HUMAN HEART CNBr TROPONIN I ISOFORM AND USE OF SAME

Abstract: This invention relates to a human cardiac Troponin I fragment generated from human recombinant Troponin I by chemical cleavage. The fragment represents 73 % of the primary structure of human cardiac Troponin I and is immunologically more reactive than recombinant Troponin I. The fragment, or isoform, is comparable in molecular weight to a major degradation product of native cardiac Troponin I in the serum of patients who have experienced myocardial infraction. The isoform can be used as a calibrator or control in cardiac Troponin Immunoassays.

Description: HUMAN HEART CNBr TROPONIN I ISOFORM AKD USE OF SAME Background of the Invention This invention relates to the field of the diagnosis of Myocardial Infarction ("MI") . One biochemical test for aiding the diagnosis of MI is the MB isoenzyme of creatine kinase ("CK-MB"). However, CK-MB is not completely specific for cardiac muscle; it can also be found in skeletal muscle and in blood after skeletal muscle injury. See, e.g., Cummins, et al. (1987), "Cardiac-Specific Troponin I Radioimmunoassay in the Diagnosis of Acute Myocardial Infarction", American Heart Journal, June 1987, Vol. 113, No. 6. Another disadvantage of the CK-MB test is that the amount of CK-MB in the skeletal muscle varies with the degree of skeletal muscle regeneration, information which may often not be known when administering a test or analyzing a test result for MI. Another disadvantage of the CK-MB test is that CK-MB remains elevated for only 2-3 days after the onset of chest pain. For patients admitted after that time, the CK-MB test will be of limited, if any, value. See, e.g., Cummins, et al. (1987). Thus, due to the lack of specificity of the CK-MB test, and the limited time frame for its use as a diagnostic tool, CK-MB is not the MI test of choice. Other enzyme assays also exist, such as lactate dehydrogenase (LDH) and glutamic oxaloacetic transaminase (GOT) , but the frequent serial measurements required in the very early hours after the onset of chest pain could present difficulties for an absolute specific diagnosis. See, e.g., Larue, et al. (1992) , "New Monoclonal Antibodies as Probes for Human Cardiac Troponin I: Epitopic Analysis With Synthetic Peptides", Molecular Immunology, Vol. 29, No. 2, pp. 271- 278 (1992) . Thus, the prior art has recognized the need for an accurate cardiac-specific biological parameter detectable in serum very soon after MI and remaining present for more than 2-3 days after the onset of MI. The cardiac isotype of the myofibrillar contractile protein, Troponin I ("Tnl") , is uniquely located in cardiac muscle. Tnl is the inhibitory sub-unit of Troponin, a thin filament regulatory protein complex, which confers calcium sensitivity to the cardiac and striated muscle. The Troponin complex consists of three subunits, Troponin T ("TnT") the tropomyosin binding subunit, Troponin C ("TnC"), the Ca++ binding subunit and Tnl, which inhibits the actomyosin Mg++-ATPase. Troponin I exists in three isoforms: two skeletal Tnl (fast and slow) isoforms (Molecular Weight = 19,800 daltons) and a cardiac Tnl ("cTnl") isoform with an additional 31 residues (human Tnl) on the N-terminus resulting in a molecular weight of 23,000 daltons. Cardiac Tnl is found in human serum rapidly (within approximately 4 to 6 hours) following a 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 cTnl are valuable to the medical community and to the public. It is desirable to use an immunologically reactive human cTnl isoform comparable to that detected in MI patient serum. We found that MI patient serum contains Tnl fragment(s) which is the result of the C-terminal processing of cTnl molecule. The high sequence homology found in the C-terminal region between cardiac Tnl and skeletal muscle Tnl (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 Tnl 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 cTnl specific antibodies have their epitopes located approximately in the first 75% of the Tnl molecule. Therefore, this portion of the Tnl molecule should function as a MI specific cTnl isoform in most immunoassay systems. Presently, Tnl immunoassays do not use human cTnl. Dade currently sells a cTnl Immunoassay Kit in Europe and U.S.A. using a synthetic peptide in the calibrators and the controls. This product is the Stratus® Cardiac Troponin-I assay. Native human cTnl is difficult to obtain because of the scarcity of human heart. Moreover, native human cTnl is highly subject to proteolytic degradation during purification. The availability of human recombinant Tnl ("r-Tnl") can facilitate the production of this cTnl isoform. The r-Tnl, unlike the native human cTnl, can be produced and purified in acceptable quantities. As expressed by Dade, the primary structure of r-Tnl contains 226 amino acids (SEQ ID NO: 1) ; 209 of them represent the Tnl sequence (SEQ ID NO: 2). (See Fig. 1.) In addition to the primary sequence of cTnl (SEQ ID NO: 2) , r-Tnl 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). (See Fig. l.) The primary structure of the r-Tnl molecule has methionine residues at positions -7, -4, 0, 153, 154, 200 and 211 (SEQ ID NO: 1) . (See Fig. 2.) It is desirable to use an immunologically reactive human cTnl isoform comparable to that detected in MI