WO2011064788A1 - A method and a kit for detection of acute coronary syndrome based on concentration of unbound iron present in a biological sample - Google Patents

A method and a kit for detection of acute coronary syndrome based on concentration of unbound iron present in a biological sample Download PDF

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Publication number
WO2011064788A1
WO2011064788A1 PCT/IN2010/000028 IN2010000028W WO2011064788A1 WO 2011064788 A1 WO2011064788 A1 WO 2011064788A1 IN 2010000028 W IN2010000028 W IN 2010000028W WO 2011064788 A1 WO2011064788 A1 WO 2011064788A1
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Prior art keywords
biological sample
standard
standard solutions
concentration
iron
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PCT/IN2010/000028
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French (fr)
Inventor
Mohan M. Rajapurkar
Suhas S. Lele
Sudhir Shah
Original Assignee
Rajapurkar Mohan M
Lele Suhas S
Sudhir Shah
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Application filed by Rajapurkar Mohan M, Lele Suhas S, Sudhir Shah filed Critical Rajapurkar Mohan M
Publication of WO2011064788A1 publication Critical patent/WO2011064788A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/90—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving iron binding capacity of blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • a method for detecting concentration of unbound iron present in a biological sample is disclosed. More particularly, a method and a kit for detection of Acute Coronary Syndrome (ACS) in a subject and prediction of adverse progress of ACS in the subject based on detection of the concentration of unbound iron present in the biological sample is disclosed.
  • ACS Acute Coronary Syndrome
  • MI Myocardial Infarction
  • UA Unstable Angina
  • biochemical markers are used to diagnose MI and UA associated with ACS.
  • serum markers such as Creatine Kinase (CK) and its cardiac specific iso-enzymes (CK-MB) are used for diagnosis of acute MI.
  • CK-MB Creatine Kinase
  • CK-MB cardiac specific iso-enzymes
  • false positive results may occur due to presence of CK-MB released from, for example, skeletal muscles or during conditions such as, myopathies, hypothyroidism, etc.
  • UA in subjects may be detected based on the amount of cardiac Troponin T and Troponin I present in the biological samples obtained from the subjects that are at the risk of having ACS.
  • slow release kinetics associated with Troponin T and Troponin I may lead to false negative findings in early stages of ACS.
  • PCT International Publication WO/2009/109982 discloses a new method of detecting ACS based on the concentration of Bleomycin Detectable Iron (BDI) present in the biological sample of a subject.
  • BDI Bleomycin Detectable Iron
  • the BDI assays that are well known in the art, for example a BDI assay described by Evans, et al., may be used for detecting the concentration of BDI in a biological sample.
  • the BDI assays known in the art are commonly associated with non- reproducible results. The reason behind the non-reproducibility of the results may be attributed to difficulties in maintaining a stable pH during the assay procedure.
  • BDI assays known in the art it is difficult to produce a linear standard graph for the assay.
  • the BDI assays known in the art are sensitive only over a narrow range of iron concentrations for example, zero ⁇ to 50 ⁇ .
  • kits for carrying out an assay are more desirable than performing the assay in a conventional manner.
  • a kit for detection of ACS is based on the BDI assays known in the art.
  • a kit for detection of ACS that is based on an improved method for carrying out the BDI assay.
  • BDI Bleomycin Detectable Iron
  • ACS Acute Coronary Syndrome
  • kit for early detection of ACS a method for detecting concentration of unbound iron in a biological sample. The method includes, preparing a set of standard solutions of iron using one or more solvents. The one or more solvents are treated with a chelating resin prior to preparing the set of standard solutions. A set of reagents is added to the biological sample and to the set of standard solutions.
  • One or more reagents of the set of reagents are treated with the chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions to produce a predetermined pH in the biological sample and the set of standard solutions. Further, adding the set of reagents to the biological sample and a standard solution of the set of standard solutions produces a sample color in the biological sample and a standard color in the standard solution of the set of standard solutions.
  • the sample_color is developed based on the concentration of unbound iron present in the biological sample.
  • the standard color is developed based on the concentration of iron present in the standard solution of the set of standard solutions. The concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample color and one or more standard colors corresponding to one or more standard solutions of the set of standard solutions.
  • one or more standard solutions of the set of standard solutions of iron are prepared by dissolving a predetermined amount of Ferric Chloride, or a solution thereof, in a predetermined amount of one or more of water and Hydrochloric Acid.
  • the one or more standard solutions of the set of standard solutions thus obtained are stable for at least six months when stored at a temperature below 4 °C in an acidic pH.
  • the one or more standard solutions may be used for detecting the concentration of iron in range of zero ⁇ /L to 1000 ⁇ /L.
  • the set of standard solutions is used for obtaining a standard graph that is linear and reproducible.
  • a method for early detection of ACS by detecting the concentration of unbound iron present in a biological sample obtained from a subject is disclosed in accordance with an embodiment of the invention.
  • a set of reagents is added to the biological sample and a set of standard solutions.
  • One or more reagents of the set of reagents are treated with a chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions.
  • the addition of the set of reagents produces a predetermined pH in the biological sample and the set of standard solutions. Further, the addition of the set of reagents produces a sample_color in the biological sample and a standard_color in the standard solution of the set of standard solutions.
  • the sample color is produced based on the concentration of unbound iron present in the biological sample.
  • the standard color is produced based on the concentration of iron present in the standard solution of the set of standard solutions.
  • the concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample color and one or more standard colors corresponding to one or more standard solutions of the set of standard solutions.
  • An instance of ACS is detected in the subject based on the concentration of the unbound iron present in the biological sample.
  • the method for detection of ACS further includes, obtaining two or more biological samples from the subject at two or more different time instances.
  • concentrations of unbound iron present in the two or more biological samples are determined to detect adverse progress of ACS in the subject.
  • kits for detection of ACS includes one or more receptacles for receiving a biological sample from a subject.
  • the kit further includes a set of standard solutions of iron and a set of reagents.
  • one or more standard solutions of the set of standard solutions are prepared by dissolving a predetermined amount of Ferric Chloride, or a solution thereof, in a predetermined amount of one or more of water and Hydrochloric Acid.
  • the one or more standard solutions of the set of standard solutions thus obtained are stable for at least six months when stored at a temperature below 4 °C in an acidic pH.
  • the one or more standard solutions may be used for detecting the concentration of iron in range of zero ⁇ /L to 1000 ⁇ /L. Further, the one or more standard solutions are used for obtaining a standard graph corresponding to the set of standard solutions that is linear and reproducible.
  • the set of reagents is added to the biological sample and the set of standard solutions. One or more reagents of the set of reagents are treated with a chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions. The addition of the set of reagents produces a predetermined pH in the biological sample and the set of standard solutions.
  • the addition of the set of reagents produces a sample color in the biological sample and a standard color in the standard solution of the set of standard solutions.
  • the sample color is produced based on the concentration of unbound iron present in the biological sample.
  • the standard color is produced based on the concentration of iron present in the standard solution of the set of standard solutions.
  • the concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample color and the standard_colors corresponding to one or more standard solutions of the set of standard solutions.
  • An instance of ACS is detected in the subject based on the concentration of the unbound iron present in the biological sample.
  • FIG. 1A illustrates a standard graph with concentrations of standard solutions, diluted with 20 ⁇ HC1, plotted along X-axis in logarithmic scale and values of measured A 532 plotted along Y-axis in linear scale.
  • FIG. IB illustrates a standard graph with concentrations of standard solutions, diluted with Chelex treated water, plotted along X-axis in logarithmic scale and values of measured A 532 plotted along Y-axis in linear scale.
  • FIG. 2A illustrates a standard graph for a Bleomycin Detectable Iron Assay performed in accordance with the invention on January 03, 2009.
  • FIG. 2B illustrates a standard graph for a Bleomycin Detectable Iron Assay performed in accordance with the invention on July 01, 2009 using standard solutions and one or more reagents prepared on January 03, 2009.
  • the invention provides a method for detecting concentration of unbound iron in a biological sample is proposed;
  • the method includes, preparing a set of standard solutions of iron using one or more solvents.
  • the one or more solvents are treated with a chelating resin prior to preparing the set of standard solutions.
  • a set of reagents is added to the biological sample and to the set of standard solutions.
  • One or more reagents of the set of reagents are treated with the chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions to produce a predetermined pH in the biological sample and the set of standard solutions.
  • sample color in the biological sample and a standard color in the standard solution of the set of standard solutions produces a sample color in the biological sample and a standard color in the standard solution of the set of standard solutions.
  • the sample color is developed based on the concentration of unbound iron present in the biological sample.
  • the standard color is developed based on the concentration of iron present in the standard solution of the set of standard solutions.
  • concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample_color and one or more standard colors corresponding to one or more standard solutions of the set of standard solutions.
  • the unbound iron includes a non- transferrin-bound iron, free iron, Bleomycin Detectable Iron (BDI) or catalytic iron that is found in a human body.
  • the unbound iron may include any form of iron that may act as a biochemical marker for diagnosing a condition, for example, but are not limited to, oxidative stress, ACS, ACS in patients with chronic kidney diseases, Myocardial Infarction (MI), Unstable Angina (UA), heart attack, heart failure, and the like.
  • a biological sample includes a fluid obtained from a human body.
  • the fluid obtained from the human body may include, for example, but are not limited to, blood, serum, plasma, urine, saliva, mucus secretion, synovial fluid, and cerebrospinal fluid.
  • the method for detecting concentration of unbound iron in the biological sample includes preparing a set of standard solutions.
  • the set of standard solutions includes one or more standard solutions of varying concentrations of iron.
  • the varying concentrations of iron in the one or more standard solutions ranges between zero ⁇ /L and 10000 ⁇ /L.
  • the set of standard solutions includes standard solutions containing, zero ⁇ /L of Iron, 1 ⁇ /L of Iron, 10 ⁇ /L of Iron, 100 ⁇ /L of Iron, and 1000 ⁇ /L of Iron.
  • the set of standard solutions includes standard solutions containing, 0 ⁇ /L of Iron, 0.1 ⁇ /L of Iron, 0.2 ⁇ /L of Iron, 0.3 ⁇ /L of Iron, 0.4 ⁇ /L of Iron and 0.5 ⁇ /L of Iron.
