Patent Application
20070082410
The invention relates to a stabilized composition of cardiac troponin protein useful as calibrator and control standards in immunoassays. Particularly, the troponin protein is stabilized in an aqueous matrix containing at least one anionic surfactant.
The Troponin complex plays a role in the calcium-dependent regulation of muscle contraction and relaxation. Three distinct proteins, or isoforms of Troponin, comprise the Troponin complex, and can be found in both cardiac and skeletal muscles. These three proteins are designated as Troponin-I, Troponin-C and Troponin-T. During myocardial infarction, cardiac muscle cells die and consequently release their intracellular contents, including the Troponin proteins, into the blood stream.
Myocardial infarction is a leading cause of death in developed countries. The World Health Organization (WHO) developed guidelines for diagnosing myocardial infarction in 1979 (See Circulation, 59:607-609 (1979)). The WHO guidelines recommend that a diagnosis of myocardial infarction be dependent on the occurrence of two of three particular criteria. The three criteria are: (1) chest pain or history of cardiac event; (2) electrocardiogram indication of cardiac event; and (3) elevated levels of the enzyme creatine kinase.
Millions of individuals enter emergency rooms each year complaining of chest pain and have non-diagnostic electrocardiograms thus making difficult a diagnosis of cardiac event. Measurement of circulating levels of creatine kinase has questionable specificity relative to occurrence of myocardial infarction since elevation of this protein in the blood could also result from skeletal muscle damage. Troponin-I and Troponin-T have shown greater specificity due to the presence of a cardiac specific form of these proteins present only in the heart. The greater specificity of Troponin-I and Troponin-T for diagnosis of myocardial infarction has led to the development of several immunoassays for the determination of the levels of these proteins in blood samples of a patient expected to exhibit elevated levels. The literature demonstrating the superior utility of troponin has resulted in an updating of the definition of myocardial infection as of 2000 (which was jointly published in the Journal of the American College of Cardiology, 36:959-969 (2000) and in the European Heart Journal, 21:1502-1513 (2000)). This updated definition places a greater emphasis on biomarkers for diagnosis. As a result, the criteria for an acute, evolving or recent myocardial infarction are the typical rise and gradual fall (troponin) or more rapid rise and fall (CK-MB) of biochemical markers of myocardial necrosis with at least one of the following: (a) ischemic syndromes; (b) development of pathologic Q waves on the ECG; (c) ECG changes indicative o ischemia (ST segment elevation or depression); or (d) coronary artery intervention (e.g. coronary angioplasty).
Quantitative immunoassays require the use of both calibration standards to define a calibration curve and control standards to test the integrity of the calibration curve. One necessary characteristic of the standards used is stability, i.e. demonstrate minimal loss of immunoactivity over a defined period of time (expiration date) under appropriate storage conditions. Stabilization techniques for Troponin-I have included freezing, lyophilization and dissolution in strong reducing agents such as guanidine. These techniques generally require additional time, specialized equipment and special handling procedures required for thawing or reconstitution. Additionally, thawing or reconstitution of the troponin protein often results in unstable material with limited shelf-life.
Due to the inherent instability of the troponin protein there exists a need for material that is resistant to temperature variations potentially subjected to during transport and storage. There is further a need for a method of preparing such stabilized troponin. The invention provides a stabilized troponin protein and a method of preparing the stabilized troponin.
In one embodiment, the invention provides a diagnostic assay standard comprising an aqueous solution of Troponin-I protein and a matrix comprising at least one anionic surfactant having a formula selected from the group consisting of:
R1O—SO3M and R1(CH2H4O)x—O—SO3M
wherein R1 is a saturated or unsaturated, branched or unbranched alkyl group having from about 8 to about 24 carbon atoms; x is an integer from 1 to 10; and M is a water-soluble cation.
In a further embodiment, the water-soluble cation in this diagnostic assay standard is ammonium, sodium, potassium, magnesium, triethanolamine, diethanolamine or monoethanolamine.
In yet a further embodiment, the anionic surfactant in this diagnostic assay standard is sodium, ammonium, potassium, magnesium, monoethanolamine, diethanolamine or triethanolamine salts of lauryl or myristyl sulfate, sodium polyoxyethylene (1) lauryl sulfate or ammonium, sodium, magnesium, potassium or monoethanolamine laureth sulfate.
In yet another further embodiment, the diagnostic assay standard is stable at room temperature for at least six (6) months.
In still yet another further embodiment, the matrix in the diagnostic assay standard further comprises porcine gelatin.
In still another further embodiment, the matrix in the diagnostic assay standard further comprises a compound selected from the group consisting of sodium chloride and sodium phosphate.
In another embodiment, the matrix of the diagnostic assay standard further comprises a pH buffer added to maintain the pH within a range of about 6.8 to about 7.2.