patient serum. The availability of r-Tnl can facilitate the production of this cTnl isoform. Moreover, since most of the known Tnl antibodies have their epitopes located approximately in the first 75% of the Tnl molecule, that portion of the Tnl molecule will function as a cTnl isoform in most immunoassays. Summary of the Invention This invention relates to the use of a human cTnl fragment generated from human r-Tnl by chemical cleavage. The cleavage of r-Tnl by cyanogen bromide (CNBr) results in a major polypeptide of 153 amino acids, hereinafter referred to as the "CNBr-cTnl isoform" (SEQ ID NO: 3) . The CNBr-cTnl isoform represents 73% of the primary structure of human cTnl and is immunologically more reactive than r- Tnl. The purified CNBr-cTnl isoform has an average of 3-4 times more reactivity than r-Tnl and lower non-specific binding, as measured by radial partition immunoassay. As demonstrated in Figure 7 the molecular size of the CNBr- cTnl isoform is comparable in molecular weight to the major degradation product of native cardiac Tnl in MI patient serum. The CNBr-cTnl isoform can be used as calibrators or controls in various cTnl immunoassays. The CNBr-cTnl isoform has increased stability over the synthetic peptide currently used in the Dade Tnl immunoassay. This invention also relates to the effect of TnC upon the immunological and biological activity and non-specific binding of the CNBr-cTnl isoform. This invention further relates to the complex formed by the CNBr - cTnl isoform, TnC and TnT. Description of the Drawings Fig. 1 depicts the alignment of the human cardiac Troponin I amino acid sequences. Single letter code has been used. Other symbols include: (r)=r-TnI (SEQ ID NO: 1); (h)=native human cTnl (SEQ ID NO: 2); (I)=CNBr-cTnI isoform (SEQ ID NO: 3); (cam)=S-carboxyamidomethylcysteine. Fig. 2 depicts the CNBr cleavage strategy of r-Tnl; (M) = methionine. Fig. 3 shows the activity of r-Tnl and CNBr-cTnl isoform in calibrator base as measured with the Stratus® II Tnl Immunoassay System. Fig. 4 depicts the activity of r-Tnl and the CNBr-cTnl isoform in human serum as measured with the Stratus® II Tnl Immunoassay System. Fig. 5 shows the non-specific binding/specific binding ratio of r-Tnl and the CNBr-cTnl isoform on various tabs as measured with the Stratus® II Tnl Immunoassay System. The specific activity of Tnl forms was measured on Tnl specific antibody tabs. The abbreviations for other (Tnl non- specific tabs) tabs are: PSA = prostate specific antigen tabs; CKMB = creatine kinase MB tabs; AFP = alpha fetal protein tabs; PAP = prostate acid phosphatase tabs; blank = blank glass fiber tabs; isoform = CNBr-cTnl isoform. Fig. 6 depicts the results of the SDS - polyacrylamide gel electrophoresis (15%) of the CNBr-cTnl isoform. Lanes 1 and 8 are the molecular weight standard, Lanes 2 and 5 are the r-Tnl, and Lanes 3, 4, 6, and 7, the CNBr-cTnl isoform. Fig. 7 depicts the Western Blot analysis of the CNBr- cTnl isoform. Lane 1 is the CNBr-cTnl fragment, Lanes 2 and 3 are the degradation fragment of cTnl extracted from MI patient serum, and Lane 4 the molecular weight standard. Fig. 8 shows the alignment of the N-terminal amino acid sequence of the CNBr-cTnl isoform ("c") (SEQ ID NO: 3) with those of human cardiac Tnl ("b") (SEQ ID NO: 2) and r- Tnl ("a") (SEQ ID NO: 1). Fig. 9 shows the results of polyacrylamide gel electrophoresis (native gel) of Tnl:TnC complexes. Lane 1 is TnC. Lane 2 is r-TnI:TnC (1 mol:l mol). Lane 3 is rTnI:TnC (2 mol:l mol). Lane 4 is rTnI:TnC (3 mol:l mol). Lane 5 is CNBr-cTnl isoform:TnC (1 mol:l mol). Lane 6 is CNBr-cTnl isoform:TnC (2 mol:l mol). Lane 7 is CNBr-cTnl isoform:TnC (3 mol:l mol). Fig. 10 depicts the activity and non-specific binding of various Tnl preparations. The activity of the various Tnl forms and their respective complexes was determined using the Stratus® II Tnl Immunoassay System. Non-specific binding was determined using Ferritin tabs. Fig. 11 demonstrates the effect of the presence of TnC on the activity of the CNBr-cTnl isoform as measured by the Stratus® II Tnl Immunoassay System. Fig. 12 compares the polyacrylamide gel electrophoresis (10% PAGE, tris-tricine buffer at pH 8.3) (native gel) results of the complex formation of the CNBr- cTnl isoform:TnC:TnT complex (Lanes 3 and 4) , and the CNBr- cTnl isoform:TnC complex (Lanes 1 and 2) . Fig. 13 depicts the polyacrylamide gel electrophoresis (native gel) results of the complex formation of r-TnI:TnC complex, the CNBr-cTnl isoform:TnC complex, and Tnl cisoform II ("cisoform II") :TnC complex. Lane l is the TnC control; Lane 2 is r-TnI:TnC at a 1:1 mol ratio; Lane 3 is CNBr-cTnl isoform:TnC at a 1:1 mol ratio; Lane 4 is cisoform II:TnC, at a 1:1 mol ratio; Lane 5 is r-TnI:TnC at a 1:2 mol ratio; Lane 6 is CNBr-cTnl isoform:TnC at a 1:2 mol ratio, and Lane 7 is cisoform II:TnC at a 1:2 mol ratio. All samples contain 2 mM CaCl 2 and were incubated at room temperature for 30 minutes. Fig. 14 shows the stability of the CNBr-cTnl isoform:TnC complex in bovine serum. The stability of the CNBr-cTnl isoform (1 ug/ml) and the CNBr-cTnl isoform:TnC complex (1:1) were followed at 4°C over three weeks. The final concentration of CNBr-cTnl isoform alone and in the complex is 0.25 ug isoform/ml. Three lots of bovine serum were used; H,Q,S represent