Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-07-31
2001-02-06
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S002000, C435S007940, C435S015000, C435S975000
Reexamination Certificate
active
06183977
ABSTRACT:
TECHNICAL FIELD
This invention relates to a method of determining the hepatic status of a subject, including a liver transplant recipient and, thereby, deciding on appropriate therapy or corrective action, if required, dependent on said hepatic status.
BACKGROUND ART
The ability to differentiate between the various types of hepatic injury is of great significance in the treatment of both transplant patients and also individuals who suffer from other hepatic diseases which may affect the biliary system.
Glutathione S-transferases (GSTs) comprise a multigene family of proteins consisting mainly of alpha (&agr;GST), mu (&mgr;GST), pi (&pgr;GST) and theta-class (&thgr;GST) isoforms as defined by isoelectric point and are responsible for the detoxification of a range of xenobiotics, mainly via conjugation to glutathione (Beckett, G. J and Hayes, J. D., Advances in Clinical Chemistry (1993); 30, 281-380). Generally, the proteins are dimeric in nature consisting of two 25-27kDa subunits and may exist in homodimeric or heterodimeric forms. Pi Glutathione S-transferase (&pgr;GST) is a homodimer, and is located in the cytoplasm of bile duct epithelial cells within the liver (Beckett G. J. and Hayes, J. D., (1993) supra). &agr;GST is known to be present in hepatocytes within the liver and exists in both homodimeric and heterodimeric states (Campbell, J. A. H., et. al., Cancer (Philadelphia) (1991) 67, 1608-1613; Howie, A. F., et. al., Clin. Chem. Acta., (1988) 177, 65-76). This heterogenous GST distribution of &agr; and &pgr;GST suggests that the different isoenzymes have unique in vivo functions in different hepatic regions (Campbell, J. A. H., et. al., (1991) supra).
EP-A 0 640 145 discloses a method which assists in the early diagnosis of rejection in a liver transplant recipient and which comprises measuring an increase in plasma or serum &agr;GST from the recipient in the absence of or preceding any change in plasma or serum transaminase. Thus, it has been conclusively demonstrated that measurement of the plasma &agr;GST level facilitates monitoring of the post-transplant hepatic status by acting as an extremely sensitive, although not totally specific marker of graft rejection.
It is notable that &pgr;GST has received no attention as a potential marker of graft rejection, a fact possibly due to the low levels of enzyme present in the biliary epithelial cells of the liver. There is some evidence, however, that &agr; and &pgr;GST are present in bile from both normal individuals and people suffering from specific cancers (e.g., cholangiocarcinoma) as measured by radio-immunoassay (Howie. A. F., et. al., Clin. Chem. Acta. (1989) 184, 269-278). Additionally, some authors have referenced the fact that measurement of serum and plasma &pgr;GST levels may facilitate diagnosis of malignant tumours since &pgr;GST appears to be specifically expressed in malignant tissue (Niitsu, Y., etal., Cancer (1989) 63, 317-323; Howie, A. F., et. al., Clin. Chem. (1990) 36(3), 453-456. and Hida, T., et al., Cancer (1994) 73(5), 1377-1382. None of the aforementioned authors allude to the fact that &pgr;GST may have a role in the prediction of transplanted liver rejection or other liver/biliary disorders.
Since it is known that primary graft rejection generally occurs in the biliary tree within the liver (Ascher, N., (1993) In ‘Immunology of liver transplantation’ Neuberger, J. and Adams, D. (eds)), it would appear that specific measurement of biliary or plasma &pgr;GST levels may allow diagnosis of early rejection or facilitate discrimination between post-transplant hepatocellular or biliary damage. The importance of distinguishing between non-specific hepatic injury and graft rejection cannot be overstated since the treatment for each condition is entirely different. Furthermore, initiation of the incorrect treatment could be extremely deleterious to the health of an individual already severely ill. For example, if graft injury occurs due to viral infection (e.g., Hepatitis C re-infection or cytomegalovirus (CMV), it is necessary to carefully monitor the levels of anti-rejection immunosuppression treatment since excess immunosuppresive agents (e.g., cyclosporin A or FK506) would significantly impair the ability to fight viral infection. Conversely, failure to recognise genuine rejection from non-specific graft injury could lead to delay in augmentation of immunosuppressive therapy and ultimately lead to graft removal.
Accordingly, there is a need for methods of determining the hepatic status of an individual in various disease states or abnormal conditions of the liver.
SUMMARY OF THE INVENTION
The invention provides a method of determining the hepatic status of a subject, which method comprises measuring the level of the pi glutathione S-transferase (&pgr;GST) isoform in a sample of a biological fluid from said subject by an immunoassay specific for the &pgr;GST isoform, comparing the level of &pgr;GST measured with the normal range of &pgr;GST in said biological fluid and, when an increase in &pgr;GST level relative to said normal range is detected, determining the hepatic status of the subject based on the level of &pgr;GST in said biological fluid.
By providing a further method for determining hepatic status based on a marker specific to a particular hepatic site greatly facilitates the treatment of patients with various disease states and other abnormal conditions of the liver as hereinafter described in greater detail.
The subject is suitably a liver transplant recipient and the hepatic status is determined post-transplantation.
The invention has particular application in the case of liver transplantation because it enables one to determine at a very early stage post-transplantation a likelihood of rejection because the primary graft rejection generally occurs in the biliary tree within the liver as stated above. Accordingly, even earlier detection of liver transplant rejection is possible with the method according to the invention relative to the method described and claimed in EP-A 0 640 145.
Preferably, the recipient is a human.
The immunoassay is preferably an enzyme immunoassay, more especially a sandwich enzyme immunoassay.
The method according to the invention can be used to measure &pgr;GST in a range of media, but especially in bile, plasma and serum.
By biological fluid herein is meant for example body fluids such as bile, plasma, serum and urine as well as tissue support media and perfusates. The biological fluids herein are also referred to generally as matrices.
The method according to the invention facilitates for the first time detection of the &pgr;GST isoenzyme level in bile.
When the biological fluid is bile, the normal &pgr;GST level is less than 15 &mgr;g/L.
When the biological fluid is plasma, the normal &pgr;GST level is less than 100 &mgr;g/L.
As demonstrated hereinbelow care should be taken when the method is carried out on plasma that the plasma is collected and stored prior to the determination in the presence of an anti-coagulant under conditions which permit substantially no haemolysis to occur during said storage period.
We have found that use of fluoro-oxylate tubes results in a high degree of haemolysis releasing &pgr;GST from erythrocytes which gives falsely elevated levels of &pgr;GST. Other GST isoenzymes are either not found in the blood or are present at extremely low levels. For example, &mgr;GST is present in leucocytes. However, it is not clear from the literature as to whether it is present in erythrocytes. In any event &mgr;GST is only present in 50% of the population. &thgr;GST expresses a similar inter-individual variability as does &mgr;GST and if present in blood is present at extremely low levels. &agr;GST is not present in blood to any great extent.
In a preferred embodiment, the sample is diluted with a diluent which contains an effective amount of a protein which optimises antibody-antigen reactions.
We have found that if the diluent includes Tween 20 conventionally used as a standard reagent in such immunometric methods that incorrect &pgr;GST concen
Doyle John Martin
Kilty Cormac Gerard
Manning Fiona Mary
Biotrin Intellectual Properties, Ltd.
Birch Stewart Kolasch & Birch, LLP.
Naff David M.
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