Covalently coupled troponin complexes

Details Covalently coupled troponin complexes Covalently coupled troponin complexes

1997-07-22

2001-07-31

Jones, Dwayne C. (Department: 1614)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C560S070000, C436S086000, C436S811000, C514S012200

Reexamination Certificate

active

06268481

ABSTRACT:

FIELD OF INVENTION
The present invention relates generally to clinical chemistry. In particular, it relates to stabilized troponin complexes useful in the diagnosis of myocardial infarction or other ischemic events.
BACKGROUND OF THE INVENTION
The determination of the presence or amount of certain constituents or analytes is useful in the diagnosis of disease and physical well-being. Compositions which behave similarly to how constituents present in human bodily fluids (e.g. blood, blood serum, plasma, spinal fluid or urine) behave are used in clinical laboratories. These compositions assist in the determination of whether the clinical instrumentation and procedures used by the laboratory to measure the constituents are accurate. These compositions are also used to calibrate the clinical devices which measure the amount or presence of the constituent in a sample. These compositions will be referred to hereinafter as control compositions or controls.
In addition, it is important that the analyte or analyte analog present in the control composition behave similarly to the corresponding analyte to be tested for in a patient's bodily fluid—that is, the control composition should mimic the patient sample.
Rapid and simple tests that can be used to accurately diagnose the occurrence of myocardial infarction (“MI”) or distinguish other ischemic events such as unstable angina are extremely important. Cardiac troponin I (cTnI) and troponin T have recently become established as the markers of choice in evaluating cardiac distress. See, New England Journal of Medicine Volume 335 No. 18, pages 1342-1349, Antman et al. and pages 1333-1341, Ohman et al.
The Troponin complex is present in both skeletal and cardiac muscles and consists of three subunits, Troponin T (“TnT”) the tropomyosin binding subunit, Troponin C (“TnC”), the Ca++ binding subunit and TnI, which inhibits the actomyosin Mg++-ATPase.
The majority of the research into the troponin complex has centered around the regulatory function and structure of the troponin complex in skeletal muscle. The troponin complex assists in muscle contraction. The TnC molecule has four binding domains to bind divalent metal ions. The Ca++/Mg++ binding sites are in the COOH terminal region and the Ca++ binding sites are in the amino terminal region. In studies of skeletal muscle, in the absence of Ca++, the amino terminus of TnI binds to the COOH terminus region of TnC and to the globular COOH terminus region of TnT. Thus, research indicates that TnI and TnC are anti-parallel and TnI and TnT are anti-parallel. The presence of calcium ion increases the TnC amino terminus domain's affinity for the inhibitory and COOH regions of TnI. In addition, there is a hydrophobic surface in the N-terminal domain of TnC that represents a Ca++ dependent binding site for TnI and TnT. It has been proposed that the Ca++ dependent reactions relate to the regulatory mechanism and Ca++ independent interactions maintain the structural integrity of the complex. In order to study structure and function of the troponin complex in its regulation of skeletal muscle, cross-linking studies have been accomplished. See, Farah, C. and Reinach, F. Review: The Troponin complex and regulation of muscle contraction. FASEB Journal 9 pp. 755-767 (1995). Covalent binding between TnC and skeletal muscle TnI has been formed between the carboxyl groups in the TnC and lysine groups in TnI using EDC. Kobayoshi et al. (1994), Structure of the troponin complex: implications of photocross-linking of troponin I to troponin C thiol mutants. J. Biol. Chem. 269, 5725-5729. In addition Leszyk et al. (1987) Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-malemidobenzophenone; identification of residues in troponin I that are close to cystein-98 of troponin C. Biochemistry 26, 7042-7047, reported that the main product of cross-linking between TnC and skeletal muscle TnI comprises segments derived from the N-terminal regulatory domain of TnC (residues 46 to 78) and the inhibitory region of skeletal TnI (residues 96-116). The Troponin complex is also referred to herein as the ternary complex.
