Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1997-04-17
2000-06-06
Minnifield, Nita
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 71, 435 74, 435 15, 435193, 4352523, 43525233, 4353201, 435325, 536 235, 536 232, 536 231, G01N 3353, G01N 33573, C12Q 148, C12N 910
Patent
active
060717068
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a stabilising medium for alpha glutathione S transferase (.alpha.GST) in urine and the use thereof in an enzyme immunoassay for .alpha.GST.
BACKGROUND ART
Glutathione transferases (GSTs) are enzymes which are found in highly varying amounts in human tissues. The enzymes form three major classes, designated .alpha., .pi. and .mu.. These three classes of enzyme are quite distinct in their properties.
.alpha.GST is found in the proximal tubule region of the kidney and is released into the urine in normal individuals, as confirmed by enzyme immunoassay and western blot analysis (Campbell, J. A. H. et al (1991) Cancer (Philadelphia), 67, 1608-1613). Any event which precipitates proximal tubule damage may cause the release of .alpha.GST into urine leading to an increase in normal urinary levels. Thus, an elevation of urinary .alpha.GST levels may be indicative of proximal tubule damage (Sherman, R. A. et al. (1985) Urermia Investigation, 8, 111-115). Recent work has shown that cisplatin induced proximal tubule damage in Wistar rats is associated with elevated levels of urinary .alpha.GST activity and decreased serum creatinine clearance (Stojanov, M. et al. (1994) Clin. Chem., 14, 1125), and that acute tubular necrosis and renal transplant infarction in humans result in a rapid increase of both .alpha. and .pi. GST levels (Sundberg, A. G. M. et al. (1994) Nephron 67, 308-316).
The ability to use urine as a sample of a body fluid for the detection and determination of an enzyme indicative of kidney damage and, in particular, a particular region of the kidney is an advantage, especially because no invasive collection of the body sample is required. In general, one wishes to estimate .alpha.GST in patients who are seriously ill and minimisation of any unnecessary trauma is very desirable.
Traditionally, radioimmunoassay has been used for estimating .alpha.GST in urine with the attendant disadvantages of using a radio-labelled substance.
Frequently, it is not possible to carry out the necessary estimation of urinary .alpha.GST for some considerable time, such as days, after a sample has been collected. Accordingly, it is often necessary to store the urine sample at very low temperatures, typically of the order of -20.degree. C. However, it is found that on storing urinary .alpha.GST at such low temperatures leads to a loss of immunoreactivity and thus a poor sensitivity of any immunoassay. This loss of immunoreactivity is most likely due to freeze-thaw denaturation.
Accordingly, there is a need for a medium which enables one to store .alpha.GST in urine at temperatures of the order of -20.degree. C. without any substantial loss of immunoreactivity of .alpha.GST in an immunoassay used to detect the .alpha.GST, when required.
DISCLOSURE OF INVENTION
Accordingly, the invention provides a stabilising medium for .alpha.GST in urine, which comprises a stabilising amount of a non-enzyme protein, a chelating agent and a buffer, such that the medium has a pH in the range 7.0-7.5, and the medium being effective to prevent loss of .alpha.GST immunological activity.
The stabilising medium according to the invention can be used to store urine samples at temperatures of the order of -20.degree. C. without any loss of immunoreactivity. However, additionally, the stabilising medium according to the invention is found to improve immunoreactivity when added to fresh urine which is stored temporarily at 2-8.degree. C. prior to assay for up to two hours.
Preferably, the non-enzyme (.alpha.GST) protein is an albumin.
The protein can be a mixture of albumin and hydrolysed gelatin. Such gelatin hydrolysates are commercially available for example from Sigma Chemicals (Code G-0262 enzymatically generated).
An especially preferred albumin (non-hydrolysed) is a serum albumin, especially bovine serum albumin (BSA).
A preferred mixture of a serum albumin and a hydrolysed gelatin is a mixture of equal amounts (w/v) of bovine serum albumin and gelatin hydrolysate.
The concentration of non-enzyme pr
REFERENCES:
patent: 4273756 (1981-06-01), Ling et al.
patent: 5217868 (1993-06-01), Kilty et al.
patent: 5264419 (1993-11-01), Horwell et al.
patent: 5278316 (1994-01-01), Horwell et al.
patent: 5817497 (1998-10-01), Goli et al.
patent: 5874248 (1999-02-01), Hillman et al.
Backman et al, Kidney International, 33:571-577, 1988.
Doyle John Martin
Kilty Cormac Gerard
Baskar Padma
Biotrin Intellectual Properties Limited
Minnifield Nita
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