  • the set of standard solutions includes standard solutions containing, zero ⁇ /L of Iron, 10 ⁇ /L of Iron, 20 ⁇ /L of Iron, 30 ⁇ /L of Iron, 40 ⁇ /L of Iron and 50 ⁇ /L of Iron. It will be appreciated by a person skilled in the art that the set of standard solutions may be prepared with standard solutions with a concentrations ranging between zero ⁇ /L and 10000 ⁇ /L of Iron, without departing from the scope of the invention.
  • the set of standard solutions is prepared by using one or more solvents.
  • the one or more solvents include water and Hydrochloric Acid (HC1).
  • the set of standard solutions is prepared by dissolving a predetermined amount of Ferric Chloride in one or more of water and HC1.
  • the concentration of iron in one or more standard solutions of the set of standard solutions ranges from zero ⁇ /L to 10000 . ⁇ /L.
  • a standard stock solution of iron may be obtained by dissolving a predetermined amount of Ferric Chloride in a predetermined amount of one or more of water and HC1.
  • the standard stock solution may have concentration of iron in range of 1 to 100000 ⁇ /L.
  • the standard stock solution may be diluted to obtain one or more standard solutions of the set of standard solutions with desired iron concentration.
  • 16 mg of Ferric Chloride is dissolved in 10 ml of 20 ⁇ HC1 to get a standard stock solution of iron with concentration of 10000 ⁇ ⁇ /L.
  • a tenfold dilution of the standard stock solution of iron is then performed by serial dilution with 20 ⁇ HC1 to yield standard solutions of the set of standard solutions with concentrations of 1 ⁇ /L, 10 ⁇ /L, 100 ⁇ /L, and 1000 ⁇ /L.
  • the standard stock solution with any concentration of iron in a range of 0.1 to 100000 ⁇ /L can be prepared by dissolving.
  • the standard stock solution may be appropriately diluted to get the one or more standard solutions of the set of standard solutions with iron concentration in range of 0.01 ⁇ /L to 10000 ⁇ /L without departing from the scope of the invention.
  • the one or more solvents used for the preparation of the set of standard solutions are treated with a chelating resin.
  • the chelating resin includes Chelex 100. Due to the treatment of the one or more solvents with Chelex 100 one or more standard solutions of the set of standard solutions with a predetermined pH may be obtained.
  • the predetermined pH corresponding to the one or more standard solutions ranges between 7 and 8. Specifically, the predetermined pH corresponding to the one or more standard solutions ranges between 7.2 and 7.8. More specifically, the predetermined pH corresponding to the one or more standard solutions ranges between 7.4 and 7.6. For example, the predetermined pH corresponding to the one or more standard solutions is 7.4.
  • the predetermined pH corresponding to the one or more standard solutions of the set of standard solutions may be attributed to the treatment of the one or more solvents with Chelex 100.
  • the treatment of the one or more solvents with Chelex 100 makes the one or more solvents alkaline.
  • the use of the one or more solvents that is acidic in nature, for example, HC1 brings the pH within the range of 7 to 8.
  • the set of standard solutions prepared according to the various embodiments of the invention are stable for at least six months when stored at a temperature below 4 °C in an acidic pH.
  • the HC1 used for preparing the set of standard solutions may provide the acidic pH.
  • the method further includes adding a set of reagents to the biological sample and the set of standard solutions.
  • the set of reagents includes DNA, Magnesium Chloride, Ascorbic Acid, Ethylenediaminetetraacetic Acid (EDTA), Thiobarbituric Acid (TBA), Bleomycin Sulfate, HC1, and Water.
  • One or more reagents of the set of reagents may be used in one of, but are not limited to, a solution form, a powder form, and a crystalline form.
  • each reagent of the set of reagents are used in the solution form.
  • any other suitable form of the set of reagents may be used without departing from the scope of the invention.
  • DNA includes a lmg/ml solution of DNA.
  • the Magnesium Chloride includes a 50 ⁇ /L solution of Magnesium Chloride.
  • the Ascorbic Acid includes a 0.75 ⁇ /L solution of Ascorbic Acid.
  • the TBA includes a solution containing 1% w/v TBA in 50 ⁇ Sodium Hydroxide.
  • the Bleomycin Sulfate includes a 1.5 Units/mL solution of Bleomycin Sulfate.
  • the water includes double distilled and deionized water treated with Chelex 100.
  • the method further includes, treating one or more reagents of the set of reagents with Chelex 100 prior to adding the one or more reagents to the sample and to the set of standard solutions. Treating the one or more reagents includes adding a predetermined amount of Chelex 100 to a predetermined amount of the one or more reagents to obtain one or more mixtures of the one or more reagents and Chelex 100.
  • the predetermined amount of Chelex 100 may range between 0.1 g and 10 g.
  • the predetermined amount of the one or more reagents of the set of reagents may range from 1 ml to 1000 ml.
  • the one or more mixtures are allowed to stand for a period ranging from few minutes to several hours.
  • the one or more mixtures are allowed to stand at room temperature for an hour. Thereafter, Chelex 100 is separated from the one or more mixtures that are allowed to stand by using centrifugation.
  • water and DNA are further treated with Chelex 100 prior to adding the water and the DNA to the biological sample and the set of standard solutions. It will be appreciated by a person skilled in the art that any reagent of the one or more reagents may be treated with Chelex 100 prior to addition to the biological sample and the set of standard solutions without departing from the scope of the invention.
  • the addition of the one or more reagents of the set of reagents to the biological sample and to the set of standard solutions produces a predetermined pH in the biological sample and the set of standard solutions.
  • the predetermined pH ranges between 7 and 8. Specifically, the predetermined pH ranges between 7.2 and 7.8. More specifically the predetermined pH ranges between 7.4 and 7.6. For example, the predetermined pH is 7.4.
  • the set reagents including the DNA, the Bleomycin, the Magnesium Chloride, the Ascorbic Acid are added to the biological sample and the set of standard solutions in a plurality of reaction tubes to obtain a plurality of reaction mixtures.
  • the DNA, the Bleomycin, the Magnesium Chloride, the Ascorbic Acid are added to a plurality of reaction tubes. Thereafter, the biological sample and the set of standard solutions may be added to the plurality of reaction tubes to obtain the plurality of reaction mixtures.
  • the pH of the plurality of the reaction mixtures may be found to be within range of 7.4 to 7.6.
  • the EDTA is added to the plurality of reaction mixtures followed by the TBA and 25% HC1.
  • the plurality of reaction mixtures are then incubated for a period ranging from few minutes to few hours.
  • the plurality of reaction mixtures is incubated at 80 °C for 20 minutes.
  • Incubating one or more reaction mixtures corresponding to the biological sample produces a sample_colour in the one or more reaction mixtures corresponding to the biological sample.
  • the sample_color produced in the one or more reaction mixtures is dependent on the concentration of unbound iron in the biological sample.
  • incubating a reaction mixture corresponding to a standard solution of the set of standard solutions produces a standard_color in the reaction mixture.
  • the standard_color produced in the reaction mixture corresponding to the standard solutions is dependent on the concentration of iron in the standard solution.
  • a standard graph corresponding to the set of standard solutions is then prepared by using values corresponding to the sample_colour and standard_colors corresponding to the set of standard solutions.
  • the values corresponding to the sample colour and the standard colors may be obtained by using spectrometry techniques or any other techniques known in the art.
  • UV spectrometry is used to obtain the values corresponding to the sample_colour and the standard colors.
  • the concentration of unbound iron in the biological sample is detected by comparing the sample color with the one or more standard colors' by using the standard graph. It will be appreciated by a person skilled in the art that the concentration of unbound iron in the biological sample may be detected by comparing the sample color with the one or more standard colors corresponding to the set of standard solutions using any art know method without departing from the scope of the invention.
  • the standard graph prepared in accordance with various embodiments disclosed herein is linear in nature and is reproducible.
  • the reproducibility of results may be attributed to the fact that the set of standard solutions and the one or more reagents of the set of reagents are stable for at least six months when stored below 4 °C in an acidic pH.
  • the reproducibility of results may also be attributed to the fact that addition of the one or more reagents to the biological sample and to the set of standard solution produces a predetermined pH.
  • the predetermined pH ranges from 7.4 to 7.6.
  • the standard graph prepared in accordance with the various embodiments disclosed herein may be used to determine iron concentration that ranges from zero to 10000 ⁇ /L in a biological sample.
  • a method for detection of ACS by detecting the concentration of unbound iron present in a biological sample obtained from a subject includes, preparing a set of standard solutions of iron using one or more solvents.
  • the one or more solvents are treated with a chelating resin prior to preparing the set of standard solutions.
  • a set of reagents is added to the biological sample and to the set of standard solutions.
  • One or more reagents of the set of reagents are treated with the chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions to produce a predetermined pH in the biological sample and the set of standard solutions.
  • sample_color is developed based on the concentration of unbound iron present in the biological sample.
  • standard color is developed based on the concentration of iron present in the standard solution of the set of standard solutions.
  • concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample color and one or more standard colors corresponding to one or more standard solutions of the set of standard solutions.
  • An instance of ACS is detected in the subject based on the concentration of the unbound iron present in the biological sample.
  • the concentration of the unbound iron in the biological sample is detected by using the method for detecting concentration of unbound iron in a biological sample, in accordance with various embodiments of the invention disclosed herein.
  • ACS may be detected in the subject when the concentration of unbound iron in the biological sample obtained from the subject is above a predetermined level.
  • the predetermined level of unbound iron in the biological sample is 0.3 ⁇ /L.
  • the concentration of unbound iron in the subject is detected to be above 0.3 ⁇ /L the subject may be diagnosed as having ACS.
  • the concentration of unbound iron in the subject is detected to be less than 0.3 ⁇ /L the subject may be diagnosed as not having ACS.
  • the method for detecting ACS in the subject may further include obtaining two or more biological samples from the subject at two or more different time instances.
  • the concentration of unbound iron in the two or more biological samples may be detected using the method for detecting concentration of unbound iron in a biological sample, in accordance with various embodiments of the invention disclosed herein. Based on the detection the concentration of unbound iron in the two or more biological samples, adverse progress of ACS in the subject may be detected. For example, a first biological sample is obtained at a first time instance. The concentration of unbound iron in the first biological sample is detected. Based on the concentration of unbound iron in the first biological sample ACS is detected in the subject. A second biological sample is obtained after a predefined time interval from the first time instance.
  • the predefined time interval being suitable to observe a consequential variation or increase in the concentration of unbound iron in the subject.
