Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1994-09-14
2000-05-16
Duffy, Patricia A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 79, 435 791, 435 793, 435 15, 435 18, G01N 3353
Patent
active
060635819
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an assay for homocysteine in clinical samples.
Homocysteine is an intermediary amino acid produced when methionine is metabolised to cysteine. Generally, homocysteine produced in the body is rapidly metabolised by one of two routes, (1) condensation with serine to form cystathione or (2) conversion to methionine, and its concentration (and that of its oxidised form homocystine) in the living body under normal conditions is virtually negligible.
Homocysteine levels in biological samples may however be of clinical significance in a number of situations as homocysteine plays an important part in the complex set of pathways which make up sulphydryl amino acid metabolism and its accumulation may be indicative of various disorders occurring in these pathways, including in particular inborn errors of metabolism. Thus, for example homocystinuria (an abnormal build-up of homocysteine in the urine) is known to be a disorder of amino acid metabolism resulting from deficiencies in the enzymes cystathione .beta. synthetase or methyltetrahydro-folic acid methyltransferase (which catalyses the methylation of homocysteine to methionine).
Sulphydryl amino acid metabolism is closely linked to that of folic acid and vitamin B.sub.12 (cobalamin), which act as substrates or co-factors in the various transformations involved. For this reason homocysteine accumulation has also been proposed as an indicator of malfunction of cobalamin or folate dependent enzymes, or other disorders or diseases related to cobalamin or folate metabolism.
Moreover since homocysteine conversion to methionine relies on a reaction requiring S-methyl tetrahydrofolate as the methyl donor, homocysteine metabolism may also be affected by anti-folate drugs, such as methotrexate, administered to combat other disorders, notably cancer. Monitoring of homocysteine has therefore also been proposed in the management of malignant disease treatment with anti-folate drugs.
More recently, elevated levels of homocysteine in the blood have been correlated with the development of atherosclerosis (see Clarke et al., New Eng. J. Med. 324:1149-1155 (1991)) and even moderate homocysteinemia is now regarded as a risk factor for cardiac and vascular diseases. Measurement of plasma or blood levels of homocysteine is thus also of importance in the diagnosis and treatment of vascular disease.
Although immunological methods of determining homocysteine directly are not available as there is no available antibody to homocysteine, a number of other methods for determining homocysteine in clinical samples have been proposed. These have all involved chromatographic separations and generally have been based on one of the three following principles: S-adenosyl-L-homocysteine hydrolase in the presence of a radioactively or otherwise labelled S-adenosine co-substrate followed by separation and quantitation of the product (S-adenosyl-L-homocysteine, SAH) formed. Generally chromatographic separation (HPLC or TLC) and radioactivity measurements are used (see Refsum et al., Clin. Chem. 31:624-628 (1985); Kredich et al., Anal. Biochem 116:503-510 (1981); Chui, Am. J. Clin. Path. 90(4):446-449 (1988); Totani et al., Biochea. Soc. 14(6):1172-9 (1988); and Schimizu et al., Biotechnol. Appl. Biochem 8:153-159 (1986)) HPLC-separation and fluorometry (see Refsum et al., Clin. Chem. 35(9); 1921-1927 (1989)).
These methods are time-consuming and cumbersome to perform and all rely on direct quantitation. More particularly, chromatographic separation is a common feature of the prior art methods and requires highly specialised and sophisticated equipment.
The use of such equipment is generally not well accepted in routine clinical laboratory practice and such processes are consequently not generally amenable to automation in typical clinical laboratory procedures.
A need therefore exists for an improved assay for homocysteine which is simple, specific, quick to perform, readily adapted for use in clinical laboratories and above all which avoids the need for costly and tim
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Bergmeyer, Methods of Enzymatic Analysis, 1985, pp. 110-117 and 357-364 and 403-409.
Refsum et al., Clinical Chemistry, vol. 31, No. 4, Apr. 1985, pp. 624-628.
Refsum et al "Radioenzymatic Determination of Homocysteine in Plasma and Urine" Clin Chem 31(4) p 624-628 (1985).
Hemmila "Fluoroimmunoassays and Immunofluorometric Assays", Clin Chem 31(1):359-370 (1985).
Harlow et al. "Antibodies, A Laboratory Manual" Chapter 14, 1988 by Cold Spring Harbor Laboratory.
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Duffy Patricia A.
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