the serum lots Hyc 2242, Quad 9058 and Sigma S7140 respectively. The Tnl activity was measured using the Stratus II Tnl Immunoassay system. Fig. 15 depicts the non-specific binding of the CNBr- cTnl isoform and the CNBr-cTnl isoform:TnC complex in bovine serum. The non-specific binding measurements were carried out on the Stratus II Immunoassay system using Ferritin tabs. The bovine serum lots H,Q,S represent Hyc 2242, Quad 9058 and Sigma S7140 respectively. Fig. 16 demonstrates the stability of the CNBr-cTnC isoform and the CNBr-cTnC isoform:TnC complex in human serum. The final concentration of CNBr-cTnl isoform alone and in the complex (1:1) is 0.25 ug/ml. The Tnl activity was measured on the Stratus II Tnl immunoassay system. Fig. 17 shows the non-specific binding of the CNBr- cTnl isoform and the CNBr-cTnl isoform:TnC complex in human serum. The non-specific binding measurements were carried out on the Stratus II Immunoassay system using Ferritin tabs and blank tabs. Fig. 18 depicts the stability of the reconstituted CNBr-cTnl isoform:TnC complex (1:1) in bovine serum. Two lots of bovine serum were used, BTI and Quad, at three levels. Measurements were carried out using the Stratus II Tnl immunoassay system. Fig. 19 depicts a Map of expression vector pTac/Gene 10/Troponin I/6x His. Detailed Description of the Invention The recombinant human cTnl was expressed in E. coli by the Dade Biology Skills Center (and we thank the Dade Biology Skills Center for providing it) . Tnl was cloned from human heart cDNA, which is commercially available through companies such as Strategene, by polymerase chain reaction (PCR) and was subcloned into the Ncol restriction site in the Dade constructed vector pTac 102-2, as shown in Fig. 19. (Purified Tnl from bovine or human heart is also commercially available.) The vector pTac 102-2 was constructed by conventional means (see Vallins et al, Molecular cloning of human cardiac troponin I using PCR, FEBS Lett. 270, 57-61 (1990)) to include a Hindll-Bam HI fragment containing p tac , a strong hybrid promoter driving gene transcription. The promoter was induced in E-coli through IPTG, a method and technique well known to those skilled in the art. The next downstream DNA sequence is an efficient ribosomal binding site (RBS) and the N-terminal five amino acids of gene 10 for translation initiation, followed by the cloning cite Ncol. The Dade designed Tnl (amino acids 1-210 (SEQ ID NO: 4) , including the initiating methionine) was inserted into Ncol in frame with the N- terminus, and with six Histidine codons at the C-terminus (HIS6) . The expressed protein contained 226 amino acids. The HIS6 C-terminus facilitated a single step purification. Cleavage of the r-Tnl molecule at the methionine residues at positions -4, 0, 153, 154, 200 and 211 by CNBr produced a major polypeptide of 153 amino acids. (SEQ ID NO: 3.) The resulting polypeptide had 73% of the human cTnl primary structure (209 amino acids) (SEQ ID NO: 3) , and retained the epitopes for the antibodies used in the Stratus® II Tnl Immunoassay System. (See Vallins et al 1990 FEBS Lett. 270, 57-61.) Cyanogen bromide cleaves at methionine residues with high specificity under acidic conditions. Cleavage of the r-Tnl by CNBr at all methionine sites should produce 6 peptides of various sizes (SEQ ID NO: 5) (SEQ ID NO: 3) (SEQ ID NO: 8) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 9) (See Fig. 2) . Generally described, the first step is to carboxymethylate the cysteine residues (there are two in the Tnl sequence) (SEQ ID NO: 1) at positions 79 and 96 in order to prevent dimerization by inter or intra molecular disulfide bridges. CNBr treatment is carried out on the carboxymethylated r-Tnl. Unlike other possible cleavage reactions (e.g. enzymatic) , the CNBr treatment removes the tail sequence, the leading sequence, and part of the Tnl C- terminal region without affecting the primary sequence of the immunogenic sites. Example I (Preparation of CNBr-cTnl Isoform) Three trial preparations of the CNBr-cTnl isoform were conducted. Recombinant Tnl (10-20 mg. , 0.25-0.3 mM) in 100 mM Na-phosphate, 10 mM tris, 8 M urea, pH 8 ("PTU buffer") was reduced by adding sufficient Dithiothreitol (DTT) freshly prepared in the same buffer (200 mM stock solution) to give a final concentration of 2.5 mM of DTT. The mixture was incubated at room temperature (approximately 23-25°C) for a time sufficient to reduce the rTnl (approximately 1 hour) . The reduced r-Tnl was treated with iodoacetamide(prepared in the PTU buffer, 400 mM stock solution) to give a final concentration of 15 mM of iodoacetamide in the reaction mixture. The mixture was then incubated for a time and under conditions sufficient to complete the carboxy ethylation reaction (approximately 1 hour) in the dark at 37°C. The mixture was transferred to 10 x 25 mm wide spectra/por(12-14 kd MWCO) dialysis tubing and dialyzed against 2xlL of 25% acetic acid for 24 hours at room temperature, with stirring. The dialyzed cTnl was lyophilized under vacuum (<1 mm Hg) between room temperature and 45°C. The lyophilized rTnl was dissolved in approximately 1.4 ml 70% formic acid and then CNBr (1 ug/uL in 70% formic acid) was added to the rTnl solution to give a final concentration of 480 mM of CNBr (approximately 160 mol CNBr/mol