U.S. Ser. No. 08/865,468, filed on May 29, 1997 and also owned by applicant, discloses that it had been discovered that the majority of native cTnI in human serum after an MI is associated with TnC and TnT. The presence of TnI in a complex with other troponin subunits in MI patient serum increases its stability and protects it from further degradation. In addition the troponin complex protects the sites where cardiac-specific antibodies bind. U.S. Ser. No. 08/865,468, filed on May 29, 1997 also discloses methods to isolate the complex from MI patient serum.
The cardiac isotype of the myofibrillar contractile protein, Troponin I (“TnI”), is uniquely located in cardiac muscle. TnI is the inhibitory sub-unit of Troponin, a thin filament regulatory protein complex, which confers calcium sensitivity to the cardiac and striated muscle. Troponin I exists in three isoforms: two skeletal TnI (fast and slow) isoforms (Molecular Weight=19,800 daltons) and a cardiac TnI (“cTnI”) isoform with an additional 31 residues (human TnI) on the N-terminus resulting in a molecular weight of 23,000 daltons.
Cardiac TnI is found in human serum rapidly (within approximately 4 to 6 hours) following an MI. It reaches a peak level after approximately 18-24 hours and remains at elevated levels in the blood stream for up to 6 to 7 days. Thus, immunoassays which can test for human cTnI are valuable to the medical community and to the public.
It is desirable to use an immunologically reactive human cTnI isoform comparable to that found in MI patient serum. We found that MI patient serum contains TnI fragment(s) which is the result of the C-terminal processing of cTnI molecule. The high sequence homology found in the C-terminal region between cardiac TnI and skeletal muscle TnI (Larue et al. 1992 Molec. Immunology 29, 271-278, Vallins et al. 1990 FEBS Lett. 270, 57-61, Leszky et al. 1988 Biochemistry 27, 2821-2827) produce TnI antibodies directed against this region having non-cardiac specificity (Larue et al. 1992). Our data and Larue et al. 1992 suggest that most of the known cTnI specific antibodies have their epitopes located approximately in the first 75% of the TnI molecule. Therefore, this portion of the TnI molecule should function as a MI specific cTnI isoform in most immunoassay systems.
Currently cTnI immunoassays are commercially available from Dade International, Behring Diagnostics, and Sanofi Pasteur Diagnostics. The Dade product is the StratusO Cardiac Troponin-I assay.
Native intact human cTnI is difficult to obtain because of the scarcity of human heart and native intact human cTnI is highly subject to proteolytic degradation during purification. Recombinant cardiac TnI (“r-TnI”), unlike the native human cTnI, can be produced and purified in acceptable quantities. As expressed by Dade, the primary structure of r-TnI contains 226 amino acids (SEQ ID NO: 1); 209 of them represent the TnI sequence (SEQ ID NO: 2). In addition to the primary sequence of cTnI (SEQ ID NO: 2), r-TnI, as expressed by Dade International, has a leading sequence of 8 amino acids (MASMTLWM) on the N-terminal, and a tail sequence of 9 amino acids (PMVHHHHHH) on the C-terminal (SEQ ID NO: 1). The primary structure of the r-TnI molecule has methionine residues at positions −7, −4, 0, 153, 154, 200 and 211 (SEQ ID NO: 1). See also FIG.
1
.
Full length cardiac troponin I is known to have the following sequence:
ADGSSDAAREPRPAPAPIRRRSSNYRAYATEPHAKKKSKISASRKLQLKTLLLQIAKQELEREAEERRGEKGRALSTRCQPLELTGLGFAELQDLCRQLHARVDKVDEERYDIEAKVTKNITEIADLTQKIFDLRGKFKRPTLRRVRISADAMMQALLGARAKESLDLRAHLKVKKEDTEKENREVGDWRKNIDALSGMEGRKKKFES (SEQ ID NO: 2) (Armour, K. L. et al.,(1993) Cloning and Expression in
Escheria Coli
of the cDNA Encoding Human Cardiac Troponin I, Gene, 131 (2):287-292).
U.S. Ser. No. 08/564,526, also owned by appli

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