Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,... – Preparation composition
Reexamination Certificate
1999-06-29
2001-02-20
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
Preparation composition
C435S007100, C435S007600, C435S183000, C435S184000, C435S219000, C435S007940, C435S013000, C435S069300, C435S967000, C435S972000, C436S008000, C436S015000, C436S017000, C436S517000, C436S518000, C436S536000, C436S811000, C424S569000, C530S350000, C530S412000, C530S414000, C530S417000, C530S402000, C530S403000, C530S391500, C530S391700, C530S807000, C530S814000
Reexamination Certificate
active
06190916
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to stable compositions useful as primary standards and calibrators and controls comprising a cardiac troponin I (cTnI) such as native, recombinant, addition and deletion forms thereof, whether or not complexed with other troponin subunits such as TnC and/or TnT, in an inactivated human serum. The compositions are obtained by incubating troponin I or troponin I complexes with human serum. The compositions are characterized by an immunodetectability ratio of epitopes on the N-terminal segment to epitopes on the C-terminal segment substantially equivalent to that of cardiac troponin I in pooled, fresh serum from patients having undergone an acute myocardial infarction.
BACKGROUND OF THE INVENTION
Early and accurate assessment of suspected acute myocardial infarction is critically dependent on the sensitive and specific detection of intracellular cardiac muscle components released into the circulation, in order to distinguish a potentially lethal event in need of emergency measures from non-life threatening conditions such as angina and non-cardiac chest pain such as dyspepsia. Early electrocardiographic changes are neither adequately specific nor sensitive, and the medical profession has come to rely on serum biochemical markers of cardiac tissue injury for early diagnosis. Initially, the serum markers creatine kinase (CK) and specifically the cardiac CK-MB isoform were used; subsequently myoglobin as a more sensitive early indicator of cardiac damage became preferred. More recently, the cardiac troponin complex and its cardiac-specific subunits have come to be preferred as markers of myocardial damage because of their high specificity. A combination of these analytes, provides a high degree of diagnostic accuracy. If performed in the emergency room, an early and accurate diagnosis of myocardial damage significantly enhances the safe recovery of a suspected heart attack victim.
Troponin is a generic term used to identify a muscle protein integrally involved in the calcium-dependent regulation of muscle contraction. Troponin exists in both cardiac and skeletal muscle as a non-covalently-bound complex of three subunits; the isoforms troponin C, the calcium-binding subunit; troponin I, the inhibitory subunit; and troponin T, which locates the troponin complex on tropomyosin. Differences exist between the amino acid sequences of the cardiac muscle and skeletal muscle troponin isoforms, and these differences are exploited in diagnostic tests which specifically measure the cardiac isoforms of the troponin to assist the diagnostician in determining if a cardiac event has taken place.
cTnI is a low molecular weight protein containing about 210 amino acid residues. When a cardiac event such as myocardial infarction occurs, cTnI together with cTnC and cTnT are released into the blood stream as a result of deterioration of cardiac muscle. cTnI and cTnT isoforms are specific to cardiac tissue and detectably distinguishable from skeletal isoforms.
It has been suggested that serum troponin may exist as complexes usually referred to as CIT, IT, CI and CT complexes. Troponin subunits, especially cTnI, undergo various degradation reactions resulting in the formation of complexes that differ in molecular weight, tertiary structure and other physical and chemical characteristics. One of these characteristics is that epitopes which may originally have been exposed for reaction with selected antibodies are no longer so exposed and may even have been destroyed. See for example:
Bodor et al., Clin. Chem. 38(11), 2203 (1992)
Adams et al., Clin. Chem. 40(7), 1291 (1994)
Adams et al., Circulation 88(1), 101 (1993)
Adams et al., New England Journal of Medicine 330(10), 670 (1994)
A number of instruments have been designed and are commercially available to measure total cTnI in the blood of suspected heart attack victims. These include the Abbott AxSYM, the Dade OPUS, the Bayer IMMUNO-1, the Beckman ACCESS, and the Dade STRATUS. Each of these instruments measure cTnI be reacting different epitopes with different antibodies. One, for example, measures an epitope near the C-terminus of the molecule whereas another measures an epitope near the middle of the molecule or a the N-terminus.
The epitope measured or the antibodies employed do not detract from the accuracy of the instrument or its sensitivity. However, it does mean that each instrument has a different reference range and each has a different value indicative of a positive reading. For example, on the Dade STRATUS a positive test is recognized by a value of >1.5 ng/mi. On the ACCESS device, it is >0.2 and on AxSYM it is >2.0. These variations sometimes cause confusion if a patient is moved from one hospital to another or the technician is working on different instruments.
Other problems with utilizing cTnl as an indication of cardiac damage is the rapid degradation of the molecules and the fact that its concentration varies among different patients. The problems can be alleviated by utilizing fresh samples. However, these are difficult to obtain because patients present at different times after the onset of chest pain and the variation amongst patients is never known with any degree of confidence.
The art has sought to deal with the problems by utilizing pooled patients'serum to calibrate the various instruments. The solution has been unsatisfactory because the pooled sample degrades during storage even at low temperatures.
The art has long searched for some method of standardization so that the reference ranges on the different instruments would be the same. The invention described and claimed herein makes such standardization possible. For convenience, the term cTnI will be used in the Specification and Claims to refer to natural cTnI, as well as addition and deletion analogs thereof whether isolated from a natural source or produced by recombinant techniques. It will include also complexes of natural cTnI and such analogs with the other troponin isoforms, cTnC and cTnT. It will refer also to those analogs of natural cTnI which have been extended at either end by the addition of other amidno acid segments. The criteria for recognizing a troponin useful in this invention is that it will act like natural ctnl to achieve the novel, stable compositions of the invention.
The ratio of immunodetectability is determined by a separate determination of a detectable epitopes in the area of the C-terminus and the area of the N-terminus of the molecule. Although separate determinations are made, they are made on the same sample at the same time. The epitope on the N-segment is measured with the STRATUS device. The epitope on the C-segment is measured with the ACCESS device.
DESCRIPTION OF THE INVENTION
The compositions of this invention when utilized in diagnostic procedures perform in a fashion similar to fresh patient serum samples even after long storage periods. They thus offer prolonged stability and when prepared from recombinant cTnI and fresh serum, better lot-to-lot consistency and unlimited availability.
Various forms of cardiac troponin I may be used in the present invention, including but not limited to native troponin I, recombinant troponin I, synthetic troponin I, and various addition or deletion forms thereof. Synthetic proteins may be made by, for example, solid-phase synthesis. An example of a recombinant form of cardiac troponin I with a 6-amino acid leader sequence is disclosed in U.S. Pat. No. 5,834,210, incorporated herein by reference. Other forms may be used which do not detract from the purposes herein.
The stable standardization compositions of this invention are obtained by mixing a cTnI with human serum at selected concentrations and allowing the mixture to incubate at about 35° C. to about 44° C., preferably 35° C. to 37° C. until the desired equilibrium composition has been obtained. The desired equilibrium composition has an immunodetectability ratio of the N-terminus to the C-term-inus of cTnI substantially equivalent to that of pooled, fresh serum from acute myoc
Liu Shigui
Shi Qinwei
Zhang Min Yuan
Gabel Gailene R.
Klauber & Jackson
Le Long V.
Spectral Diagnostics Inc.
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