  • concentration of unbound iron in the second biological sample is detected.
  • adverse progress of ACS is diagnosed. For example, when the concentration of unbound iron in the second biological sample is greater than the concentration of unbound iron in the first biological sample, adverse progress of ACS in the subject is diagnosed.
  • kits for detection of ACS includes one or more receptacles for receiving a biological sample from a subject, a set of standard solutions, and a set of reagents. Additionally, the kit may include a plurality of reaction tubes for carrying out reactions involved in the method for detection of ACS and a literature for guiding a chemist, biochemist, doctor, pathologist or the like in carrying out the method for detection of ACS.
  • one or more standard solutions of the set of standard solutions of iron are prepared by dissolving a predetermined amount of Ferric Chloride, or a solution thereof, in a predetermined amount of one or more of water and Hydrochloric Acid.
  • the one or more standard solutions of the set of standard solutions thus obtained are stable for at least six months when stored at a temperature below 4 °C in an acidic pH.
  • the one or more standard solutions may be used for detecting the concentration of iron in range of zero ⁇ /L to 1000 ⁇ /L.
  • the set of standard solutions is used for obtaining a standard graph that is linear and is reproducible.
  • a set of reagents is added to the biological sample and to the set of standard solutions.
  • One or more reagents of the set of reagents are treated with the chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions to produce a predetermined pH in the biological sample and the set of standard solutions.
  • the predetermined pH ranges between 7.4 and 7.6.
  • sample_color in the biological sample and a standard color in the standard solution
  • standard color is developed based on the concentration of iron present in the standard solution of the set of standard solutions.
  • the concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample_color and the one or more standard colors corresponding to one or more standard solutions of the set of standard solutions.
  • An instance of ACS is detected in the subject based on the concentration of the unbound iron present in the biological sample.
  • the methods and the kit disclosed herein provide for detection of the concentration of unbound iron present in the biological sample.
  • the methods and the kit disclosed herein can be used with reproducible results.
  • the set of standard solutions and the one or more reagents of the set of reagents disclosed herein are stable for at least six months when stored below 4 ° C in an acidic pH.
  • a predetermined pH ranging between 7.4 and 7.6 may be obtained and maintained while carrying out the methods and using the kit disclosed herein.
  • a standard graph that is linear in nature may be obtained in accordance with various embodiments of the invention.
  • the standard graph may be used for detecting the concentration of unbound iron in the biological sample that ranges between zero ⁇ /L to 1000 ⁇ /L.
  • the methods and the kit disclosed herein may be used for accurate and early detection of ACS and for detection of adverse progress of ACS in the subject.
  • Example 1 [0058] The Bleomycin Detectable Iron (BDI) Assay: [0059] Source of reagents:
  • the Chelex 100 was purchased from Bio Rad, USA and the HC1 (UN 1789) was purchased from J.T. Baker.
  • the reagents, mentioned below were purchased from Sigma, USA: DNA (Calf Thymus DNA-type I, D-1501), Bleomycin Sulfate (15 Unit, B5507), Magnesium Chloride (M-2670), Ascorbic Acid (A-1417), Ferric Chloride (F-7134), Sodium Hydroxide (S-8045), EDTA (E-9884) and Thiobarbituric Acid (TBA-T5500).
  • DNA Calf Thymus DNA-type I, D-1501
  • Bleomycin Sulfate (15 Unit, B5507)
  • Magnesium Chloride M-2670
  • Ascorbic Acid A-1417
  • Ferric Chloride F-7134
  • S-8045 Sodium Hydroxide
  • E-9884 E-9884
  • TSA-T5500 Thiobarbituric Acid
  • Chelex 100 One gram of Chelex 100 was added to 100 ml of double distilled and demonized water. The mixture of Chelex 100 and water was shaken well. Thereafter, the mixture was allowed to stand overnight below 4 C. A primary Chelex treated water was obtained by filtering the mixture through 0.22 ⁇ filter to remove particles of Chelex 100.
  • the primary Chelex treated water and the DNA solution was treated again with Chelex 100.
  • 50-60 mg of Chelex 100 was added to 4 ml of each of the primary Chelex treated water and the DNA solution.
  • the resulting mixtures were mixed by gentle inversion and then allowed to stand for one hour at room temperature.
  • Chelex 100 was isolated from the mixtures by centrifugation at 2000 g for 20 minutes.
  • the retreated Chelex water and retreated DNA solution thus obtained were carefully aspirated out for further use.
  • 50 ⁇ solution of Magnesium Chloride and 0.75 ⁇ solution of Ascorbic acid were prepared and diluted with the freshly retreated Chelex water and used immediately for the assay.
  • 10000 ⁇ /L iron standard stock solution was prepared by dissolving 16 mg of Ferric Chloride in 10 ml of 20 ⁇ solution of HC1. Thereafter, a tenfold dilution of the 10000 ⁇ /L iron standard stock solution was done by serial dilution with 20 ⁇ HC1 to yield standard stock solutions of iron with concentrations, 1000 ⁇ /L, 100 ⁇ /L, 10 ⁇ /L and 1 ⁇ ⁇ /L, respectively. The standard stock solutions of iron thus obtained were found to be stable for six months when stored below 4 °C.
  • the standard graph was obtained after adding reagents to the tubes in order mentioned below: (i) 250 ⁇ , of 1 mg/ml solution of DNA, (ii) 25 ⁇ , of 1.5 U/ml solution of Bleomycin Sulfate, (iii) 50 ⁇ , of 50 ⁇ solution of Magnesium Chloride, and (iv) 50 ⁇ . of 0.75 ⁇ solution of Ascorbic acid, to get a final volume of 425 ⁇ in each tube. After the addition of the above-mentioned reagents to the tubes, the pH in each tube was measured. The pH in each tube was found to be between 7.4 and 7.6. 50 ⁇ , of biological sample was then added to a properly labeled tube.
  • a control for standard solutions was then obtained by adding 20 ⁇ , of 20 ⁇ solution of HCl to the 180 ⁇ , of the retreated Chelex water. 150 ⁇ , of the resultant solution was discarded and the remainder 50 ⁇ , was used for the assay. This step was performed to obtain background 532. In addition, this step was found to be responsible for removing interference of possible contaminant iron. Thereafter, a sample control was obtained wherein the sample was added to a tube containing each of the reagent except Bleomycin to detect any possible TBA-reactive material that might be present in the biological sample.
  • each tube was then incubated for 90 minutes at 37 °C in a water bath.
  • 50 ⁇ _ of 0.1 M solution of EDTA, followed by 500 ⁇ , of 1% w/v solution of TBA in 50 ⁇ NaOH, and 500 ⁇ of 25% solution of HCl were added to each tube.
  • the tubes were then incubated at 80 °C in a water bath for 20 minutes to develop the chromogen.
  • the precipitated proteins in the biological sample were then removed by centrifugation at 2500 g for 20 minutes using Remi-India (CPR 30) refrigerated centrifuge.
  • the A 532 of supernatant layer was determined in Beckman Coulter -USA UV- Vis Spectrophotometer (DU-800) and the iron concentration in the biological sample was calculated by using the standard graph.
  • FIG. 1A illustrates a standard graph with concentrations of standard solutions, diluted with 20 ⁇ HC1, plotted along X-axis in logarithmic scale and values of measured A 532 plotted along Y-axis in linear scale. It was found that the values of measured A 532 were found to be linearly increasing for concentration of iron between 0.1 ⁇ /L and 10 ⁇ /L. However, the values of measured A 532 were found to decrease for concentrations of iron between 100 ⁇ /L and 1000 ⁇ /L.
  • FIG. IB illustrates a standard graph with concentrations of standard solutions, diluted with Chelex treated water, plotted along X-axis in logarithmic scale and values of measured A 532 plotted along Y-axis in linear scale. It was found that the values of measured A 532 were found to be linearly increasing for concentration of iron between 0.1 ⁇ /L and 10 ⁇ /L. However, the corresponding optical density values obtained were much lower for each concentration of iron between 0.1 ⁇ /L and 100 ⁇ /L compared to standard solutions diluted with 20 ⁇ HC1. [0082] Based on the standard graphs illustrated in FIG. 1A and FIG.
  • FIG. 2A illustrates a standard graph for a Bleomycin Detectable Iron Assay performed in accordance with the invention on January 03, 2009. Freshly prepared reagents and standard solutions were used for the assay. The concentrations of the standard solutions were plotted along X-axis in logarithmic scale and values of measured A 532 plotted along Y-axis in linear scale. Approximately, six months later, on July 01, 2009, the assay was repeated with the standard solutions and one or more reagents prepared on January 03, 2009.
  • FIG. 2B illustrates a standard graph for a Bleomycin Detectable Iron Assay performed in accordance with the invention on July 01, 2009 using the standard solutions and the one or more reagents prepared on January 03, 2009.
  • the standard graph obtained on July 01, 2009 [FIG. 2B] was found to be similar to the standard graph obtained on January 03, 2009 [FIG. 2A]. Further, the measured values of A 532 were found to be linear for concentration of iron between 0.1 ⁇ /L to 1000 ⁇ /L.
  • the Kit includes six tubes (Tarson PP tubes) for receiving standard solutions, one or more regeneration tubes with Chelex 100 (R), one or more tubes for receiving the biological sample, one or more tubes for sample blank, Reagent A, Reagent B, Reagent C, Reagent D, Reagent E, Reagent F, Reagent G, Reagent H, literature regarding the instructions to use and store the kit, and a semi-log graph paper for preparing a standard graph.
  • Tarson PP tubes for receiving standard solutions
  • R Chelex 100
  • Reagent A, Reagent B, Reagent C, Reagent D, Reagent E, Reagent F, Reagent G, Reagent H literature regarding the instructions to use and store the kit
  • a semi-log graph paper for preparing a standard graph.
  • Reagent A that includes primary Chelex treated DNA
  • Reagent B that includes primary Chelex treated water
  • Reagent C that includes Magnesium Chloride
  • Reagent D that includes powder of Ascorbic Acid
  • Reagent E that includes solution of Bleomycin Sulfate
  • Reagent F that includes solution of EDTA
  • Reagent G that includes solution of TBA
  • Reagent H that includes 25 % v/v solution of HC1.