In a further embodiment, the matrix contains sodium polyoxyethylene (1) lauryl sulfate as the anionic surfactant.
In a further embodiment, when sodium polyoxyethylene (1) lauryl sulfate is present in this diagnostic assay standard as the anionic surfactant, it is present in a concentration of about 0.1% to about 0.25%.
In another embodiment, the invention provides a method for stabilizing Troponin in an aqueous solution, the method comprising the steps of:
(a) preparing an aqueous solution of at least one anionic surfactant having a formula selected from the group consisting of:
R1O—SO3M and R1(CH2H4O)x—O—SO3M
wherein R1 is a saturated or unsaturated, branched or unbranched alkyl group having from about 8 to about 24 carbon atoms; x is an integer from 1 to 10; and M is a water-soluble cation;
(b) adding Troponin-I protein to the aqueous solution; and
(c) storing the solution at room temperature.
In yet another embodiment, the invention relates to a diagnostic assay standard comprising an aqueous solution of a complex of a Troponin-I protein and a Troponin-C protein and a matrix comprising at least one anionic surfactant having a formula selected from the group consisting of:
R1O—SO3M and R1(CH2H4O)x—O—SO3M
wherein R1 is a saturated or unsaturated, branched or unbranched alkyl group having from about 8 to about 24 carbon atoms; x is an integer from 1 to 10; and M is a water-soluble cation.
In a further embodiment, the water-soluble cation in this diagnostic assay standard is ammonium, sodium, potassium, magnesium, triethanolamine, diethanolamine or monoethanolamine.
In yet a further embodiment, the anionic surfactant in this diagnostic assay standard is sodium, ammonium, potassium, magnesium, monoethanolamine, diethanolamine or triethanolamine salts of lauryl or myristyl sulfate, sodium polyoxyethylene (1) lauryl sulfate or ammonium, sodium, magnesium, potassium or monoethanolamine laureth sulfate.
In a further embodiment, this diagnostic assay standard is stable at room temperature for at least six (6) months.
In still yet another further embodiment, the matrix in the diagnostic assay standard further comprises porcine gelatin.
In still another further embodiment, the matrix in the diagnostic assay standard further comprises a compound selected from the group consisting of sodium chloride and sodium phosphate.
In another embodiment, the matrix of the diagnostic assay standard further comprises a pH buffer added to maintain the pH within a range of about 6.8 to about 7.2.
In a further embodiment of this diagnostic assay standard, the matrix contains sodium polyoxyethylene (1) lauryl sulfate as the anionic surfactant.
In a further embodiment, when sodium polyoxyethylene (1) lauryl sulfate is present in this diagnostic assay standard as the anionic surfactant, it is present in a concentration of about 0.1% to about 0.25%.
In another embodiment, the invention provides a method for stabilizing Troponin in an aqueous solution, the method comprising the steps of:
(a) preparing an aqueous solution of at least one anionic surfactant having a formula selected from the group consisting of:
R1O—SO3M and R1(CH2H4O)x—O—SO3M
wherein R1 is a saturated or unsaturated, branched or unbranched alkyl group having from about 8 to about 24 carbon atoms; x is an integer from 1 to 10; and M is a water-soluble cation;
(b) adding Troponin-I protein and Troponin-C protein, either individually or together as a single complex to the aqueous solution; and
(c) storing the solution at room temperature.
The present invention is directed to a stabilized Troponin-I protein in a matrix comprising at least one anionic surfactant that is useful as calibration and control standards in immunoassays intended to determine the levels of troponin protein in blood samples of patients suspected of having elevated troponin levels. The present invention also relates to methods of preparing a stabilized troponin protein in a matrix comprising at least one anionic surfactant.
The at least one anionic surfactant used in the matrix and methods of the present invention have a formula selected from the group consisting of:
R1O—SO3M and R1(CH2H4O)x—O—SO3M
wherein R1 is a saturated or unsaturated, branched or unbranched alkyl group having from about 8 to about 24 carbon atoms; x is an integer from 1 to 10; and M is a water-soluble cation. The water soluble cation can be ammonium, sodium, potassium, magnesium, triethanolamine (TEA), diethanolamine (DEA) or monoethanolamine (MEA).
Examples of anionic surfactants that have the above described formula include, but are not limited to, sodium, ammonium, potassium, magnesium, monoethanolamine, diethanolamine or triethanolamine salts of lauryl or myristyl sulfate (such as sodium lauryl sulfate, ammonium lauryl sulfate, TEA-lauryl sulfate, MEA-lauryl sulfate, magnesium lauryl sulfate, potassium lauryl sulfate, sodium myristyl sulfate, ammonium myristyl sulfate, DEA-myristyl sulfate, etc.), sodium polyoxyethylene (1) lauryl sulfate or ammonium, sodium, magnesium, potassium or monoethanolamine laureth sulfate (such as ammonium laureth sulfate, MEA-laureth sulfate, sodium laureth sulfate, potassium laureth sulfate, magnesium laureth sulfate etc).
