Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4716—Muscle proteins, e.g. myosin, actin

Definitions

• This invention relates to the field of the diagnosis of Myocardial Infarction ("MI") .
• MI Myocardial Infarction
• One biochemical test for aiding the diagnosis of MI is the MB isoenzyme of creatine kinase (“CK-MB”).
• 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.
• CK-MB test 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.
• Tnl 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.
• 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.
• 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.
• immunoassays which can test for human cTnl are valuable to the medical community and to the public.
• MI patient serum contains Tnl fragment(s) which is the result of the C-terminal processing of cTnl molecule.
• 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) .
• 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.
• 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.
• 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.
• 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).
• MASMTLWM leading sequence of 8 amino acids
• PMVHHHHHH tail sequence of 9 amino acids
• 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) .
• 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.
• 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.
• 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
• 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
• 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.
• 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.
• 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
• Fig. 19 depicts a Map of expression vector pTac/Gene 10/Troponin I/6x His.
• 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.
• amino acids 1-210 SEQ ID NO: 4
• HIS6 Histidine codons at the C-terminus
• 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
• 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.
• the mixture was incubated at room temperature
• 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
• 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.
• 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.
• 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).
• the reaction mixtures were prepared as follows.
• 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.
• 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 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.
• the CNBr-cTnl isoform can only be generated from human cTnl or human r- Tnl.
• the CNBr cleavage of cTnl from other species 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.
• 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.
• 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.
• Tnl binds TnC in the presence of calcium ions.
• TnC 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Fig. 5 shows that the CNBr-cTnl isoform has lower non ⁇ specific binding than r-Tnl.
• 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.
• EndoAsp endoproteinase Asp-N
• 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).
• 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.
• 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
• 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:
• CNBr-cTnl isoform is spiked into serum, diluted serum, plasma, diluted plasma or base at the designated level.
• 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.
• Lys Phe Lys Arg Pro Thr Leu Arg Arg Val Arg lie Ser Ala Asp Ala 145 150 155 160
• Lys Thr Leu Leu Leu Gin lie Ala Lys Gin Glu Leu Glu Arg Glu Ala 50 55 60
• Gin Lys lie Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu Arg 130 135 140
• Val Arg lie Ser Ala Asp Ala Met Met Gin Ala Leu Leu Gly Ala 145 150 155 160
• Lys Thr Leu Leu Leu Gin lie Ala Lys Gin Glu Leu Glu Arg Glu Ala 50 55 60
• Gin Lys lie Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu Arg 130 135 140
• Arg Arg Val Arg lie Ser Ala Asp Ala Met Met Gin Ala Leu Leu Gly 145 150 155 160
• Lys Asn lie Asp Ala Leu Ser Gly Met Glu Gly Arg Lys Lys Lys Phe 195 200 205
• 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

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