  • each reagent A and reagent B are taken in the one or more regeneration tubes (R) supplied with the kit, half an hour prior to the assay. Reagent A and Reagent B are thoroughly mixed and left allowed to stand for 10 minutes. The one or more regeneration tubes are centrifuged at 4000 g for 15 minutes. The desired volume is aspirated out from the supernatant of the one or more regeneration tubes without disturbing the bottom pellet to obtain Working Reagent A and Working Reagent B. [0095] Preparation of Working Reagent C:
  • Reagent E, Reagent F, Reagent G, and Reagent H are ready to use and stable for at least six months when stored below 4 °C. [00101] Assay Method:
  • the six tubes for receiving the standard solutions are appropriately labeled.
  • the standard solutions have following concentrations of iron: zero ⁇ /L (S 0 ), 1 ⁇ /L (Si), 10 L (S 2 ), 100 ⁇ ⁇ /L (S 3 ), and 1000 ⁇ /L (S 4 ).
  • the tubes for receiving the sample blank are appropriately labeled indicating the tubes without Reagent E.
  • each tube is removed from the water bath, Reagent F is added to each of the tubes. Then 500 ⁇ ., of Reagent G is added to each of the tube followed by 500 ⁇ , of Reagent H. Each of the tubes is then thoroughly mixed and kept in a water bath with boiling water. Thereafter, each of the tube is removed from the water bath and allowed to cool.
  • the colours (Optical Density) produced in the standard solutions is read at 532 nm against the standard solution zero ⁇ /L (So), as standard blank.
  • a colour (Optical Density) produced in the sample tube is read at 532 nm against the colour produced in the tubes without Reagent E.
  • the biological sample may include one of serum and urine. In such cases, before reading the colors of the standard solutions and the biological sample, each tubes is centrifuged at 4000 g for 10 minutes to remove turbidity that may be present in the tubes.
  • a standard graph is prepared by plotting the values of the concentration of the standards solutions in ⁇ /L along X-axis against the corresponding optical density values along Y-axis on the semi-log graph paper provided with the kit. The standard graph is used to find out the amount of unbound iron in the biological sample.

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Abstract

A method for detecting concentration of unbound iron in a biological sample is disclosed in accordance with an embodiment of the invention. One or more reagents of a set of reagents are treated with a chelating resin and then added to the biological sample and a set of standard solutions to produce a predetermined pH in the biological sample and the set of standard solutions. Further, addition of the set of reagents to the biological sample and a standard solution of the set of standard solutions produces a sample color in the biological sample and a standard color in the standard solution. The concentration of the unbound iron in the biological sample is detected based on a comparison between the sample color and one or more standard colors corresponding to one or more standard solutions of the set of standard solutions. A method and a kit for detection of ACS are also disclosed.

Description

A METHOD AND A KIT FOR DETECTION OF ACUTE CORONARY SYNDROME BASED ON CONCENTRATION OF UNBOUND IRON PRESENT
IN A BIOLOGICAL SAMPLE
FIELD OF INVENTION
[00011 A method for detecting concentration of unbound iron present in a biological sample is disclosed. More particularly, a method and a kit for detection of Acute Coronary Syndrome (ACS) in a subject and prediction of adverse progress of ACS in the subject based on detection of the concentration of unbound iron present in the biological sample is disclosed.
BACKGROUND
[0002] Apart from chest pain, the two major manifestations of ACS are Myocardial Infarction (MI) and Unstable Angina (UA). Various biochemical markers are used to diagnose MI and UA associated with ACS. For example, serum markers such as Creatine Kinase (CK) and its cardiac specific iso-enzymes (CK-MB) are used for diagnosis of acute MI. However, false positive results may occur due to presence of CK-MB released from, for example, skeletal muscles or during conditions such as, myopathies, hypothyroidism, etc.
[0003] The other biochemical markers such as, cardiac Troponin T and Troponin I are not present in smooth muscles and in the blood of healthy human subjects. Therefore, Troponin T and Troponin I are considered highly specific for cardiac problems e.g. MI. Elevated levels of one or more of Troponin T and Troponin I may indicate much higher chances of adverse cardiac outcomes. However, the tests involving one or more of Troponin T and Troponin I show positive results only after six to twelve hours of the onset of the chest pain even if there is muscle necrosis. Thus, Troponin T and Troponin I are not helpful in early detection of ACS. [0004] Further, UA in subjects may be detected based on the amount of cardiac Troponin T and Troponin I present in the biological samples obtained from the subjects that are at the risk of having ACS. However, slow release kinetics associated with Troponin T and Troponin I may lead to false negative findings in early stages of ACS. Thus, there is a need in the art for methods that employ biological markers for accurate early detection of ACS.
[0005] PCT International Publication WO/2009/109982, which is incorporated herein by reference, discloses a new method of detecting ACS based on the concentration of Bleomycin Detectable Iron (BDI) present in the biological sample of a subject. The BDI assays that are well known in the art, for example a BDI assay described by Evans, et al., may be used for detecting the concentration of BDI in a biological sample. [0006] However, the BDI assays known in the art are commonly associated with non- reproducible results. The reason behind the non-reproducibility of the results may be attributed to difficulties in maintaining a stable pH during the assay procedure. Further, in BDI assays known in the art it is difficult to produce a linear standard graph for the assay. In addition, the BDI assays known in the art are sensitive only over a narrow range of iron concentrations for example, zero μπιοΐ to 50 μιτιοΐ.
[0007] Thus, there is need in the art for an improved method for carrying out a BDI assay, wherein the results of the BDI assay are reproducible, the standard solutions are stable, and a stable pH is obtained and maintained during the assay procedure.
[0008] Further, it will be appreciated by a person skilled in the art that a kit for carrying out an assay is more desirable than performing the assay in a conventional manner. A kit for detection of ACS is based on the BDI assays known in the art. Thus, there is a need for a kit for detection of ACS that is based on an improved method for carrying out the BDI assay. SUMMARY OF THE INVENTION
[0009] It is an object of the invention to provide a method for detecting the concentration of unbound iron present in a biological sample. More specifically, it is an object of the invention to provide an improved method for carrying out a Bleomycin Detectable Iron (BDI) assay, wherein a predetermined pH is obtained and maintained during the BDI assay. [0010] Further, it is an object of the invention to provide an improved method for carrying out a BDI assay, wherein a standard graph corresponding to the set of standard solutions is linear and is easily reproducible.
[0011] Furthermore, it is an object of the invention to provide an improved method for carrying out a BDI assay, wherein the BDI assay can be performed to obtain reproducible results.
[0012] Still further, it is an object of the invention to provide an improved method for carrying out a BDI assay, wherein a standard graph is linear, stable and sensitive for concentrations of iron between zero μπιοΙ/L and 1000 μπιοΙ/L.
[0013] In addition, it is an object of the invention to provide an improved method for carrying out a BDI assay, wherein a set of standard solutions and one or more reagents of a set of reagents is stable for an appreciable period.
[0014] It is another object of the invention to provide a method for early detection of Acute Coronary Syndrome (ACS) by detecting the concentration of unbound iron present in a biological sample obtained from a subject. [0015] It is yet another object of the invention to provide a kit for early detection of ACS. [0016] In order to achieve the above-mentioned objects of the invention, a method for detecting concentration of unbound iron in a biological sample is proposed. The method includes, preparing a set of standard solutions of iron using one or more solvents. The one or more solvents are treated with a chelating resin prior to preparing the set of standard solutions. A set of reagents is added to the biological sample and to the set of standard solutions. One or more reagents of the set of reagents are treated with the chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions to produce a predetermined pH in the biological sample and the set of standard solutions. Further, adding the set of reagents to the biological sample and a standard solution of the set of standard solutions produces a sample color in the biological sample and a standard color in the standard solution of the set of standard solutions. The sample_color is developed based on the concentration of unbound iron present in the biological sample. Whereas, the standard color is developed based on the concentration of iron present in the standard solution of the set of standard solutions. The concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample color and one or more standard colors corresponding to one or more standard solutions of the set of standard solutions.
[0017] In an embodiment of the invention, one or more standard solutions of the set of standard solutions of iron are prepared by dissolving a predetermined amount of Ferric Chloride, or a solution thereof, in a predetermined amount of one or more of water and Hydrochloric Acid. The one or more standard solutions of the set of standard solutions thus obtained are stable for at least six months when stored at a temperature below 4 °C in an acidic pH. The one or more standard solutions may be used for detecting the concentration of iron in range of zero μπιοΙ/L to 1000 μιηοΙ/L. The set of standard solutions is used for obtaining a standard graph that is linear and reproducible.
[0018] A method for early detection of ACS by detecting the concentration of unbound iron present in a biological sample obtained from a subject is disclosed in accordance with an embodiment of the invention. A set of reagents is added to the biological sample and a set of standard solutions. One or more reagents of the set of reagents are treated with a chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions. The addition of the set of reagents produces a predetermined pH in the biological sample and the set of standard solutions. Further, the addition of the set of reagents produces a sample_color in the biological sample and a standard_color in the standard solution of the set of standard solutions. The sample color is produced based on the concentration of unbound iron present in the biological sample. Whereas, the standard color is produced based on the concentration of iron present in the standard solution of the set of standard solutions. The concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample color and one or more standard colors corresponding to one or more standard solutions of the set of standard solutions. An instance of ACS is detected in the subject based on the concentration of the unbound iron present in the biological sample.
[0019] In an embodiment, the method for detection of ACS further includes, obtaining two or more biological samples from the subject at two or more different time instances. The concentrations of unbound iron present in the two or more biological samples are determined to detect adverse progress of ACS in the subject.
[0020] A kit for detection of ACS is disclosed in accordance with an embodiment of the invention. The kit for detection of ACS includes one or more receptacles for receiving a biological sample from a subject. The kit further includes a set of standard solutions of iron and a set of reagents. In an embodiment of the invention, one or more standard solutions of the set of standard solutions are prepared by dissolving a predetermined amount of Ferric Chloride, or a solution thereof, in a predetermined amount of one or more of water and Hydrochloric Acid. The one or more standard solutions of the set of standard solutions thus obtained are stable for at least six months when stored at a temperature below 4 °C in an acidic pH. In addition, the one or more standard solutions may be used for detecting the concentration of iron in range of zero μιηοΙ/L to 1000 μηιοΙ/L. Further, the one or more standard solutions are used for obtaining a standard graph corresponding to the set of standard solutions that is linear and reproducible. [0021] In an embodiment, the set of reagents is added to the biological sample and the set of standard solutions. One or more reagents of the set of reagents are treated with a chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions. The addition of the set of reagents produces a predetermined pH in the biological sample and the set of standard solutions. Further, the addition of the set of reagents produces a sample color in the biological sample and a standard color in the standard solution of the set of standard solutions. The sample color is produced based on the concentration of unbound iron present in the biological sample. Whereas, the standard color is produced based on the concentration of iron present in the standard solution of the set of standard solutions. The concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample color and the standard_colors corresponding to one or more standard solutions of the set of standard solutions. An instance of ACS is detected in the subject based on the concentration of the unbound iron present in the biological sample.