Troponin proteins, particularly cardiac Troponin-I (cTnI), are unstable molecules that rapidly lose immunoactivity in aqueous environments. Immunoactivity stability is an essential characteristic for an analyte that is to be used to prepare calibrators or controls for a diagnostic assay. The inventors of the present invention found that anionic surfactants having the above described formulas are capable of imparting stability to cTnI molecules. More specifically, the inventors discovered that the addition of at least one anionic surfactant having one of the above-described formulas, such as, but not limited to, sodium polyoxyethylene (1) lauryl sulfate, to cTnI, imparts thermal stability and robustness to formation of aqueous standard solutions used in calibrators and test assay controls. Robustness refers to the strength of the properties of the troponin. Robustness also may refer to the way in which the troponin has been constructed.
Anionic surfactants having the above described formulas, such as sodium polyoxyethylene (1) lauryl sulfate, provide stability to Troponin-I allowing it to be robust to elevated temperatures, wide ranges in ionic strengths, and pH. Anionic surfactants having one of the above described formulas are commercially available. For example, sodium lauryl sulfate is available as Tainolin AS-30, Tainolin AS-95N, Tainolin AS-97P from Jarchem Industries, Inc. (Newark, N.J.) (hereinafter “Jarchem”), sodium laureth sulfate as Tainolin AES-28-2N, Tainolin AES-70-2N, Tainolin AES-70-2NC from Jarchem, ammonium lauryl sulfate is available as Tainolin ASA-25, Tainolin ASA-28, Tainolin ASA-30 and Tainolin ASA-70 from Jarchem, ammonium laureth sulfate is available as Tainolin AESA-25, Tainolin AESA-70 from Jarchem, TEA-lauryl sulfate is available as Tainolin AST-40 from Jarchem, MEA-lauryl sulfate is available as Tainolin ASM-28 from Jarchem, MEA-laureth sulfate is available as Tainolin AESM-28 from Jarchem, magnesium lauryl sulfate is available as Tainolin ASMG-30 from Jarchem, magnesium laureth sulfate is available as Tainolin AESMG-27 from Jarchem, potassium lauryl sulfate is available as Tainolin ASK-30 from Jarchem, potassium laureth sulfate is available as Tainolin AESK-26 from Jarchem, sodium polyoxyethylene (1) lauryl sulfate is available as STANDAPOL® ES-1 from Cognis Corporation, Hoboken, N.J.
Anionic surfactants having one of the formulas described above can be used as a diluent component for solutions containing troponin and do not require any additional processing. The matrix containing the at least one anionic surfactant and troponin proteins have no special handling requirements by the user and the user may store the matrix at temperatures of between 2-8° C. or at room temperatures of up to 31° C. for periods of at least six (6) months, one (1) year or more.
The anionic surfactants having the above described formulas are believed to be readily soluble in aqueous conditions and can be added and mixed until dissolved. Concentration ranges of the at least one anionic surfactant in the matrix is between about 0.1% and 0.25% at a pH in the range of 6.8-7.2 and preferably, 7.0.
The preferred anionic surfactant is sodium polyoxyethylene (1) lauryl sulfate. When sodium polyoxyethylene (1) lauryl sulfate is used in the matrix, this anionic surfactant stabilizes the immunoactivity of cTnI and recombinant Troponin-I/Troponin-C complexes that are formed as a single protein molecule. The recombinant complex can be used in the formulation of calibrators and controls for the assays conducted with, for example, AxSYM Troponin-I ADV and ARCHITECT Troponin-I assays. The present invention also contemplates that the matrix containing at least one anionic surfactant, such as, sodium polyoxyethylene (1) lauryl sulfate, can also be used to stabilize the immunoactivity of native or recombinant Troponin-I/Troponin-C complexes that are formed by adding each of the Troponin-I and Troponin-C together to form a complex.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Troponin-I (Code 67911, Lot 63821P101, Abbott Laboratories, Abbott Park, Ill.) was diluted in matrices of 0.1% porcine gelatin (Code 35745, Lot 74281P100, Abbott Laboratories, Abbott Park, Ill.) at pH 4.0 and 0.1% porcine gelatin (Code 35745, Lot 74281P100) containing STANDAPOL® ES-1 (Lot 1 2L019 from Cognis Corporation, Hoboken, N.J.) at pH 6.8 to a final concentration of between 40-50 ng/ml (AxSYM Units are ng/ml). Porcine gelatin is added as a source of protein in order to minimize the loss of troponin during manufacture.
