as well as in patients of non-insulin-dependent diabetes mellitus (Ducloux, et al.,
Nephrol. Dial. Transplantl
, 13:2890-2893 (1998)). The accumulating evidence linking elevated homocysteine with cardiovascular disease has prompted the initiation of double-blind, randomized and placebo controlled multicenter clinical trials to demonstrate the efficacy of lowering plasma Hcy in preventing or halting the progress of vascular disease (Diaz-Arrastia, et al.,
Arch. Neurol
, 55:1407-1408 (1998)).
Determination of plasma homocysteine levels may become a common clinical practice in the near future. Today, cardiologists have already started to recommend their patients to examine their homocysteine levels especially for those who have family history in cardiovascular disease, or who have cardiovascular problem but with normal levels of cholesterol and other risk factors, and those who are older than 60 years-old.
The assay of total Hcy in plasma or serum is complicated by the fact that 70% of plasma Hcy is protein-bound, 20-30% exists as free symmetric or mostly asymmetric mixed disulfides, free reduced Hcy exists in only trace amounts (Stehouwer, et al.,
Kidney International
, 55308-314 (1999)). As a risk factor for cardiovascular disease, the determination of total plasma Hcy levels (reduced, oxidized and protein-bound) has been recommended in clinical setting(Hornberger, et al.,
American J. of Public Health
, 88:61-67 (1998)). Since 1982, several methods for determining total plasma Hcy have been described (Mansoor, et al.,
Anal. BioChem
., 200:218-229 (1992); Steir, et al.,
Arch. Intern. Med
., 158:1301-1306 (1998); Ueland, et al.,
Clin. Chem
., 39:1764-1779 01993); and Ueland, et al., “Plasma homocysteine and cardiovascular disease” in Francis, R. B

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