[0022] The above-mentioned objects and other objects of the invention will become apparent to those skilled in the art upon reading the detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1A illustrates a standard graph with concentrations of standard solutions, diluted with 20 μΜ HC1, plotted along X-axis in logarithmic scale and values of measured A532 plotted along Y-axis in linear scale.
[0024] FIG. IB illustrates a standard graph with concentrations of standard solutions, diluted with Chelex treated water, plotted along X-axis in logarithmic scale and values of measured A532 plotted along Y-axis in linear scale.
[0025] FIG. 2A illustrates a standard graph for a Bleomycin Detectable Iron Assay performed in accordance with the invention on January 03, 2009. [0026] FIG. 2B illustrates a standard graph for a Bleomycin Detectable Iron Assay performed in accordance with the invention on July 01, 2009 using standard solutions and one or more reagents prepared on January 03, 2009.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Various embodiments of the invention are disclosed herein. However, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific structural, functional, formulation, process or method details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the invention in virtually any appropriately detailed method, structure, application, usage, process or formulation. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention.
[0028] In this document, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms "comprises," "comprising," "has", "having," "includes", '.'including," "contains", "containing" or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, or apparatus that comprises, has, includes, contains a list of elements/steps does not include only those elements/steps but may include other elements not expressly listed or inherent to such process, method or apparatus. An element/step proceeded by "comprises ...a", "has ...a", "includes ...a", "contains ...a" does not, without more constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises, has, includes, contains the element. The terms "a" and "an" are defined as one or more unless explicitly stated otherwise herein. The terms "substantially", "essentially", "approximately", "about" or any other version thereof, are defined as being close to as understood by one of ordinary skill in the art. [0029] Further, in this document, the terms "standard solution" and "Standard solution of iron" are used interchangeably. Furthermore, in this document, the terms, "Bleomycin Detectable Iron assay, BDI assay and assay are used interchangeably. [0030] The invention provides a method for detecting concentration of unbound iron in a biological sample is proposed; The method includes, preparing a set of standard solutions of iron using one or more solvents. The one or more solvents are treated with a chelating resin prior to preparing the set of standard solutions. A set of reagents is added to the biological sample and to the set of standard solutions. One or more reagents of the set of reagents are treated with the chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions to produce a predetermined pH in the biological sample and the set of standard solutions. Further, adding the set of reagents to the biological sample and a standard solution of the set of standard solutions produces a sample color in the biological sample and a standard color in the standard solution of the set of standard solutions. The sample color is developed based on the concentration of unbound iron present in the biological sample. Whereas, the standard color is developed based on the concentration of iron present in the standard solution of the set of standard solutions. The concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample_color and one or more standard colors corresponding to one or more standard solutions of the set of standard solutions.
[0031] In various embodiments of the invention, the unbound iron includes a non- transferrin-bound iron, free iron, Bleomycin Detectable Iron (BDI) or catalytic iron that is found in a human body. The unbound iron may include any form of iron that may act as a biochemical marker for diagnosing a condition, for example, but are not limited to, oxidative stress, ACS, ACS in patients with chronic kidney diseases, Myocardial Infarction (MI), Unstable Angina (UA), heart attack, heart failure, and the like. [0032] In various embodiments of the invention, a biological sample includes a fluid obtained from a human body. The fluid obtained from the human body, may include, for example, but are not limited to, blood, serum, plasma, urine, saliva, mucus secretion, synovial fluid, and cerebrospinal fluid.
[0033] According to an exemplary embodiment, the method for detecting concentration of unbound iron in the biological sample includes preparing a set of standard solutions. The set of standard solutions includes one or more standard solutions of varying concentrations of iron. The varying concentrations of iron in the one or more standard solutions ranges between zero μηιοΙ/L and 10000 μιηοΙ/L. In an embodiment of the invention, the set of standard solutions includes standard solutions containing, zero μηιοΙ/L of Iron, 1 μηιοΙ/L of Iron, 10 μηιοΙ/L of Iron, 100 μηιοΙ/L of Iron, and 1000 μπιοΙ/L of Iron. Alternatively, the set of standard solutions includes standard solutions containing, 0 μηιοΙ/L of Iron, 0.1 μπιοΙ/L of Iron, 0.2 μηιοΙ/L of Iron, 0.3 μπιοΙ/L of Iron, 0.4 μπιοΙ/L of Iron and 0.5 μιηοΙ/L of Iron. In yet another embodiment of the invention, the set of standard solutions includes standard solutions containing, zero μιηοΙ/L of Iron, 10 μηιοΙ/L of Iron, 20 μηιοΙ/L of Iron, 30 μπιοΙ/L of Iron, 40 μπιοΙ/L of Iron and 50 μιηοΙ/L of Iron. It will be appreciated by a person skilled in the art that the set of standard solutions may be prepared with standard solutions with a concentrations ranging between zero μηιοΙ/L and 10000 μπιοΙ/L of Iron, without departing from the scope of the invention.
[0034] In an embodiment, the set of standard solutions is prepared by using one or more solvents. The one or more solvents include water and Hydrochloric Acid (HC1). The set of standard solutions is prepared by dissolving a predetermined amount of Ferric Chloride in one or more of water and HC1. The concentration of iron in one or more standard solutions of the set of standard solutions ranges from zero μηιοΙ/L to 10000 . μπιοΙ/L. Alternatively, a standard stock solution of iron may be obtained by dissolving a predetermined amount of Ferric Chloride in a predetermined amount of one or more of water and HC1. The standard stock solution may have concentration of iron in range of 1 to 100000 μπιοΙ/L. Further, the standard stock solution may be diluted to obtain one or more standard solutions of the set of standard solutions with desired iron concentration. [0035] For example, 16 mg of Ferric Chloride is dissolved in 10 ml of 20 μΜ HC1 to get a standard stock solution of iron with concentration of 10000 μη οΙ/L. A tenfold dilution of the standard stock solution of iron is then performed by serial dilution with 20 μΜ HC1 to yield standard solutions of the set of standard solutions with concentrations of 1 μηιοΙ/L, 10 μιηοΙ/L, 100 μιηοΙ/L, and 1000 μιηοΙ/L. It will be appreciated a person skilled in the art that the standard stock solution with any concentration of iron in a range of 0.1 to 100000 μπιοΙ/L can be prepared by dissolving. a predetermined amount of Ferric Chloride in one or more of water and HC1 without departing from the scope of the invention. Further, the standard stock solution may be appropriately diluted to get the one or more standard solutions of the set of standard solutions with iron concentration in range of 0.01 μηιοΙ/L to 10000 μπιοΙ/L without departing from the scope of the invention.
[0036] Prior to preparation of the set of standard solutions, the one or more solvents used for the preparation of the set of standard solutions are treated with a chelating resin. The chelating resin includes Chelex 100. Due to the treatment of the one or more solvents with Chelex 100 one or more standard solutions of the set of standard solutions with a predetermined pH may be obtained. In an embodiment, the predetermined pH corresponding to the one or more standard solutions ranges between 7 and 8. Specifically, the predetermined pH corresponding to the one or more standard solutions ranges between 7.2 and 7.8. More specifically, the predetermined pH corresponding to the one or more standard solutions ranges between 7.4 and 7.6. For example, the predetermined pH corresponding to the one or more standard solutions is 7.4. The predetermined pH corresponding to the one or more standard solutions of the set of standard solutions may be attributed to the treatment of the one or more solvents with Chelex 100. The treatment of the one or more solvents with Chelex 100 makes the one or more solvents alkaline. Further, the use of the one or more solvents that is acidic in nature, for example, HC1, brings the pH within the range of 7 to 8.
[0037] The set of standard solutions prepared according to the various embodiments of the invention are stable for at least six months when stored at a temperature below 4 °C in an acidic pH. In an exemplary embodiment, the HC1 used for preparing the set of standard solutions may provide the acidic pH. [0038] The method further includes adding a set of reagents to the biological sample and the set of standard solutions. The set of reagents includes DNA, Magnesium Chloride, Ascorbic Acid, Ethylenediaminetetraacetic Acid (EDTA), Thiobarbituric Acid (TBA), Bleomycin Sulfate, HC1, and Water. One or more reagents of the set of reagents may be used in one of, but are not limited to, a solution form, a powder form, and a crystalline form. In an embodiment, each reagent of the set of reagents are used in the solution form. However, it will be appreciated by the person skilled in the art that any other suitable form of the set of reagents may be used without departing from the scope of the invention.
[0039] In an embodiment, DNA includes a lmg/ml solution of DNA. The Magnesium Chloride includes a 50 μπιοΙ/L solution of Magnesium Chloride. The Ascorbic Acid includes a 0.75 μπιοΙ/L solution of Ascorbic Acid. The TBA includes a solution containing 1% w/v TBA in 50 μΜ Sodium Hydroxide. The Bleomycin Sulfate includes a 1.5 Units/mL solution of Bleomycin Sulfate. The water includes double distilled and deionized water treated with Chelex 100.
[0040] The method further includes, treating one or more reagents of the set of reagents with Chelex 100 prior to adding the one or more reagents to the sample and to the set of standard solutions. Treating the one or more reagents includes adding a predetermined amount of Chelex 100 to a predetermined amount of the one or more reagents to obtain one or more mixtures of the one or more reagents and Chelex 100. The predetermined amount of Chelex 100 may range between 0.1 g and 10 g. The predetermined amount of the one or more reagents of the set of reagents may range from 1 ml to 1000 ml. The one or more mixtures are allowed to stand for a period ranging from few minutes to several hours. For example, the one or more mixtures are allowed to stand at room temperature for an hour. Thereafter, Chelex 100 is separated from the one or more mixtures that are allowed to stand by using centrifugation. In an embodiment, water and DNA are further treated with Chelex 100 prior to adding the water and the DNA to the biological sample and the set of standard solutions. It will be appreciated by a person skilled in the art that any reagent of the one or more reagents may be treated with Chelex 100 prior to addition to the biological sample and the set of standard solutions without departing from the scope of the invention.