Each formulation was stored at both 2-8° C. and 37° C. as follows:
The concentration of Troponin-I in each sample was measured using a standard curve generated on day 0 using AxSYM Troponin-I standard calibration (No. 3C29-01, Abbott Laboratories, Abbott Park, Ill.) on the AxSYM instrument system (Clinical Chemistry, 45, Nov. 12, 1999). Over the following ten days, the concentration of Troponin-I was measured and compared to the concentration observed on day 0. The percent activity remaining on each day was calculated by dividing the concentration measured for each sample at the various time points by the concentration measured on day 0. Results are tabulated in Table 1 and depicted in
The addition of 0.1% STANDAPOL® ES-1 to the matrix containing 0.1% porcine gelatin and adjustment of the pH from 4 to 6.8 has a significant effect on thermal stability of Troponin-I as observed after storing the samples at 37° C. for ten days. Similar stabilities were observed for the samples stored at 2-8° C. with or without STANDAPOL® ES-1.
Three vendor lots of STANDAPOL® ES-1 were used in the preparation of analyte matrices. All matrices were based on a formulation: 0.025% porcine gelatin (Code 35745, Lot 74281P100, Abbott Laboratories, Abbott Park, Ill.), 0.175% of the selected STANDAPOL® ES-1, 2.5 mM NaPO4 (as a buffer to maintain pH of the solution) and 0.1% ProClin 300 (primary active ingredient—methylchloroisothiazolone) at pH 7.0. ProClin 300 is an antimicrobial agent used to prohibit bacterial growth.
The three vendor lots of STANDAPOL® ES-1 were:
Lot 1—Code 88965, Lot 39267X102 (Abbott Laboratories)
Lot 2—Code 88965, Lot 74497P100 (Abbott Laboratories)
Lot 3—Code 88965, Lot 74498P101 (Abbott Laboratories)
The three matrices (manufactured in Abbott Laboratories R&D facilities) were compared to a preparation manufactured in Bulk Solutions (Department 864, Abbott Laboratories). To this preparation was added Lot 1 to a concentration of 0.175%. Each of the matrices was used to dilute cardiac Troponin-I to a concentration between 15 ng/ml-20 ng/ml. The samples were stored under three different temperature conditions: 2-8° C., room temperature and 45° C. The Troponin-I concentration of each standard was measured (via the AxSYM Troponin-I assay) on days 0, 1, 2 and 7. The percent Troponin-I activity remaining was determined. The results are tabulated in Tables 2-7 and depicted in
After 7 days of storage at 2-8° C., the percent Troponin-I activity remaining varied with the lot of STANDAPOL® ES-1 used from 96% to 105%.
After 7 days of storage at room temperature, the percent of Troponin-I activity remaining varied with the lot of STANDAPOL® ES1 used from 99% to 103%.
After 7 days of storage at 45° C., the percent Troponin-I activity remaining varied with the lot of STANDAPOL® ES-1 used from 93% to 101%.
Calibrators are standard formulations containing known concentrations of Troponin-I and are used to define the calibration curve used in the assay. Controls are standard formulations also containing known concentrations of Troponin-I and were used to check the integrity of the calibration curve across the dynamic range of the assay.
Standard calibrators were prepared using the matrix formulation containing STANDAPOL® ES-1:
Similarly, three control standards were prepared at the following Troponin-I concentrations:
The complete set of standard calibrators and controls were each stored frozen, at 2-8° C. and at 31° C. The temperature 31° C. was chosen as a worst case room temperature condition. Freshly thawed controls were used to evaluate the stability of the calibrators stored under various temperature conditions on days 0, 1, 3, 7 and 14. The results are tabulated in Tables 8-10 and depicted in
Calibrator stability, stored under various temperature conditions, was assessed over 14 days using freshly thawed controls. Troponin-I activity remaining after 14 days ranged between 97-106%. Calibrators stored at 2-8° C. and 31° C. demonstrated similar Troponin-I % activity remaining ranging from 95-102% and 97-102%, respectively after 14 days.
Control stability stored under frozen, 2-8° C. and 31° C. conditions showed comparable retention of activity. Frozen controls retained between 97-106% activity after 14 days. Controls stored at 2-8° C. and 31° C. yielded Troponin-I activities from 101-109% after 14 days as compared to activity measured on day 0. The medium control stored under frozen, 2-8° C. and 31° C. conditions retained 97, 102 and 93% activity, respectively after 184 days.
The sodium polyoxyethylene (1) lauryl sulfate was found not to cause any carryover effects from sample to sample in the AxSYM instrument system.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
While some potential advantages and objects have been expressly identified herein, it should be understood that some embodiments of the invention may not provide all, or any, of the expressly identified advantages and objects.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This application claims benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Ser. No. 60/675,542, filed Apr. 28, 2005.
| Number | Date | Country | |
|---|---|---|---|
| 60675542 | Apr 2005 | US |