methionine) . The tube containing the reaction mixture was purged with nitrogen and then incubated at least 16 hours at room temperature in the dark with rocking. The reaction was stopped by adding distilled water to give 1:10 dilution of the digest. The digest was lyophilized under vacuum (<1 mm Hg) between room temperature and 45°C. The lyophilized digest was dissolved in a minimum volume of 88% formic acid. The digest was applied on a Sephadex G-200 (1.6 x 100 cm) column equilibrated with 25% acetic acid. The CNBr-cTnl isoform was eluted with 25% acetic acid. The first major peak, which represented the CNBr-cTnl isoform, was pooled and tested for purity by SDS-PAGE and for immunoreactivity by the Stratus® II Tnl Immunoassay System. As measured by the Stratus® II Tnl Immunoassay System, the purified CNBr-cTnl isoform has 3-4 fold higher immunological activity than r-Tnl (See Figs. 3 and 4.) The following data are graphed in Fig. 3. Tnl (Lot) r-Tnl CNBr-cTnl isoform RD 4 853 3872 PP 1 949 3816 PP 2 1148 3720 Values are expressed in mv/ in, which is a standard unit used to measure rate of change. The r-Tnl or CNBr- cTnl isoform was spiked into Calbase in a total volume of 1 ml. Tnl concentration was 0.5 mg/ml. The ratio between non-specific binding and specific binding of the CNBr-cTnl isoform is lower than that of r-Tnl. (See Fig. 5.) The following data are graphed in Fig. 5. Tnl Forms Specific Non-Specific Binding Binding CNBr-cTnl isoform 3426.6 1328.6 CNBr-CTnl isoform: TnC complex 42,333.6 1890.0 r-Tnl 491.3 334.2 r-TnI:TnC complex 3749.4 1742.3 Values are expressed in v/min. An aliquot of the r- Tnl or CNBr-cTnl isoform was spiked in Calbase to give a final concentration of 20 nM (3.5 ul of CNBr-cTnl isoform 0.1 mg/ml is spiked into 996.5 ul Calbase; 5.2 ul of r-Tnl 0.1 mg/ml is spiked into 994.8 ul Calbase). For the complexes with TnC, the reaction mixtures were prepared as follows. For the r-TnI:TnC complex, 5.2 ul r- Tnl (0.1 mg/ml) plus 3.65 ul TnC (0.165 mg/ml) plus 2 ul CaCl 2 (25 mM) plus 14.15 ul PTU buffer. The total volume measured 25 ul. The reaction was incubated at room temperature for approximately 15 minutes and then spiked into 975 ul Calbase. For the CNBr-cTnl isoform: TnC complex, 3.5 ul Tnl (0.1 mg/ml) plus 3.65 ul TnC (0.165 mg/ml) plus 2 ul CaCl 2 (25 mM) plus 15.85 ul PTU buffer. The total volume was 25 ul. The reaction was incubated at room temperature for approximately 15 minutes and then spiked into 975 ul Calbase. The purified CNBr-cTnl isoform migrates on SDS-PAGE gel electrophoresis as a single band with an apparent molecular weight of 21,000 daltons. Western blot analysis of the CNBr-cTnl isoform has a molecular weight close to that of a major degradation fragment of cTnl in MI patient serum. (See Fig. 7) The N-terminal sequence analysis of the isoform gave the sequence Ala-Asp-Gly-Ser-Ser-Asp-Ala- Ala-Ala-Arg-Glu, which is identical to the N-terminal sequence of human cTnl (SEQ ID NO: 2) (See Fig. 8) . The amino acid analysis (See Table 1) confirms that the purified CNBr-cTnl isoform represents the first 153 amino acids of the cTnl molecule. (SEQ ID NO: 1.) Rabbit skeletal muscle TnC was purified as described by Potter, J.D. (1982) in Methods Enzymology 85, 241-263. TnC from other tissue sources can be used as well. TnT was obtained commercially. The CNBr treatment proved specific, with no evidence of side or non-specific reactions. Other chemical and proteolytic means lack specificity and the experimental conditions are hard to control. Even using very specific proteolytic enzymes, the possibility is quite high of affecting the epitopes of various cTnl antibodies including those used in the Stratus® Tnl Immunoassay System. Another procedure, complicated and costly, would be to make the 153 amino acid cTnl isoform (SEQ ID NO: 3) through cDNA and expression in E. coli or other expression systems after changing the Cys codons if desired. The carboxymethylation of the cysteine residues is not a pre-requisite for the generation of the 153 amino acid isoform. (SEQ ID NO: 3.) Rather, the carboxymethylation facilitates the process by minimizing the complications during or after CNBr digestion. Using the CNBr cleavage procedure, the CNBr-cTnl isoform can only be generated from human cTnl or human r- Tnl. The CNBr cleavage of cTnl from other species (bovine, rabbit, etc.) does not generate the 153 amino acid isoform because in such species, the non-human cTnl has a methionine residue at position 53 in the amino acid sequence, which would also undergo the cleavage reaction with CNBr. In human cTnl or r-Tnl, leucine replaces the methionine at position 53. Longer or shorter form(s) of the CNBr-cTnl isoform can be produced by adding or deleting a few amino acids to/from the N terminal, the C-terminal or any part of the Tnl isoform sequence. Human cTnl cDNA cloned into a vector can be modified by site directed mutagenesis (oligonucleotides) and/or PCR (Guo et at., 1994 J. Biol. Chemistry 269, 15210- 15216, Farah et al. 1994 J. Biol. Chem. 269, 5230-5240, Sheng et al. 1992 J. Biol. Chem. 267, 25407-25413) to produce CNBr-cTnl isoform or its modified forms. The modified cDNA can be subcloned into a vector to give rise to the expression construct for CNBr-cTnl isoform or its modified form(s) . The protein expression can be carried