Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2000-07-12
2002-09-17
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S004000, C436S066000, C436S064000
Reexamination Certificate
active
06451550
ABSTRACT:
Haptoglobin (Hp) is a protein which is present in the blood of man and animals. The concentration of Hp in plasma or serum, following separation of the blood cells, varies in an individual animal and is related to the health status of the animal. Hp is one of a group of proteins the concentration of which increases dramatically following infection, inflammation or trauma. These proteins are known as the acute phase proteins.
Measurement of the concentration of Hp in plasma gives valuable diagnostic information to clinicians in human and veterinary medicine. In veterinary medicine, measurement of Hp is particularly important in assessing the health status of cattle and sheep as in these species Hp gives a particularly strong response to infection, with the concentration increasing in the circulation over 100 times. In other species, such as with man, the dog, the cat and the pig the measurement of Hp is also important as the plasma concentration increases 2 to 3 fold, which is sufficient to provide diagnostic information. Additionally in these other species a fall in Hp concentration may have value in diagnosis of haemolysis. Moreover, the measurement of Hp is of equal importance as a marker of inflammation or infection in laboratory animals such as rodents or mice.
Presently assays for Hp are based on either immunoassay or on the ability of Hp to bind to haemoglobin (Hb):
Hp in human plasma is measured in clinical biochemistry laboratories as a routine test for the acute phase response by antibody based methods with antiserum specific for human Hp. The commonest approach is by immunoturbidimetry, where the formation of a precipitate of antibody-Hp complex in solution can be measured and related to Hp concentration. However immunoturbidimetric assays are expensive when compared to routine biochemical tests as they require the preparation of a continuing supply of suitable antiserum. In addition, for use in veterinary diagnostic laboratories, tests based on antiserum to one species have to be validated for each separate species and the validation should be repeated for each new batch of antiserum used.
The original assays based on Hp-Hb binding depended on the finding that formation of the Hp-Hb complex alters the spectrophotometric absorption characteristic of Hb in proportion to the concentration of Hp in a plasma sample. But this has been replaced by making use of the innate peroxidase activity of the complex, which can be detected at a slightly acidic pH when it is proportional to the Hp content as the peroxidase activity of free haemoglobin is inhibited, allowing assays to be quantified by calibration with standard samples of Hp.
This assay is used in the majority of veterinary diagnostic laboratories which currently perform Hp assays and is preferred to immunoassay systems as it can be performed on all species with only a modest requirement for validation and it is also considerably cheaper than antibody based methods as the reagents are inexpensive. However, the automated version of this test utilises a reagent guaiacol which has a noxious odour and is not accepted by staff in many laboratories. The assay has not generally been adopted by commercial reagent suppliers probably for this reason. Attempts have been made to use other substrates for the peroxidase reaction, and while this can be achieved for manual methods, using tetra methyl benzidine (TMB), see Conner J. et al Research in Vet. Sci. (1988) 44, 82-88 a successful automated assay for Hp is still not available. This may be due to the sensitivity of TMB. It has been observed by the present inventors that serum samples without any haptoglobin (zero blanks) display a significant level of peroxidase activity capable of causing false positive results with TMB. It will be appreciated that such spurious peroxidase activity is undesirable when conducting an automated or semi-automated assay.
U.S. Pat. No. 4,695,552 describes a process for the determination of the Hp-Hb complex similar to the processes described above. However, it is identified that an acidic pH may not be sufficient to completely inactivate the peroxidase activity of free haemoglobin and that a detergent is added to substantially eliminate any peroxidase activity of the free haemoglobin. Nevertheless, there is no suggestion that components present in blood serum or plasma samples may effect the accuracy of such an assay.
It is amongst the objects of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages.
The present invention is based in part on the discovery by the present inventors that albumin and possibly other proteins present in blood samples has an undesirable “peroxidase effect” on assays of the type mentioned above.
The present invention therefore provides an assay for determining a level of haptoglobin in a sample, wherein the assay comprises the steps of:
a) forming a reaction mixture comprising the sample to be tested, haemoglobin and at least one reagent for reducing a peroxidase effect due to any albumin and/or any other protein(s) present in the sample,
b) allowing the sample, haemoglobin and said at least one reagent to react, so as to allow formation of an haptoglobin/haemoglobin complex; and
c) determining a level of peroxidase activity of said haptoglobin/haemoglobin complex, wherein the determination is carried out at an acidic pH sufficient to significantly reduce or substantially inactivate any peroxidase activity of uncomplexed haemoglobin.
It has been previously reported that uncomplexed haemoglobin displays a peroxidase activity at an acidic pH, but that this activity could be inactivated at pH 4.1. Makimura, S. and Suzuki, N. (1982) Jpn. J. Ven. Sci. 44, p15-21. U.S. Pat. No. 4,695,552 suggests however that a low level of peroxidase activity due to free haemoglobin may remain even at such a pH. Thus, the present assay is preferably carried out at a pH less than 4.1, for example pH 3.6-4.0, especially pH 3.8.
Typically the sample may be a blood sample generally of plasma or serum. The sample may be obtained from any animal, particularly mammalian animals, including rodentine, bovine, ovine, canine, feline, porcine and equine animals, as well as primates including humans. The sample may also be other body fluids such as milk, or ascitic fluid, or even in vitro incubation medium.
The sample may require to be diluted, if the haptoglobin concentration in the sample is above about 2 g/l, since the concentration of haptoglobin may not be accurately determined due to the non-linearity of a standard plot above such concentrations. However, the skilled addressee will readily understand that assay protocols could be developed with increased assay linearity to obviate any requirement for dilution. For example, a smaller sample volume may be used in appropriate circumstances.
The haemoglobin may be of the same origin as the animal from which the blood sample is being tested. That is, if the animal to be tested is bovine in origin, bovine haemoglobin may be employed. However, advantageously it has been found that the assay may be performed using haemoglobin of a species different from the species where the sample comes from. Thus the assay may be performed on a great variety of species while only utilising a single source of haemoglobin. Preferably also the haemoglobin is met-haemoglobin, although optionally oxy-haemoglobin may be used.
Serum and plasma from all species contains albumin, so this protein will be present in the sample to be tested at concentrations of up to 40 g/l. The present inventors have found through assessment of the best medium to use as a zero sample, for running as a blank, that there is significant interference with the assay from albumin. This interference has been detected at albumin concentrations of 1, 5, or 10% (10, 50, 100 g/l).
It has been observed that albumin does not have an innate peroxidase activity. Without wishing to be bound by theory it is thought that albumin and possibly other proteins present in blood serum, plasma or other samples to be tested may be bind
Chaudhry Mahreen
Gitomer Ralph
Myers Bigel & Sibley & Sajovec
The University Court of the University of Glasgow
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