EP2158491A2 - Anticorps et procedes ameliores de manipulation d'un echantillon test destines a etre utilises dans des dosages pour myeloperoxidase - Google Patents

Anticorps et procedes ameliores de manipulation d'un echantillon test destines a etre utilises dans des dosages pour myeloperoxidase

Info

Publication number
EP2158491A2
EP2158491A2 EP08827464A EP08827464A EP2158491A2 EP 2158491 A2 EP2158491 A2 EP 2158491A2 EP 08827464 A EP08827464 A EP 08827464A EP 08827464 A EP08827464 A EP 08827464A EP 2158491 A2 EP2158491 A2 EP 2158491A2
Authority
EP
European Patent Office
Prior art keywords
mpo
test sample
antibody
concentration
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08827464A
Other languages
German (de)
English (en)
Other versions
EP2158491A4 (fr
Inventor
Saul A. Datwyler
David J. Hawksworth
Stephen C. Hsu
Matthew S. Matias
David P. Pacenti
Mary S. Pinkus
Jessie W. Shih
Bryan C. Tieman
Joan D. Tyner
Robert N. Ziemann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US11/750,507 external-priority patent/US20080286818A1/en
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of EP2158491A2 publication Critical patent/EP2158491A2/fr
Publication of EP2158491A4 publication Critical patent/EP2158491A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present disclosure relates to isolated antibodies that can be used in an assay to determine the concentration levels of myeloperoxidase (MPO) in a test sample.
  • MPO myeloperoxidase
  • the present disclosure further relates to an assay for determining the concentration levels of MPO in a test sample using one or more of the isolated antibodies of the present disclosure.
  • the present disclosure also relates to the use of improved test sample handling methods in assays in order to preserve the original MPO levels in the test sample.
  • the present disclosure relates to an improved assay which employs a test sample wherein the original MPO levels in the a test sample have been preserved to allow for the accurate detection of MPO levels in said assay.
  • assays are used for detecting analytes in biological samples.
  • analytes that can be detected include, drugs, hormones, infectious agents, microorganisms, antibodies and the like.
  • the identification of one or more analytes in a biological sample can be used to diagnose cancer, heart disease, etc.
  • a specific type of assay involves a specific binding reaction between the analyte to be detected and at least one specific binding partner.
  • the specific binding partner (which can be an antibody, antigen, etc) specifically binds to the analyte or reacts with it.
  • the analyte and the specific binding partner form a specific binding pair complex.
  • An example of such a specific binding pair complex is an antibody (or antibody fragment) and antigen.
  • more than one analyte or more than one specific binding partner can react with each other during each reaction.
  • the specific binding pair complex can then be detected.
  • at least one specific binding partner is labeled with a detectable label such as a chromogen, fluorophore, substances capable of chemi- or electrochemiluminescence, radioisotopes, haptens, enzymes labels or substances that can form another specific binding pair such as biotin and streptavidin.
  • a detectable label such as a chromogen, fluorophore, substances capable of chemi- or electrochemiluminescence, radioisotopes, haptens, enzymes labels or substances that can form another specific binding pair such as biotin and streptavidin.
  • the specificity and sensitivity of the antibodies used in such immunoassays is very important. If one or more antibodies used in an immunoassay exhibits poor specificity or sensitivity this could lead to false positive or false negative results.
  • One way in which to improve or increase the specificity and sensitivity of one or more antibodies is to improve the binding affinity of said antibodies for their intended target (i.e., an antigen). Antibodies having an improved binding affinity for their intended targets should exhibit increased specificity and sensitivity.
  • MPO myeloperoxidase
  • ACS acute cardiac syndrome
  • MPO donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7
  • pi 10 oxidoreductase
  • MPO is a tetrameric, heavily glycosylated, basic (pi 10) heme protein of approximately 150 kDa. It is comprised of two identical disulfide- linked protomers, each of which possesses a protoporphyrin-containing 59-64 kDa heavy subunit and a 14 kDa light subunit (See, Nauseef, W. M, et al, Blood, 67:1504-1507 (1986)).
  • MPO is abundant in neutrophils and monocytes, accounting for 5% and 1 to 2%, respectively, of the dry weight of these cells (See, Nauseef, W. M, et al., Blood 67: 1504-1507 (1986)).
  • the heme protein is stored in primary azurophilic granules of leukocytes and secreted into both the extracellular milieu and the phago lysosomal compartment following phagocyte activation by a variety of agonists (See, Klebanoff, S. J, et al., The Neutrophil: Functions and Clinical Disorders. Amsterdam: Elsevier Scientific Publishing Co. (1978)).
  • Increased concentrations of plasma MPO levels determined in patient blood samples have been shown to be linked with coronary disease.