out in E. Coli or other expression system. Changes in some amino acids of the CNBr-cTnl isoform sequence might not affect its performance except those occurring at the epitope(s) where the specific assay antibodies bind. The buffer used for the carboxymethylation of r-Tnl can be replaced by other buffers with a pH of about 8. Dithiothreitol can be substituted with other reducing agents, particularly those which are effective and work maximally at a pH of around 8, such as glutathione, DTE, acetyl cysteine. Alkylating reagents other than iodoacetamide can be used (e.g. iodoacetic acid, NEM, etc.) in order to block cysteine residues. The time needed for the cleavage of Tnl by CNBr could vary between 10-24 hours at room temperature in the dark. The CNBr cleavage must be carried out under acidic conditions, because the selectivity of the reaction of CNBr with amino acids depends on pH. Acids other than formic acid, such as trifluoroacetic acid, can be used. The method of purification of the isoform is not critical. It may be purified by various chromatographic methods. Size exclusion columns such as the Sephacryl S-200, Separose 12, and Sephadex G 100, 150 and 200, are useful at large scale. The isoform can also be purified by TnC affinity column such as TnC-sepharose affinity column, as well as other TnC affinity columns. Example II (Binding Properties of the CNBr-cTnl Isoform with TnC) Tnl binds TnC in the presence of calcium ions. For complete formation of the CNBr-cTnl:TnC complex at least one mole of TnC is needed per mole of the CNBr-cTnl isoform. The time required to form the complex is flexible. The CNBr-cTnl isoform is able to form a complex with TnC from various species. The complex can be formed best at pH range of 4-8.5 in the absence or presence of urea. The binding properties of the CNBr-cTnl isoform with TnC have been studied using polyacrylamide gel electrophoresis (native gel) . The CNBr-cTnl isoform was incubated with TnC at molar ratios of 1:1, 2:1 and 3:1 in PTU buffer (100 mM Na-phosphate, 10 mM tris buffer containing 8 M urea pH 8) . All samples were incubated for 30 minutes at room temperature in the presence of up to 2 mM CaCl2• (A shorter time, such as 15 minutes, or possibly less, may also be sufficient.) As depicted in Figure 9, Lane 6, one mole CNBr-cTnl isoform is required to bind one mole of TnC. The formation of the CNBr-cTnl isoform:TnC complex is accompanied by the disappearance of TnC (Lane 6). However, as depicted in Figure 9, at least two moles of r-Tnl is required to bind one mole TnC for the formation of the r-TnI:TnC complex (Lane 4). These results suggest that the CNBr-cTnl isoform binds TnC more efficiently than does r-Tnl. Example III (Immunological Activity of the CNBr-cTnl Isoform:TnC complex) The immunological activity of the CNBr-cTnl isoform:TnC complex was measured using the Stratus® Tnl Immunoassay System. The CNBr-cTnl isoform was incubated with TnC in PTU buffer at a molar ratio of (1:1) in the presence of 2 mM CaCl 2 for 30 minutes at room temperature. The complex was then added to calibrator base to give a final Tnl concentration of 20 nM. (The calibrator base used to measure the complex contains no EDTA but has a concentration of 2 mM CaCl 2 •) CNBr-cTnl isoform alone was added to calibrator base to give a final concentration of the CNBr-cTnl isoform of 20 nM and incubated separately. Similarly, r-Tnl was incubated with TnC at a molar ratio of (1:1) in the presence of 2 mM CaCl 2 for 30 minutes at room temperature. The complex was then added to calibrator base to give a final Tnl concentration of 20 nM. Recombinant Tnl alone was added to calibrator base to give a final Tnl concentration of 20 nM and incubated separately. As shown in Fig. 10, binding the CNBr-cTnl isoform to TnC in the presence of calcium ions enhances the activity of the CNBr- cTnl isoform several times over that of the CNBr-cTnl isoform alone. The non-specific binding of the CNBr-cTnl isoform:TnC complex is low, less than 5% of the total activity. In comparison, binding of r-Tnl to TnC increases its activity but not nearly to the extent observed with the CNBr-cTnl isoform:TnC complex. The effect of TnC on the activity of the CNBr-cTnl isoform was examined using the Stratus® II Tnl Immunoassay System. The CNBr-cTnl isoform was incubated with TnC in PTU buffer containing 2 mM CaCl2 at room temperature for 30 minutes. The CNBr-cTnl isoform:TnC complex was prepared at ratios of 1:4, 1:2, 1:1, 1:0.5, 1:0.25 and 1:0.00 (mol/mol) . An aliquot of each reaction mixture was spiked into calibrator base (without EDTA, but containing 2 mM CaCl 2 ) to give a final concentration of 6.5 nM of CNBr-cTnl isoform. Measurements were carried out on the Stratus® II Immunoassay System. As shown in Fig. 11, the 1:1 ratio of CNBr-cTnl isoform:TnC complex produces maximum activity. Addition of more TnC slightly increases the Tnl activity, but has no effect on the non-specific binding of the complex. Fig. 9 shows that at the 1:1 ratio, the CNBr-cTnl isoform is totally complexed with TnC. Example IV (Reconstitution of Troponin Complex using CNBr- cTnl isoform) The complex between the CNBr-cTnl isoform, TnC and TnT was formed by mixing stoichiometric amounts of each subunit in 100 mM sodium phosphate buffer pH 7.5 containing 10 mM tris, 1 mM CaCl 2 , 7 mM