[0041] The addition of the one or more reagents of the set of reagents to the biological sample and to the set of standard solutions produces a predetermined pH in the biological sample and the set of standard solutions. The predetermined pH ranges between 7 and 8. Specifically, the predetermined pH ranges between 7.2 and 7.8. More specifically the predetermined pH ranges between 7.4 and 7.6. For example, the predetermined pH is 7.4. [0042] In an embodiment, the set reagents including the DNA, the Bleomycin, the Magnesium Chloride, the Ascorbic Acid are added to the biological sample and the set of standard solutions in a plurality of reaction tubes to obtain a plurality of reaction mixtures. Alternatively, the DNA, the Bleomycin, the Magnesium Chloride, the Ascorbic Acid are added to a plurality of reaction tubes. Thereafter, the biological sample and the set of standard solutions may be added to the plurality of reaction tubes to obtain the plurality of reaction mixtures. The pH of the plurality of the reaction mixtures may be found to be within range of 7.4 to 7.6.
[0043] Thereafter, the EDTA is added to the plurality of reaction mixtures followed by the TBA and 25% HC1. The plurality of reaction mixtures are then incubated for a period ranging from few minutes to few hours. In an embodiment, the plurality of reaction mixtures is incubated at 80 °C for 20 minutes. Incubating one or more reaction mixtures corresponding to the biological sample, produces a sample_colour in the one or more reaction mixtures corresponding to the biological sample. The sample_color produced in the one or more reaction mixtures is dependent on the concentration of unbound iron in the biological sample. Whereas, incubating a reaction mixture corresponding to a standard solution of the set of standard solutions produces a standard_color in the reaction mixture. The standard_color produced in the reaction mixture corresponding to the standard solutions is dependent on the concentration of iron in the standard solution.
[0044] A standard graph corresponding to the set of standard solutions is then prepared by using values corresponding to the sample_colour and standard_colors corresponding to the set of standard solutions. The values corresponding to the sample colour and the standard colors may be obtained by using spectrometry techniques or any other techniques known in the art. In an embodiment, UV spectrometry is used to obtain the values corresponding to the sample_colour and the standard colors. The concentration of unbound iron in the biological sample is detected by comparing the sample color with the one or more standard colors' by using the standard graph. It will be appreciated by a person skilled in the art that the concentration of unbound iron in the biological sample may be detected by comparing the sample color with the one or more standard colors corresponding to the set of standard solutions using any art know method without departing from the scope of the invention.
[0045] The standard graph prepared in accordance with various embodiments disclosed herein is linear in nature and is reproducible. The reproducibility of results may be attributed to the fact that the set of standard solutions and the one or more reagents of the set of reagents are stable for at least six months when stored below 4 °C in an acidic pH. The reproducibility of results may also be attributed to the fact that addition of the one or more reagents to the biological sample and to the set of standard solution produces a predetermined pH. The predetermined pH ranges from 7.4 to 7.6. Further, the standard graph prepared in accordance with the various embodiments disclosed herein, may be used to determine iron concentration that ranges from zero to 10000 μπιοΐ /L in a biological sample.
[0046] A method for detection of ACS by detecting the concentration of unbound iron present in a biological sample obtained from a subject is disclosed in accordance with an embodiment of the invention. The method includes, preparing a set of standard solutions of iron using one or more solvents. The one or more solvents are treated with a chelating resin prior to preparing the set of standard solutions. A set of reagents is added to the biological sample and to the set of standard solutions. One or more reagents of the set of reagents are treated with the chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions to produce a predetermined pH in the biological sample and the set of standard solutions. Further, adding the set of reagents to the biological sample and a standard solution of the set of standard solutions produces a sample color in the biological sample and a standard color in the standard solution of the set of standard solutions. The sample_color is developed based on the concentration of unbound iron present in the biological sample. Whereas, the standard color is developed based on the concentration of iron present in the standard solution of the set of standard solutions. The concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample color and one or more standard colors corresponding to one or more standard solutions of the set of standard solutions. An instance of ACS is detected in the subject based on the concentration of the unbound iron present in the biological sample.
[0047] The concentration of the unbound iron in the biological sample is detected by using the method for detecting concentration of unbound iron in a biological sample, in accordance with various embodiments of the invention disclosed herein. In an embodiment, ACS may be detected in the subject when the concentration of unbound iron in the biological sample obtained from the subject is above a predetermined level. The predetermined level of unbound iron in the biological sample is 0.3 μηιοΐ /L. When the concentration of unbound iron in the subject is detected to be above 0.3 μιηοΐ /L the subject may be diagnosed as having ACS. Whereas, in cases, where the concentration of unbound iron in the subject is detected to be less than 0.3 μπιοΐ /L the subject may be diagnosed as not having ACS.
[0048] In an embodiment, the method for detecting ACS in the subject may further include obtaining two or more biological samples from the subject at two or more different time instances. The concentration of unbound iron in the two or more biological samples may be detected using the method for detecting concentration of unbound iron in a biological sample, in accordance with various embodiments of the invention disclosed herein. Based on the detection the concentration of unbound iron in the two or more biological samples, adverse progress of ACS in the subject may be detected. For example, a first biological sample is obtained at a first time instance. The concentration of unbound iron in the first biological sample is detected. Based on the concentration of unbound iron in the first biological sample ACS is detected in the subject. A second biological sample is obtained after a predefined time interval from the first time instance. The predefined time interval being suitable to observe a consequential variation or increase in the concentration of unbound iron in the subject. Thereafter, concentration of unbound iron in the second biological sample is detected. Based on analysis of the amount of unbound iron in the second biological sample and the first biological sample, adverse progress of ACS is diagnosed. For example, when the concentration of unbound iron in the second biological sample is greater than the concentration of unbound iron in the first biological sample, adverse progress of ACS in the subject is diagnosed.
[0049] A kit for detection of ACS is disclosed in accordance with an embodiment of the invention. The kit includes one or more receptacles for receiving a biological sample from a subject, a set of standard solutions, and a set of reagents. Additionally, the kit may include a plurality of reaction tubes for carrying out reactions involved in the method for detection of ACS and a literature for guiding a chemist, biochemist, doctor, pathologist or the like in carrying out the method for detection of ACS.
[0050] In an embodiment of the invention, one or more standard solutions of the set of standard solutions of iron are prepared by dissolving a predetermined amount of Ferric Chloride, or a solution thereof, in a predetermined amount of one or more of water and Hydrochloric Acid. The one or more standard solutions of the set of standard solutions thus obtained are stable for at least six months when stored at a temperature below 4 °C in an acidic pH. The one or more standard solutions may be used for detecting the concentration of iron in range of zero μπιοΙ/L to 1000 μιηοΙ/L. The set of standard solutions is used for obtaining a standard graph that is linear and is reproducible. [0051] In an embodiment, a set of reagents is added to the biological sample and to the set of standard solutions. One or more reagents of the set of reagents are treated with the chelating resin prior to adding the one or more reagents to the biological sample and the set of standard solutions to produce a predetermined pH in the biological sample and the set of standard solutions. The predetermined pH ranges between 7.4 and 7.6.
[0052] Further, adding the set of reagents to the biological sample and a standard solution of the set of standard solutions produces a sample_color in the biological sample and a standard color in the standard solutionThe sample color is developed based on the concentration of unbound iron present in the biological sample. Whereas, the standard color is developed based on the concentration of iron present in the standard solution of the set of standard solutions.
[0053] The concentration of the unbound iron present in the biological sample is detected based on a comparison between the sample_color and the one or more standard colors corresponding to one or more standard solutions of the set of standard solutions. An instance of ACS is detected in the subject based on the concentration of the unbound iron present in the biological sample.
[00541 In accordance with various embodiments of the invention, the methods and the kit disclosed herein provide for detection of the concentration of unbound iron present in the biological sample. The methods and the kit disclosed herein can be used with reproducible results. Further, the set of standard solutions and the one or more reagents of the set of reagents disclosed herein are stable for at least six months when stored below 4 ° C in an acidic pH. A predetermined pH ranging between 7.4 and 7.6 may be obtained and maintained while carrying out the methods and using the kit disclosed herein. Furthermore, a standard graph that is linear in nature may be obtained in accordance with various embodiments of the invention. Still further, the standard graph may be used for detecting the concentration of unbound iron in the biological sample that ranges between zero μηιοΙ/L to 1000 μηιοΙ/L. In addition, the methods and the kit disclosed herein may be used for accurate and early detection of ACS and for detection of adverse progress of ACS in the subject.
[0055] In the foregoing disclosure, exemplary embodiments of the invention have been described. However, one of ordinary skill in the art appreciates that various modifications and changes may be made without departing from the scope of the invention. Accordingly, the complete specification is to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the invention. The benefits, advantages, solutions to problems, and any element(s)/feature(s)/step(s)/usage/application that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential features or elements with reference to the disclosure.
[0056] Examples:
The examples and experiments are presented solely for the purpose of illustration and should not be construed as limiting the invention.
[0057] Example 1: [0058] The Bleomycin Detectable Iron (BDI) Assay: [0059] Source of reagents:
[0060] The Chelex 100 was purchased from Bio Rad, USA and the HC1 (UN 1789) was purchased from J.T. Baker. The reagents, mentioned below were purchased from Sigma, USA: DNA (Calf Thymus DNA-type I, D-1501), Bleomycin Sulfate (15 Unit, B5507), Magnesium Chloride (M-2670), Ascorbic Acid (A-1417), Ferric Chloride (F-7134), Sodium Hydroxide (S-8045), EDTA (E-9884) and Thiobarbituric Acid (TBA-T5500). [0061] Preparation of Water:
[0062] One gram of Chelex 100 was added to 100 ml of double distilled and demonized water. The mixture of Chelex 100 and water was shaken well. Thereafter, the mixture was allowed to stand overnight below 4 C. A primary Chelex treated water was obtained by filtering the mixture through 0.22 μιη filter to remove particles of Chelex 100.