  • increased concentrations of blood MPO levels can also be used to predict risk in patients with acute coronary syndromes, including, but not limited to, heart failure (See, Tang, W.H. et al, J. Am. College Card., 49(24): 2364-2370 (2007); Tang, W.H., et al., Am. J. Card., 98:796- 799 (2006); Zhang, R., et al., JAMA, 286(17):2136-2142 (2001); Meuwese, M.C., et al., J. Am. College Card, 50(2): 159-165 (2007); G. Ndrepepa, et al., Eur. J. CHn.
  • MPO MPO is known to be present in leukocytes as well as free in the plasma.
  • current pre-analytical patient blood sample handling does not involve any specific steps aimed at preservation of original MPO levels. Rather, current MPO assays follow the blood storage procedures used with troponin measurements, namely collecting samples as lithium heparin plasma or serum.
  • the CardioMPOTM a trademark of PrognostiX, Inc. (Cleveland, Ohio)
  • the CardioMPOTM assay is an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the CardioMPOTM package insert states that the patient blood sample "should be stored in lithium heparin collection tubes", and that the tubes should be placed on ice or at 2 to 8°C immediately and then stored at 2 to 8°C until processed (See, CardioMPOTM package insert at page 7).
  • MPO levels may also increase if anticoagulant collection tubes (such as lithium heparin tubes) are used as part of the sample handling process. Additionally, the original MPO levels may increase in those patients being treated prior to blood draw with an anticoagulant (such as heparin or bivalirudin).
  • the current blood sample handling procedures for MPO do not take into account the reality that most blood samples will be exposed to at least some time at room temperature storage.
  • the room temperature storage may also be for an extended time. Exposure to room temperature conditions can occur at any point before the analysis. Transportation of the sample to the laboratory from the location of blood draw (such as
  • Leukocyte MPO can be released into the plasma depending on how the sample is handled and the specimen tube used to collect the sample.
  • the inventors have determined that pre-analytical sample handling methods currently used in advance of a MPO assay allows MPO to leak out of the leukocytes during clotting of serum and preparation of lithium heparin plasma, which causes an elevation in MPO levels which can lead to inaccurate stratification of ACS patients, and thus consequently, the wrong treatment selection.
  • one object of the present disclosure is to provide antibodies capable of binding to complimentary epitopes on MPO in an assay. These antibodies exhibit good binding affinity and can be used in assays, such as, for example, immunoassays.
  • Another object of the present disclosure is to provide for use with MPO assays methods for patient test sample handling specifically intended to preserve original patient MPO levels for accurate assay.
  • the present disclosure relates to a murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437.
  • the present disclosure relates to an antibody made from DNA extracted from murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437.
  • the present disclosure relates to a monoclonal antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA- 8437. In a yet another embodiment, the present disclosure relates to a murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438.
  • the present disclosure relates to an antibody made from DNA extracted from murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438. In yet another embodiment, the present disclosure relates to a monoclonal antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438.
  • the present disclosure relates to an immunoassay for determining the concentration of MPO in a test sample.
  • the immunoassay comprises the steps of:
  • step (c) determining the amount of the capture antibody-MPO-detection complexes formed in step (b) by detecting the detectable label, wherein the amount of the second complexes formed is the amount of MPO contained in the test sample, wherein either the first capture antibody or the second antibody is an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-
  • the first capture antibody can optionally be immobilized on a solid phase to produce an immobilized antibody.
  • the present disclosure relates to an immunoassay for determining the concentration of MPO in a test sample.
  • the immunoassay comprises the steps of:
  • step (c) determining the amount of the capture antibody-MPO-detection complexes formed in step (b) by detecting the detectable label, wherein the amount of the second complexes formed is the amount of MPO contained in the test sample, wherein either the first capture antibody or the second antibody is an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-
  • the first capture antibody can optionally be immobilized on a solid phase to produce an immobilized antibody.
  • the present disclosure relates to an immunoassay for determining the concentration of MPO in a test sample.
  • the immunoassay comprising the steps of:
  • first capture antibody (a) contacting a first capture antibody that binds to MPO with a test sample suspected of containing MPO to form a first capture antibody- MPO complex, wherein the first capture antibody is an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437 or an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438;
  • the first capture antibody can optionally be immobilized on a solid phase to produce an immobilized antibody.
  • the first capture antibody is an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437 and the second antibody is an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438.
  • the present disclosure relates to a kit for use in an immunoassay.
  • the kit for use in the immunoassay contains an antibody produced by murine hybridoma cell line 1-1175-509 and instructions for using said kit.
  • the present disclosure relates to a kit for use in an immunoassay.
  • the kit for use in the immunoassay contains an antibody produced by murine hybridoma cell line 1-2169-715 and instructions for using said kit.
  • the present disclosure relates to a kit for use in an immunoassay.
  • the kit for use in the immunoassay contains an antibody produced by murine hybridoma cell line 1-1175-509, an antibody produced by murine hybridoma cell line 1-2169-715 and instructions for using said kit.
  • the present disclosure relates to a method for determining concentration of myeloperoxidase (MPO) in a test sample.