mercaptoethanol and 4 M urea. The mixture was incubated at room temperature for 3 hours and then either used for analysis or dialyzed into the buffer of interest for storage. Formation of the CNBr-cTnl isoform:TnC:TnT complex was examined by using polyacrylamide gel electrophoresis (native gel) . Figure 12 shows that the CNBr-cTnl isoform:TnC:TnT complex, in Lanes 3 and 4, has different mobility than the CNBr-cTnl isoform:TnC complex in Lanes 1 and 2. The activity of the CNBr-cTnl isoform:TnC:TnT complex was measured using the Stratus® II Tnl Immunoassay System. The activity of the CNBr-cTnl isoform:TnC:TnT complex is several times higher than that of the CNBr-cTnl isoform and comparable to the activity of the CNBr-cTnl isoform:TnC complex. These results suggest that the CNBr-cTnl isoform is able to form a ternary complex with both TnC and TnT subunits, a property of the Tnl molecule. Further, the results suggest that the addition of TnT to the CNBr-cTnl isoform:TnC complex does not interfere with the CNBr-cTnl isoform activity on the Stratus II Tnl Immunoassay System. The results also suggest that the high Tnl activity of the CNBr-cTnl isoform: nC:TnT complex is due to the binding to TnC. Example V (Stability of the CNBr cTnl Isoform:TnC Complex) The effect of the presence of TnC on the stability of the CNBr-cTnl isoform in serum was studied. The presence of TnC was found to increase the stability of the CNBr-cTnl isoform. The CNBr-cTnl isoform and TnC were incubated at a 1:1 ratio in PTU buffer containing 2 mM CaCl 2 for 30 minutes. The stability was studied in 3 lots of bovine serum obtained from three different vendors (Hyc 2242, Quad 9058 and Sigma S7140) (Fig. 14) , a human serum pool (Fig. 15) and followed over a three week period. Once added to the serum, the temperature was kept at 4°C. The final concentration of the CNBr-cTnl isoform and the CNBr-cTnl isoform:TnC complex, in serum, was 1 ug/ml and 0.25:0.25 ug/ml respectively. Fig. 16 shows the stability of the CNBr-cTnl isoform:TnC complex (1:1) spiked into serum at three distinct levels (manufacturing ranges) , lyophilized in small vials (3ml) , and stored at 4°C until use. The lyophilized preparations of the CNBr-Tnl isoform:TnC complex were reconstituted and assayed at time intervals as shown in Fig. 16. Rvam ia VT (Non-speci ic binding of the CNBr-cTnl isoform: nC Complex) We have studied the effects of the presence of TnC on the non-specific binding of the CNBr-cTnl isoform in serum. Fig. 5 shows that the CNBr-cTnl isoform has lower non¬ specific binding than r-Tnl. As demonstrated in Fig. 17, the non-specific binding of the CNBr-cTnl isoform:TnC complex in bovine serum was lower than that of the CNBr- cTnl isoform. Similar results were also obtained in human serum (Fig. 18) . Example VII (Preparation of and Properties of a Tnl 88 Amino Acid—cisoform II) cisoform II was generated from r-Tnl (see Fig. 1) using the endoproteinase Asp-N ("EndoAsp") . EndoAsp, a metalloprotease, cleaves at the N-terminus of aspartic acid. Recombinant Tnl was incubated with EndoAsp at a ratio of 100:1 (r-Tnl:EndoAsp, w/w) in 50 mM sodium phosphate pH 8 containing 1 M urea for 20 hours at 37°C. The major cleavage product consisted of 88 amino acids starting at position 6 (Aspartic acid, D) and ending at position 95 (Glutamine, Q) (Fig. 1) (SEQ ID NO: 10). Once purified, the cisoform II was tested for purity and activity on SDS-polyacrylamide gel electrophoresis and the Stratus® II Tnl Immunoassay System, respectively. As shown in Figure 10, the cisoform II has higher activity (2-fold) and higher non-specific binding (2-fold) than the CNBr-cTnl isoform. However, little enhancement in Tnl activity of cisoform II was seen when it was incubated with TnC in the presence of 2 mM CaCl 2 « The ability of the cisoform II to form a complex with TnC was examined. Fig. 13 shows that the binding between the cisoform II and TnC is weaker than that between the CNBr-cTnl isoform and TnC. A faint band representing the cisoform II:TnC complex is seen in Lane 4. The majority of the TnC remains unbound. The addition of an excess of TnC does not increase the cisoform II:TnC complex formation. The results suggest that the minimal effect of TnC on Tnl activity of the cisoform II is due to the inability of the cisoform II to form a stable complex with TnC. Fig. 13, Lane 3, shows that the CNBr-cTnl isoform effectively binds TnC with better efficiency than the r-Tnl and cisoform II. Example VIII (The CNBr-cTnl Isoform as a Calibrator and a Control) Preparation of control: A stock solution of the CNBr- cTnl isoform (1 mg/ml) is prepared in 100 mM sodium phosphate buffer pH 8 containing 10 mM tris and 8 M urea using polypropylene tubes. A liquid tri-level assay control containing the CNBr-cTnl isoform is prepared in serum, diluted serum, plasma, diluted plasma or base using plastic labware. The levels of controls are: Manufacturing CNBr-cTnl isoform Ranges range (ng/ml) Level 1 3 - 6 Level 2 17-22 Level 3 35-44 CNBr-cTnl isoform is spiked into serum, diluted serum, plasma, diluted plasma or base at the designated level. The mixtures are filtered and tested on the Stratus® II Immunoassay System for the matching level of Tnl concentration. Preparations of each level are placed (3 ml each) in plastic vials which are either stored at 