Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-02-17
2001-12-25
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S380000, C530S388150, C530S864000, C530S866000, C435S007100, C436S547000, C436S548000
Reexamination Certificate
active
06333397
ABSTRACT:
The invention concerns specific antibodies to cardiac muscle troponin T, their production and use in an immunological reagent for the determination of myocardial necrosis.
The myofibrils of the striated muscle consist of two protein filaments which act in combination, the thick filaments have myosin as their main component and the thin filaments contain actin, tropomyosin and troponins. Troponin, a regulatory structural protein, aggregates to a complex in the cells and consists of three different proteins:
Troponin C (MW 18000) which binds calcium ions
Troponin I (MW 24000) a sub-unit binding actin
Troponin T (MW 37000) which complexes with tropomyosin.
The comparable nomenclature has historical reasons since originally the complex as such was purified and was looked upon as a single protein and denoted troponin. Later it was proven that troponin really consists of three different proteins. This nomenclature was then retained because of the spacial relationship between the proteins on the thin filament of the contractile apparatus and because of their cooperation with respect to the regulation of muscle contraction. They are, however, three different proteins which are functionally related to the other proteins of the contractile apparatus such as myosin or actin but which have different amino acid sequences.
During long lasting severe ischaemia or muscle cell necrosis troponin I reaches the blood plasma and is thus a parameter for such diseases which can also be used for diagnosis and monitoring analogous to the known infarction enzymes CK, CK-MB, GOT and LDH.
It has been shown that the determination of CK and CK-MB is not absolutely specific for an infarction and is only increased in the serum 80 to 90 hours after the infarction. GOT and LDH are also not specific for cardiac muscle since increased amounts are also found in the blood in many other diseases.
The disadvantage of a determination of cardiac troponin I is that normally serum already contains concentrations of troponin I at a level of 10 ng/ml (cf. B. Cummins, J. Mol. Cell. Card. 19 (1987), 999-1010 and B. Cummins, Clin. Invest. 113 (1987) 1333-1344). It turns out that a biphasic serum concentration occurs in a transmural infarction. on average, troponin I was increased from the 4th to the 168th hour after the onset of pain in 37 patients with acute transmural infarction. Similar results were obtained in an animal model. Accordingly it follows that for troponin I the 10th to 50th hour after the occurrence of an infarction is the time interval for the absolute diagnostic sensitivity. Thus, apart from the limited sensitivity, due to variable serum levels of troponin I the clinically important monitoring for 10 days and more after the occurrence of the infarction cannot be achieved by the determination of troponin I.
It was therefore the object of the present invention to eliminate these disadvantages and to provide a method for the determination with which monitoring-is possible for at least 150 hours (duration of absolute diagnostic sensitivity) in myocardial infarctions and other injuries to the cardiac muscle.
This object is achieved by a method for the determination of myocardial necrosis according to the immunoassay principle which is characterized in that a serum or plasma sample is incubated with at least one antibody to troponin T and a binding partner B for troponin T or for the antibody, in which either the antibody to troponin T or the binding partner B is labelled with a determinable group, the immunological complex which thereby forms is isolated and the determinable group is determined in the isolated or in the remaining phase as a measure for troponin T from the sample.
The binding partner B has to bind to the antibody to troponin T or to troponin T. B can be for example a second antibody to troponin T or troponin T from humans or animals or an analogue thereof which is bound by the antibody.
The sample is preferably incubated with an antibody to troponin T and a conjugate of a further antibody to troponin T and a determinable group, the immunological complex formed is isolated by separation of the phases and the determinable group is determined in one of the phases.
It is furthermore preferred to incubate the sample with an antibody to troponin T and a conjugate of troponin T and a determinable group, to isolate the immunological complex formed by separation of the phases and to determine the determinable group in one of the phases.
Surprisingly, it turned out that a significantly higher sensitivity can be obtained by a troponin T immunoassay in the determination of myocardial necroses (such as e.g. by cardiac infarction, ischaemia or angina pectoris) than by the determination of other parameters such as CK, CK-MB, GOT, LDH or troponin I. As established by the inventors the reason for this is that in contrast to other proteins of the contractile apparatus no serum concentration can be measured for troponin T up to the detection limit of the test (0.25 ng/ml) in normal patients (who have not suffered myocardial necroses).
This is particularly surprising since, because of the functional relationship between the troponins, a similar serum concentration to that for troponin I would be expected for troponin T. Furthermore, the serum concentration curve of troponin T differs significantly, for example in a transmural infarction, from the curve for troponin I. In contrast to troponin I the curve of the time course is in three phases instead of two phases and troponin T is found to be increased on average for up to 300 hours after the onset of pain. The time interval for absolute diagnostic sensitivity lasts from the 6th to the 195th hour. The time interval for the absolute diagnostic sensitivity is thus nearly four times as long as that known for troponin I.
All common immunoassays are in principle suitable for the immunolgical method of determination according to the present invention such as radioimmunoassay, enzyme-immunoassay, fluorescence immunoassay etc. Furthermore, all variants of these procedures such as competitive immunoassay, IEMA procedure etc. are applicable. A sandwich test has proven to be particularly effective for the determination of troponin T. In this test procedure an immobilized antibody to troponin T and a conjugate of an antibody to troponin T and a determinable group is used. The different variants of this test method as well as details for carrying out these procedures are described at length in the literature. However, other immunological methods of determination are also possible using the antibodies according to the present invention such as those described e.g. in the German patent application DE-A 38 34 766 and/or DE-A 38 22 750.
Within the scope of the invention an antibody is understood as a complete antibody, chimeric antibody, bivalent antibody or fragments thereof. The troponin T antibodies used can be polyclonal or monoclonal. Monoclonal antibodies are preferably used.
In the sandwich test the sample solution is incubated with at least two antibodies to troponin T as a preferred embodiment. The first antibody thereby mediates the binding to the solid phase. For this, this antibody can either be bound directly or via a spacer to the solid phase or it can be present in a soluble form and only be immobilized after the immunological reaction has been carried out. The second antibody can either be labelled directly with a certain group or it can be bound to the determinable group by a functional bond. For this the antibody can be bound to one partner of a specific binding pair and the determinable group can be bound to the other partner of the specific binding pair. A complex which contains the second antibody as well as the determinable group then forms during the reaction. The binding of the first antibody to the carrier (immobilization) is carried out according to methods known to the expert by adsorptive, chemical binding or by functional binding via a specific binding pair. In this case one partner of the binding pair is immobilized while the other partner is chemically
Borgya Anneliese
Hallermayer Klaus
Katus Hugo
Looser Siegfried
Amick Marilyn L.
Nguyen Bao-Thuy L.
Roche Diagnostics Corporation
Roche Diagnostics GmbH
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