Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1997-10-03
2000-06-27
Wortman, Donna
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 15, G01N 33573, C12Q 148
Patent
active
06080551&
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a rapid assay format for the detection and/or determination of glutathione S-transferases (GSTs) for use in the assessment of organ status.
BACKGROUND ART
GSTs represent a multigene family of proteins consisting mainly of .alpha., .mu., .theta., and .pi.-class isoforms and are responsible for the detoxification of a range of xenobiotics, mainly via conjugation to glutathione. Alpha GST (.alpha.GST) is the major hepatic form and is uniformly distributed throughout the liver. In contrast, PiGST (.pi.GST) is located mainly in the epithelial cells lining the bile ducts. A similar unique distribution also exists with respect to the renal situation where .alpha.GST is located in the proximal tubules and .pi.GST is confined to the distal tubule region of the nephron. It can therefore be concluded that measurement of either plasma or urinary GST levels will facilitate the monitoring of either the hepatic or renal status of an individual. Numerous authors have, indeed, used GST measurements to evaluate both liver and renal damage in individuals who have undergone organ transplant or who have suffered organ damage by virtue of exposure to either hepatic or renal toxins (see, for example, Trull, A. K. et al., Transplantation (1994) 58, 1345-1351).
Currently such measurements are most usually performed using radioimmunoassays (RIA). However, recently a number of microtitre plate-based enzyme immunoassays (EIAs) have become commercially available from Biotrin International Limited, Mount Merrion, County Dublin, Ireland, which allow measurement of .alpha. and .pi.GST levels in biological fluids. These EIAs are now gaining widespread acceptance among the scientific community despite some initial scepticism (c.f. Beckett, G. J. and Hayes, J. D. (Advances in Clinical Chemistry (1993) 30, p.325)). Thus, at present RIA represents the detection method of choice for many workers in the field. Both of these techniques have certain limitations insofar as they require both specialised equipment and highly trained and competent personnel to generate meaningful and accurate results. Such assays are relatively slow requiring on the average 2 hours or more from start to finish. Accordingly, a more rapid assay format for GSTs is required.
Methods for the detection of GST isoenzymes are tending to become increasingly complicated as evidenced by the use of Time-Resolved ImmunoFluorometric Assay (TR-IFMA). Tiainen, P et al. (Clin. Chem. (1996) 42 2, 334-335) compared enzyme immunoassay (EIA) and TR-IFMA for .alpha.GST. The EIA used was Hepkit (trade mark of Biotrin International, Dublin). It was found that the inter-assay precision was better in Hepkit than in TR-IFMA. Also TR-IFMA is stated to have been somewhat more laborious than Heptkit because more replicates (four) and more washing cycles were needed to complete the assay procedure. Thus, it is clear that current research in to the detection and quantitation of GST has focussed on the development of more elaborate and complicated detection systems such as TR-IFMA.
Individuals who have undergone organ transplant or who have suffered organ damage by virtue of having been exposed to hepatic or renal toxins must be carefully monitored. This is especially true for individuals who have undergone organ transplant and who undergo a long post-transplant period of therapy with immunosuppressive drugs, in particular cyclosporin. Once such individuals are discharged from hospital they must be continuously monitored, which at present necessitates frequent returns to the relevant hospital unit, so that the requisite GST and other assays can be carried out. Accordingly, it would be most desirable if such persons had a means of self-monitoring which would reduce the number of hospital visits but, more importantly, would give an early indication to the patient and physician that intervention is required, in the event that an above-normal level of a GST is noted.
Thus, a method which would allow a faster and simpler format for GST detection in plasma, ur
REFERENCES:
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patent: 5427917 (1995-06-01), Hosada et al.
Hara et al. J. Cancer Res. Clin Oncol 119: 493-496, 1993.
Trull et al. Transplantation 58(12): 1345-1351, 1994.
Trull et al., Transplantation (1994) 58, 1345-1351.
Beckett and Hayes, Advances in Clinical Chemistry (1993) 30, p. 281-380.
Tiainen et al., Clin. Chem. (1996) 42 2, 334-335.
Howie et al., Clinica Chimica Acta (1989) 184, 269-278.
Tiainen and Karhi, Clin. Chem. (1994) 40-2, 184-189.
Doyle John Martin
Kilty Cormac Gerard
Biotrin Intellectual Properties, Ltd.
Brumback Brenda
Wortman Donna
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