4° C or lyophilized. The lyophilized material is reconstituted using 3 ml of water upon use. Preparation of Calibrators: The calibrators can be made by adding a sufficient amount of CNBr-cTnl isoform stock solution to serum, plasma or base, to give final concentrations ranging from 2 to 50 ng/ml. The CNBr-cTnl isoform calibrator concentrations are 0 ng/ml, 2 ng/ml, 8 ng/ml, 15 ng/ml, 25 ng/ml and 50 ng/ml. Each calibrator level is filtered and analyzed on the Stratus® II Immunoassay System using Tnl immunoassay and matched against the reference level. The calibrators are then filled into their designated vials and either lyophilized or stored at 4°C. The lyophilized calibrators are reconstituted to the pre-lyophilization volume using water. The Examples provided herein are for the purposes of illustration only and are not intended to limit the scope of the invention. SEQUENCE LISTING (1) GENERAL INFORMATION: (I) APPLICANTS: Dade International Inc. Morjana, Nihmat A. DeMarco, Curtis (ii) TITLE OF INVENTION: HUMAN HEART CNBr TROPONIN I ISOFORM AND USE OF SAME (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: DADE INTERNATIONAL INC. (B) STREET: 1717 Deerfield Road (C) CITY: Deerfield (D) STATE: Illinois (E) COUNTRY: US (F) ZIP: 60015 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 26 November 1996 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/564,526 (B) FILING DATE: 29 November 1995 (viii) ATTORNE /AGENT INFORMATION: (A) NAME: WINSTON, Lois K. (B) REGISTRATION NUMBER: 39,074 (C) REFERENCE/DOCKET NUMBER: DA-5250 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (708) 267-5364 (B) TELEFAX: (708) 267-5376 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 226 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Met Ala Ser Met Thr Leu Trp Met Ala Asp Gly Ser Ser Asp Ala Ala 1 5 10 15 Arg Glu Pro Arg Pro Ala Pro Ala Pro lie Arg Arg Arg Ser Ser Asn 20 25 30 Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys Lys Ser Lys lie 35 40 45 Ser Ala Ser Arg Lys Leu Gin Leu Lys Thr Leu Leu Leu Gin lie Ala 50 55 60 Lys Gin Glu Leu Glu Arg Glu Ala Glu Glu Arg Arg Gly Glu Lys Gly 65 70 75 80 Arg Ala Leu Ser Thr Arg Cys Gin Pro Leu Glu Leu Thr Gly Leu Gly 85 90 95 Phe Ala Glu Leu Gin Asp Leu Cys Arg Gin Leu His Ala Arg Val Asp 100 105 110 Lys Val Asp Glu Glu Arg Tyr Asp lie Glu Ala Lys Val Thr Lys Asn 115 120 125 lie Thr Glu lie Ala Asp Leu Thr Gin Lys lie Phe Asp Leu Arg Gly 130 135 140 Lys Phe Lys Arg Pro Thr Leu Arg Arg Val Arg lie Ser Ala Asp Ala 145 150 155 160 Met Met Gin Ala Leu Leu Gly Ala Arg Ala Lys Glu Ser Leu Asp Leu 165 170 175 Arg Ala His Leu Lys Gin Val Lys Lys Glu Asp Thr Glu Lys Glu Asn 180 185 190 Arg Glu Val Gly Asp Trp Arg Lys Asn lie Asp Ala Leu Ser Gly Met 195 200 205 Glu Gly Arg Lys Lys Lys Phe Glu Ser Pro Met Val His His His His 210 215 220 His His 225 2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 209 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu Pro Arg Pro Ala Pro Ala 1 5 10 15 Pro lie Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu Pro 20 25 30 His Ala Lys Lys Lys Ser Lys lie Ser Ala Ser Arg Lys Leu Gin Leu 35 40 45 Lys Thr Leu Leu Leu Gin lie Ala Lys Gin Glu Leu Glu Arg Glu Ala 50 55 60 Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys Gin 65 70 75 80 Pro Leu Glu Leu Thr Gly Leu Gly Phe Ala Glu Leu Gin Asp Leu Cys 85 90 95 Arg Gin Leu His Ala Arg Val Asp Lys Val Asp Glu Glu Arg Tyr Asp 100 105 110 lie Glu Ala Lys Val Thr Lys Asn lie Thr Glu lie Ala Asp Leu Thr 115 120 125 Gin Lys lie Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu Arg 130 135 140 Arg Val Arg lie Ser Ala Asp Ala Met Met Gin Ala Leu Leu Gly Ala 145 150 155 160 Arg Ala Lys Glu Ser Leu Asp Leu Arg Ala His Leu Lys Gin Val Lys 165 170 175 Lys Glu Asp Thr Glu Lys Glu Asn Arg Glu Val Gly Asp Trp Arg Lys 180 185 190 Asn lie Asp Ala Leu Ser Gly Met Glu Gly Arg Lys Lys Lys Phe Glu 195 200 205 Ser (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 153 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu Pro Arg Pro Ala Pro Ala 1 5 10 15 Pro lie Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu Pro 20 25 30 His Ala Lys Lys Lys Ser Lys lie Ser Ala Ser Arg Lys Leu Gin Leu 35 40 45 Lys Thr Leu Leu Leu Gin lie Ala Lys Gin Glu Leu Glu Arg Glu Ala 50 55 60 Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys Gin 65 70 75 80 Pro Leu Glu Leu Thr Gly Leu Gly Phe Ala Glu Leu Gin Asp Leu Cys 85 90 95 Arg Gin Leu His Ala Arg Val Asp Lys Val Asp Glu Glu Arg Tyr Asp 100 105 110 lie Glu Ala Lys Val Thr Lys Asn lie Thr Glu lie Ala Asp Leu Thr 115 120 125 Gin Lys lie Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu Arg 130 135 140 Arg Val Arg lie Ser Ala Asp Ala Met 145 150 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 211 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 : Met Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu Pro Arg Pro Ala Pro 1 5 10 15 Ala Pro lie Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu 20 25 30 Pro His Ala Lys Lys Lys Ser Lys lie Ser Ala Ser Arg Lys Leu Gin 35 40 45 Leu Lys Thr Leu Leu Leu Gin lie Ala Lys Gin Glu Leu Glu Arg Glu 50 55 60 Ala Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys 65 70 75 80 Gin Pro Leu Glu Leu Thr Gly Leu Gly Phe Ala Glu Leu Gin Asp Leu 85 90 95 Cys Arg Gin Leu His Ala Arg Val Asp Lys Val Asp Glu Glu Arg Tyr 100 105 110 Asp lie Glu Ala