[0063] Preparation of DNA Solution:
[0064] The finely chopped DNA was added to the primary Chelex treated water at a concentration of lmg/ml. The resulting mixture was incubated below 4 C in a shaking water bath for two hours. The DNA solution thus obtained was finally stored at 4 °C. [0065] Preparation of Bleomycin:
[0066] 15 units of Bleomycin sulfate were dissolved in 15 mL of the primary Chelex treated water. The Bleomycin sulfate solution thus obtained was stored in amber colored glass bottle at 4 °C.
[0067] Preparation of Other Reagents:
[0068] 0.1M solution of EDTA, solution of Thiobarbituric Acid (l%w/v in 50 μΜ NaOH), and 25% HC1 were prepared using the primary Chelex treated water.
[0069] Preparation of Retreated Chelex Water and DNA:
[0070] Before performing the assay, the primary Chelex treated water and the DNA solution was treated again with Chelex 100. For this purpose, 50-60 mg of Chelex 100 was added to 4 ml of each of the primary Chelex treated water and the DNA solution. The resulting mixtures were mixed by gentle inversion and then allowed to stand for one hour at room temperature. Chelex 100 was isolated from the mixtures by centrifugation at 2000 g for 20 minutes. The retreated Chelex water and retreated DNA solution thus obtained were carefully aspirated out for further use. 50 μΜ solution of Magnesium Chloride and 0.75 μΜ solution of Ascorbic acid were prepared and diluted with the freshly retreated Chelex water and used immediately for the assay.
[0071] Preparation of Iron Standard Stock Solutions:
[0072] 10000 μηιοΙ/L iron standard stock solution was prepared by dissolving 16 mg of Ferric Chloride in 10 ml of 20 μΜ solution of HC1. Thereafter, a tenfold dilution of the 10000 μιηοΙ/L iron standard stock solution was done by serial dilution with 20 μΜ HC1 to yield standard stock solutions of iron with concentrations, 1000 μηιοΙ/L, 100 μηιοΙ/L, 10 μηιοΙ/L and 1 μη οΙ/L, respectively. The standard stock solutions of iron thus obtained were found to be stable for six months when stored below 4 °C.
[0073] Assay Procedure: [0074] The standard stock solutions of iron with concentrations, 10000 μηιοΙ/L, 1000 μηιοΙ/L, 100 μηιοΙ/L, 10 μηιοΙ/L and 1 μηιοΙ/L were diluted tenfold with the freshly retreated Chelex water to get standard solutions with concentrations, 1000 μη οΙ/L, 100 μηιοΙ/L, 10 μη οΙ/L, 1 μηιοΙ/L and 0.1 μηιοΙ/L. 20 μΐ, of each the standard solutions was added to 180 μΐ, of the retreated Chelex water (with pH > 8.5) in adequately labeled tubes. The pH of each standard solution was measured and found to be 7.4. The contents of the tubes were thoroughly mixed and 150 μΐ, of the contents were discarded. The remainder 50 μΐ. of the contents of each tube were used for the assay to obtain a standard graph.
[0075] The standard graph was obtained after adding reagents to the tubes in order mentioned below: (i) 250 μΐ, of 1 mg/ml solution of DNA, (ii) 25 μΐ, of 1.5 U/ml solution of Bleomycin Sulfate, (iii) 50 μΐ, of 50 μΜ solution of Magnesium Chloride, and (iv) 50 μΐ. of 0.75 μΜ solution of Ascorbic acid, to get a final volume of 425 μΐ in each tube. After the addition of the above-mentioned reagents to the tubes, the pH in each tube was measured. The pH in each tube was found to be between 7.4 and 7.6. 50 μί, of biological sample was then added to a properly labeled tube. [0076] A control for standard solutions was then obtained by adding 20 μΐ, of 20 μΜ solution of HCl to the 180 μΐ, of the retreated Chelex water. 150 μΐ, of the resultant solution was discarded and the remainder 50 μΐ, was used for the assay. This step was performed to obtain background 532. In addition, this step was found to be responsible for removing interference of possible contaminant iron. Thereafter, a sample control was obtained wherein the sample was added to a tube containing each of the reagent except Bleomycin to detect any possible TBA-reactive material that might be present in the biological sample.
[0077] Each tube was then incubated for 90 minutes at 37 °C in a water bath. 50 μΙ_, of 0.1 M solution of EDTA, followed by 500 μΐ, of 1% w/v solution of TBA in 50 μΜ NaOH, and 500 μΐ of 25% solution of HCl were added to each tube. The tubes were then incubated at 80 °C in a water bath for 20 minutes to develop the chromogen. [0078] The precipitated proteins in the biological sample were then removed by centrifugation at 2500 g for 20 minutes using Remi-India (CPR 30) refrigerated centrifuge. The A532 of supernatant layer was determined in Beckman Coulter -USA UV- Vis Spectrophotometer (DU-800) and the iron concentration in the biological sample was calculated by using the standard graph.
[0079] Results of the Assay: [0080] FIG. 1A illustrates a standard graph with concentrations of standard solutions, diluted with 20 μΜ HC1, plotted along X-axis in logarithmic scale and values of measured A532 plotted along Y-axis in linear scale. It was found that the values of measured A532 were found to be linearly increasing for concentration of iron between 0.1 μηιοΙ/L and 10 μηιοΙ/L. However, the values of measured A532 were found to decrease for concentrations of iron between 100 μηιοΙ/L and 1000 μπιοΙ/L.
[0081] FIG. IB illustrates a standard graph with concentrations of standard solutions, diluted with Chelex treated water, plotted along X-axis in logarithmic scale and values of measured A532 plotted along Y-axis in linear scale. It was found that the values of measured A532 were found to be linearly increasing for concentration of iron between 0.1 μπιοΙ/L and 10 μπιοΙ/L. However, the corresponding optical density values obtained were much lower for each concentration of iron between 0.1 μπιοΙ/L and 100 μπιοΙ/L compared to standard solutions diluted with 20 μΜ HC1. [0082] Based on the standard graphs illustrated in FIG. 1A and FIG. IB, it was concluded that when standard stock solutions diluted with HC1 are used for preparing the standard graph, linearity up to 5 μηιοΙ/L may be achieved. Whereas, when standard stock solutions diluted with Chelex treated water are used for preparing the standard graph, an extended linearity of the standard graph is achieved that has a poor sensitivity for low concentrations of iron, as illustrated in FIG. IB. [0083] Example 2:
[0084] FIG. 2A illustrates a standard graph for a Bleomycin Detectable Iron Assay performed in accordance with the invention on January 03, 2009. Freshly prepared reagents and standard solutions were used for the assay. The concentrations of the standard solutions were plotted along X-axis in logarithmic scale and values of measured A532 plotted along Y-axis in linear scale. Approximately, six months later, on July 01, 2009, the assay was repeated with the standard solutions and one or more reagents prepared on January 03, 2009. FIG. 2B illustrates a standard graph for a Bleomycin Detectable Iron Assay performed in accordance with the invention on July 01, 2009 using the standard solutions and the one or more reagents prepared on January 03, 2009. The standard graph obtained on July 01, 2009 [FIG. 2B] was found to be similar to the standard graph obtained on January 03, 2009 [FIG. 2A]. Further, the measured values of A532 were found to be linear for concentration of iron between 0.1 μηιοΙ/L to 1000 μιηοΙ/L.
[0085] Based on the standard graphs illustrated in FIG. 2A and FIG. 2B, it was discovered that, the standard solutions and the one or more reagents prepared in accordance with the various embodiments of the invention are stable for at least six months when stored below 4 °C. Further, it was concluded that the methods in accordance with the invention could be used to detect concentration of iron in range of 0.1 μπιοΙ/L to 1000 μιηοΙ/L.
[0086] Example 3:
[0087] BDI Assay Kit (the Kit) and Method of Using the Kit: [0088] The Kit: [0089] The BDI Assay kit includes six tubes (Tarson PP tubes) for receiving standard solutions, one or more regeneration tubes with Chelex 100 (R), one or more tubes for receiving the biological sample, one or more tubes for sample blank, Reagent A, Reagent B, Reagent C, Reagent D, Reagent E, Reagent F, Reagent G, Reagent H, literature regarding the instructions to use and store the kit, and a semi-log graph paper for preparing a standard graph. [0090] List of Reagents Supplied with the Kit:
[0091] Following reagents are supplied with the kit:
[0092] (i) Reagent A that includes primary Chelex treated DNA; (ii) Reagent B that includes primary Chelex treated water; (iii) Reagent C that includes Magnesium Chloride; (iv) Reagent D that includes powder of Ascorbic Acid; (v) Reagent E that includes solution of Bleomycin Sulfate; (vi) Reagent F that includes solution of EDTA; (vii) Reagent G that includes solution of TBA; and (viii) Reagent H that includes 25 % v/v solution of HC1.
[0093] Preparation of Working Reagent A and Working Reagent B:
[0094] 4 ml each reagent A and reagent B are taken in the one or more regeneration tubes (R) supplied with the kit, half an hour prior to the assay. Reagent A and Reagent B are thoroughly mixed and left allowed to stand for 10 minutes. The one or more regeneration tubes are centrifuged at 4000 g for 15 minutes. The desired volume is aspirated out from the supernatant of the one or more regeneration tubes without disturbing the bottom pellet to obtain Working Reagent A and Working Reagent B. [0095] Preparation of Working Reagent C:
[0096] TO of Reagent C is aspirated out and 1 ml of working reagent B is added to obtain Working Reagent C. Working Reagent C needs to be freshly prepared for an assay.
[0097] Preparation of Working Reagent D: [0098] The powder supplied with the kit is added to 500 μί of Working Reagent B, just prior to assay. The resultant mixture is diluted 50 fold with Working Reagent B to obtain Working Reagent D. Working Reagent D needs to be freshly prepared for an assay. [0099] Reagents E, F, G, and H:
[00100] Reagent E, Reagent F, Reagent G, and Reagent H are ready to use and stable for at least six months when stored below 4 °C. [00101] Assay Method:
[00102] The six tubes for receiving the standard solutions are appropriately labeled. The standard solutions have following concentrations of iron: zero μιηοΙ/L (S0), 1 μπιοΙ/L (Si), 10 L (S2), 100 μη οΙ/L (S3), and 1000 μπιοΙ/L (S4). In assays involving urine or serum as the biological sample, the tubes for receiving the sample blank are appropriately labeled indicating the tubes without Reagent E.