Lys Val Thr Lys Asn lie Thr Glu lie Ala Asp Leu 115 120 125 Thr Gin Lys lie Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu 130 135 140 Arg Arg Val Arg lie Ser Ala Asp Ala Met Met Gin Ala Leu Leu Gly 145 150 155 160 Ala Arg Ala Lys Glu Ser Leu Asp Leu Arg Ala His Leu Lys Gin Val 165 170 175 Lys Lys Glu Asp Thr Glu Lys Glu Asn Arg Glu Val Gly Asp Trp Arg 180 185 190 Lys Asn lie Asp Ala Leu Ser Gly Met Glu Gly Arg Lys Lys Lys Phe 195 200 205 Glu Ser Pro 210 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Ala Ser Met 1 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Thr Leu Trp Met 1 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Gin Ala Leu Leu Gly Ala Arg Ala Lys Gly Ser Leu Asp Leu Arg Ala 1 5 10 15 His Leu Lys Gin Val Lys Lys Glu Asp Thr Glu Lys Glu Asn Arg Glu 20 25 30 Val Gly Asp Trp Arg Lys Asn lie Asp Ala Leu Ser Gly Met 35 40 45 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Glu Gly Arg Lys Lys Lys Phe Glu Ser Pro Met 1 5 10 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Val His His His His His His 1 5 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 88 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: Asp Ala Ala Arg Glu Pro Arg Pro Ala Pro Ala Pro lie Arg Arg Arg 1 5 10 15 Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys Lys 20 25 30 Ser Lys lie Ser Ala Ser Arg Lys Leu Gin Leu Lys Thr Leu Leu Leu 35 40 45 Gin lie Ala Lys Gin Glu Leu Glu Arg Glu Ala Glu Glu Arg Arg Gly 50 55 60 Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys Gin Pro Leu Glu Leu Thr 65 70 75 80 Gly Leu Gly Phe Ala Glu Leu Gin 85

Claims: WE CLAIM: 1. A human cardiac Troponin I fragment of human native cardiac Troponin I or human recombinant cardiac Troponin I having approximately 153 amino acids in substantially the following sequence: A-D-G-S-S-D-A-A-R-E-P-R-P-A-P-A-P-I-R-R-R-S-S-N-Y-R-A-Y-A- T-E-P-H-A-K-K-K-S-K-I-S-A-S-R-K-L-Q-L-K-T-L-L-L-Q-I-A-K-Q- E-L-E-R-E-A-E-E-R-R-G-E-K-G-R-A-L-S-T-R-C-Q-P-L-E-L-T-G-L- G-F-A-E-L-Q-D-L-C-R- | I cam cam Q-L-H-A-R-V-D-K-V-D-E-E-R-Y-D-I-E-A-K-V-T-K-N-I-T-E-I-A-D- L-T-Q-K-I-F-D-L-R-G-K-F-K-R-P-T-L-R-R-V-R-I-S-A-D-A-M (SEQ ID NO: 3) . 2. A method for the preparation of a human cardiac Troponin I fragment of human native cardiac Troponin I or human recombinant cardiac Troponin I having approximately 153 amino acids in substantially the following sequence: A-D-G-S-S-D-A-A-R-E-P-R-P-A-P-A-P-I-R-R-R-S-S-N-Y-R-A-Y-A- T-E-P-H-A-K-K-K-S-K-I-S-A-S-R-K-L-Q-L-K-T-L-L-L-Q-I-A-K-Q- E-L-E-R-E-A-E-E-R-R-G-E-K-G-R-A-L-S-T-R-C-Q-P-L-E-L-T-G-L- G-F-A-E-L-Q-D-L-C-R- | I cam cam Q-L-H-A-R-V-D-K-V-D-E-E-R-Y-D-I-E-A-K-V-T-K-N-I-T-E-I-A-D- L-T-Q-K-I-F-D-L-R-G-K-F-K-R-P-T-L-R-R-V-R-I-S-A-D-A-M (SEQ ID NO: 3) , comprising: a) reducing human cardiac Troponin I selected from the group consisting of human native cardiac Troponin I and human recombinant cardiac Troponin I; b) cleaving the human cardiac Troponin I of step a) with CNBr; and c) recovering the resulting human cardiac Troponin I fragment. 3. A calibrator for a Tnl immunoassay comprising: a) a known amount of human cardiac Troponin I fragment of human native cardiac Troponin I or human recombinant cardiac Troponin I having approximately 153 amino acids in substantially the following sequence: A-D-G-S-S-D-A-A-R-E-P-R-P-A-P-A-P-I-R-R-R-S-S-N-Y-R-A-Y-A- T-E-P-H-A-K-K-K-S-K-I-S-A-S-R-K-L-Q-L-K-T-L-L-L-Q-I-A-K-Q- E-L-E-R-E-A-E-E-R-R-G-E-K-G-R-A-L-S-T-R-C-Q-P-L-E-L-T-G-L- G-F-A-E-L-Q-D-L-C-R- | I cam cam Q-L-H-A-R-V-D-K-V-D-E-E-R-Y-D-I-E-A-K-V-T-K-N-I-T-E-I-A-D- L-T-Q-K-I-F-D-L-R-G-K-F-K-R-P-T-L-R-R-V-R-I-S-A-D-A-M (SEQ ID NO: 3) ; and b) serum or calibrator base. 4. The calibrator of claim 3 further comprising a known amount of Troponin C. 5. A control for a Tnl immunoassay comprising: a) a known amount of human cardiac Troponin I fragment of human native cardiac Troponin I or human recombinant cardiac Troponin I having approximately 153 amino acids in substantially the following sequence: A-D-G-S-S-D-A-A-R-E-P-R-P-A-P-A-P-I-R-R-R-S-S-N-Y-R-A-Y-A- T-E-P-H-A-K-K-K-S-K-I-S-A-S-R-K-L-Q-L-K-T-L-L-L-Q-I-A-K-Q- E-L-E-R-E-A-E-E-R-R-G-E-K-G-R-A-L-S-T-R-C-Q-P-L-E-L-T-G-L- G-F-A-E-L-Q-D-L-C-R- | I cam cam Q-L-H-A-R-V-D-K-V-D-E-E-R-Y-D-I-E-A-K-V-T-K-N-I-T-E-I-A-D-L- T-Q-K-I-F-D-L-R-G-K-F-K-R-P-T-L-R-R-V-R-I-S-A-D-A-M (SEQ ID NO: 3) ; and b) serum or calibrator base. 6. The control of claim 5 further comprising a known amount of Troponin C. 7. A peptide derived from native or recombinant human cardiac Tnl having immunological activity to antibodies against Tnl, said peptide made by the process comprising: a) reducing human cardiac Troponin I; b) cleaving the human cardiac Troponin I of step b) with CNBr; and c) recovering the resulting human cardiac Troponin I peptide. 8. A calibrator for a Tnl immunoassay comprising: a peptide made by the process of claim 7 and serum or calibrator base. 9. The calibrator of claim 8 further comprising: a known amount of Troponin C. 10. A control for a Tnl immunoassay comprising: a peptide made by the process of claim 7 and serum or calibrator base. 11. The control of claim 10 further comprising: a known amount of Troponin C.

Inventors: DEMARCO CURTIS, MORJANA NIHMAT A, DEMARCO, CURTIS, MORJANA, NIHMAT, A.
Applicants: DADE INT INC, DADE INTERNATIONAL INC.
Application year: 1996
Main classification: C07K14
IPC codes: C07K14, G01N33