[00103] 180 μΐ, of Working Reagent B is added in each of the standard tubes. 20 μΐ. each of the standard solutions with concentrations zero μηιοΙ/L, 1 μη οΙ/L, 10 μπιοΙ/L, 100 μιηοΙ/L and 1000 μηιοΙ/L are added into the correspondingly labeled tubes and mixed thoroughly. 150 μΐ, of the resultant mixture is aspirated from each of the tubes. 50 μΐ^ of the biological sample, for example, serum, urine, etc., is added in the correspondingly labeled tubes and in the respective tubes with label indicating the tubes without Reagent E.
[00104] 250 μΐ, of Working Reagent A is added in each of the tube followed by the addition of 25 μΐ of Reagent E in each of the tube. However, Reagent E is not added in the tubes with label indicating the tubes without Reagent E. 50 μΐ^ of Working Reagent C is added to each tube and then 50 μΕ of freshly prepared Working Reagent D is added to each of the tube. [00105] Thereafter, each tubes are vortexed properly. The mouth of each tube containing the standard solutions is sealed properly with a Para film. The Para film is punctured in multiple places with a fine needle. Each tube is incubated at 37 °C for 90 minutes in a water bath. After each tubes is removed from the water bath, Reagent F is added to each of the tubes. Then 500 μΐ., of Reagent G is added to each of the tube followed by 500 μΐ, of Reagent H. Each of the tubes is then thoroughly mixed and kept in a water bath with boiling water. Thereafter, each of the tube is removed from the water bath and allowed to cool. [00106] The colours (Optical Density) produced in the standard solutions is read at 532 nm against the standard solution zero μιηοΙ/L (So), as standard blank. A colour (Optical Density) produced in the sample tube is read at 532 nm against the colour produced in the tubes without Reagent E. In some instances, the biological sample may include one of serum and urine. In such cases, before reading the colors of the standard solutions and the biological sample, each tubes is centrifuged at 4000 g for 10 minutes to remove turbidity that may be present in the tubes.
[00107] A standard graph is prepared by plotting the values of the concentration of the standards solutions in μιηοΙ/L along X-axis against the corresponding optical density values along Y-axis on the semi-log graph paper provided with the kit. The standard graph is used to find out the amount of unbound iron in the biological sample.

Claims

is claimed is:
A method for detecting concentration of unbound iron in a biological sample, the method comprises:
a. preparing a set of standard solutions of iron using at least one solvent, wherein the at least one solvent is treated with a chelating resin prior to preparing the set of standard solutions;
b. adding a set of reagents to the biological sample and to the set of standard solutions, wherein at least one reagent of the set of reagents is treated with the chelating resin prior to adding the at least one reagent to the biological sample and to the set of standard solutions, wherein addition of the set of reagents to the biological sample and to the set of standard solutions produces a predetermined pH in the biological sample and the set of standard solutions, wherein as a result of addition of the set of reagents to the biological sample and to the set of standard solutions, a standard solution of the set of standard solutions develops a standard_color based on a corresponding concentration of iron in the standard solution, and the biological sample develops a sample_color based on concentration of unbound iron present in the biological sample;
c. comparing the sample color with at least one standard color corresponding to the set of standard solutions; and
d. establishing concentration of unbound iron in the biological sample in response to comparing the sample_color with the at least one standard color.
2. The method of claim 1, wherein preparing the set of standard solutions comprises dissolving a predetermined quantity of Ferric Chloride in a predetermined quantity of the at least one solvent to obtain at least one standard solution of the set of standard solutions with concentration of iron between zero μηιοΙ/L to 10,000 μιηοΙ/L.
3. The method of claim 2, wherein a solvent comprises at least one of a Hydrochloric acid and water.
4. The method of claim 1 , wherein the set of reagents comprise DNA, Magnesium Chloride, Ascorbic Acid, EDTA, Thiobarbituric acid, Bleomycin Sulfate, Hydrochloric acid and water.
5. The method claim 1, wherein the chelating resin is Chelex 100.
6. The method of claim 1, wherein at least one reagent of the set of reagents and the set of standard solutions is stable for at least six months when stored below 4°C in an acidic pH.
7. The method of claim 1 , wherein the predetermined pH ranges between 7 and 8.
8. The method of claim 7, wherein the predetermined pH is 7.4.
9. The method of claim 1, wherein the biological sample comprises a fluid obtained from a human body.
10. The method of claim 1, wherein the fluid obtained from the human body comprises at least one of blood, serum, plasma, urine, saliva, mucus secretion, synovial fluid, and cerebrospinal fluid.
1 1. The method of claim 1 further comprising detecting a cardiac condition based on the concentration of unbound iron in the biological sample, wherein the cardiac condition is at least one of Acute Coronary Syndrome, Acute Coronary Syndrome in patients with chronic kidney diseases, Heart Failure, Arrhythmia, Myocardial Infarction, Angina Pectoris, Congestive Heart Failure, and onset of Congestive Heart Failure.
12. A method for detecting Acute Coronary Syndrome in a subject by using a biological sample obtained from the subject, the method comprising:
a. adding a set of reagents to the biological sample and to a set of standard solutions, wherein at least one reagent of the set of reagents is treated with the chelating resin prior to adding the at least one reagent to the biological sample and to the set of standard solutions, wherein addition of the set of reagents to the biological sample and to the set of standard solutions produces a predetermined pH in the biological sample and the set of standard solutions, wherein as a result of addition of the set of reagents to the biological sample and to the set of standard solutions, a standard solution of the set of standard solutions develops a standard_color based on a corresponding concentration of iron in the standard solution, and the biological sample develops a sample color based on concentration of unbound iron present in the biological sample;
b. comparing the sample_color with at least one standard_color corresponding to the set of standard solutions;
c. establishing a concentration of unbound iron present in the biological sample in response to comparing the sample color with the at least one standard color; and
d. detecting Acute Coronary Syndrome in the subject based on the concentration of unbound iron present in the biological sample.
13. The method of claim 12, wherein at least one standard solution of the set of standard solutions has a concentration of iron between zero μηιοΙ/L to 10,000 μηιοΙ/L.
14. The method of claim 13, wherein the at least one standard solution of the set of standard solutions is prepared by dissolving a predetermined amount of Ferric Chloride in a predetermined amount of at least one solvent to obtain concentration of iron between zero μηιοΙ/L to 10,000 μηιοΙ/L in the at least one standard solution.
15. The method of claim 14, wherein the at least one solvent comprise at least one of a Hydrochloric acid and water.
16. The method of claim 12, wherein the set of reagents comprise DNA, Magnesium Chloride, Ascorbic Acid, EDTA, Thiobarbituric acid, Bleomycin Sulfate, Hydrochloric acid and water.
17. The method claim 12, wherein the chelating resin is Chelex 100.
18. The method of claim 12, wherein the biological sample comprises at least one of blood, serum, plasma, urine, saliva, mucus secretion, synovial fluid, and cerebrospinal fluid obtained from a human body.
19. The method of claim 12, wherein an instance of Acute Coronary Syndrome in the subject is detected when the concentration of unbound iron in the biological sample obtained from the subject is greater than a predetermined concentration.
20. The method of claim 12, wherein the method further comprises obtaining at least two biological samples from the subject for at least two different instances and determining the concentration of unbound iron in the at least two biological samples obtained from the subject for the at least two instances to detect adverse progression of Acute Coronary Syndrome iri the subject.
21. A kit for detection of Acute Coronary Syndrome (ACS), the kit comprising:
a. at least one receptacle for receiving a biological sample from a subject; b. a set of standard solutions of iron, wherein the set of standard solutions is prepared by using at least one solvent, wherein the at least one solvent is treated with a chelating resin prior to preparing the set of standard solutions; c. a set of reagents, wherein at least one reagent of the set of reagents is treated with the chelating resin prior to adding the at least one reagent to the biological sample and to the set of standard solutions, wherein addition of the set of reagents to the biological sample and to the set of standard solutions produces a predetermined pH in the biological sample and the set of standard solutions; and wherein as a result of adding the set of reagents to the biological sample and to the set of standard solutions, a standard solution of the set of . standard solutions develops a standard color based on a corresponding concentration of iron in the standard solution, and wherein the biological sample develops a sample color based on concentration of unbound iron present in the biological sample;
whereby ACS is detected based on the concentration of unbound iron present in the biological sample, wherein the concentration of unbound iron is determined by comparing the sample color with the at least one standard color corresponding to the set of standard solutions.
22. The kit of claim 21, wherein at least one standard solutions one of the set of standard solutions has a concentration of iron between zero μιηοΙ/L to 10,000 μιηοΙ/L.
23. The kit of claim 22, wherein the at least one standard solution is prepared by dissolving a predetermined amount of Ferric Chloride in a predetermined amount of the at least one solvent to obtain concentration of iron between zero μηιοΐε/ΐ, to 10,000 μη οΙ/L in the at least one standard solution.
24. The kit of claim 23, wherein the at least one solvent comprises at least one of a Hydrochloric acid and water.
25. The kit of claim 21, wherein the set of reagents comprises DNA, Magnesium Chloride, Ascorbic Acid, EDTA, Thiobarbituric acid, Bleomycin Sulfate, Hydrochloric acid and water.
26. The kit of claim 21, wherein the chelating resin is Chelex 100.
27. The kit of claim 21, wherein the biological sample comprises- at least one of blood, serum, plasma, urine, saliva, mucus secretion, synovial fluid, and cerebrospinal fluid obtained from a human body.
PCT/IN2010/000028 2009-11-25 2010-01-18 A method and a kit for detection of acute coronary syndrome based on concentration of unbound iron present in a biological sample WO2011064788A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113252660A (en) * 2021-05-13 2021-08-13 中航金属材料理化检测科技有限公司 Method for analyzing chemical component content of high-temperature alloy corrosive liquid

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009109982A1 (en) * 2008-03-05 2009-09-11 Mohan Rajapurkar Methods and kit for early detection of acute coronary syndrome and prediction of adverse cardiac events

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009109982A1 (en) * 2008-03-05 2009-09-11 Mohan Rajapurkar Methods and kit for early detection of acute coronary syndrome and prediction of adverse cardiac events

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LELE S ET AL.: "Serum catalytic iron as a novel biomarker of vascular injury in acute coronary syndromes.", EUROLNTERVENTION., vol. 5, no. 3, August 2009 (2009-08-01), pages 336 - 42 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113252660A (en) * 2021-05-13 2021-08-13 中航金属材料理化检测科技有限公司 Method for analyzing chemical component content of high-temperature alloy corrosive liquid

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