  • the method comprises the steps of:
  • the MPO secretion inhibitor can be a salt of ethylene diamine tetraacetic acid.
  • the test sample used in the above method can be whole blood or a plasma sample.
  • Step (b) in the above method can be performed using a method selected from the group consisting of: a competitive immunoassay, a sandwich immunoassay, an enzyme- linked immunosorbent assay, an enzymatic assay and a clinical chemistry assay.
  • the test sample can be stored at room temperature for a period of time up to about 8 hours. After storage at room temperature, the test sample can then be further stored at a temperature of from about 2°C to about 8°C for a period of up to about seven (7) days after processing.
  • the MPO secretion inhibitor can comprise a salt of citrate and the test sample can be stored at a temperature of from about 2°C to about 8°C for a period of up to about eight (8) hours prior to processing.
  • said test sample can still further be stored at a temperature of from about 2°C to about 8°C for a period of up to about seven (7) days after processing.
  • the present disclosure relates to an improved method for determining the concentration of MPO in a human blood sample. Specifically, the improvement in this method comprises storing said sample in a sample collection tube containing a MPO secretion inhibitor at room temperature for a period of up to about 8 hours.
  • the MPO secretion inhibitor can be a salt of ethylene diamine tetraacetic acid.
  • the human blood sample used in the above method can be whole blood or a plasma sample.
  • the concentration of MPO in the human blood sample can be determined by performing a method selected from the group consisting of: a competitive immunoassay, a sandwich immunoassay, an enzyme-linked immunosorbent assay, an enzymatic assay and a clinical chemistry assay. Additionally, after storage of the human blood sample at room temperature, the sample can then be further stored at a temperature of from about 2°C to about 8°C for a period up of from up to about seven (7) days after processing.
  • the present disclosure relates to an improved method for determining the concentration of MPO in a human blood sample.
  • the improvement in this method comprises storing said sample in a sample collection tube containing a salt of citrate at a temperature of from about 2°C to about 8°C for a period of up to eight (8) hours prior to processing.
  • said test sample can still further be stored at a temperature of from about 2°C to about 8°C for a period of up to about seven (7) days after processing.
  • the present disclosure relates to a kit.
  • the kit can comprise:
  • the present disclosure relates to an immunoassay for determining the concentration of MPO in a human peripheral blood sample.
  • the immunoassay comprises the steps of:
  • first capture antibody that binds to MPO with the human peripheral blood sample stored in a sample collection tube containing a MPO secretion inhibitor to form a first capture antibody-MPO complex
  • the first capture antibody is an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437 or an antibody produced by murine hybridoma cell line 1-2169- 715 having A.T.C.C. Accession No. PTA-8438;
  • step (d) determining the amount of the capture antibody-MPO-detection complexes formed in step (b) by detecting the detectable label, wherein the amount of the second complexes formed is the amount of MPO contained in the human peripheral blood sample.
  • the first capture antibody can be immobilized on a solid phase to produce an immobilized antibody.
  • the first capture antibody can be an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437 and the second antibody can be an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA- 8438.
  • the MPO secretion inhibitor can be a salt of ethylene diamine tetraacetic acid.
  • the test sample can be whole blood or a plasma sample.
  • the test sample can be stored at room temperature for a period of time up to about 8 hours. After storage at room temperature, the test sample can be further stored at a temperature of from about 2°C to about 8 0 C for a period up to about seven (7) days after processing.
  • the test sample can be stored at room temperature for a period of time up to about 8 hours. After storage at room temperature, the test sample can be further stored at a temperature of from about 2°C to about 8 0 C for a period up to about seven (7) days after processing.
  • the test sample can be stored at room temperature for a period of time up to about 8 hours.
  • the test sample can be further stored at a temperature of from about 2°C to about 8 0 C for a period up to about seven (7) days after processing.
  • MPO secretion inhibitor can comprise a salt of citrate and the test sample can be stored at a temperature of from about 2°C to about 8 0 C for a period of up to about eight (8) hours prior to processing.
  • said test sample can still further be stored at a temperature of from about 2°C to about 8 0 C for a period of up to about seven (7) days after processing.
  • the present disclosure relates to a method of determining whether or not a subject is at risk of developing cardiovascular disease. Specifically, such a method can comprise the steps of:
  • step (c) comparing the concentration of MPO in the test sample determined in step (b) with a predetermined level, wherein if the concentration of MPO determined in step (b) is lower than the predetermined level, then the subject is considered not to be at risk of developing cardiovascular disease and further wherein, if the concentration of MPO in the test sample determined in step (b) is higher then the predetermined level, then the subject is considered to be at risk of developing cardiovascular disease.
  • the concentration of MPO in the test sample in step (b) can be determined using any of the previously described methods (e.g., methods that use an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession
  • the present disclosure relates to a method of diagnosing cardiovascular disease in a subject.
  • the method can comprise the steps of: (a) providing a test sample stored in a sample collection tube containing a MPO secretion inhibitor;
  • the concentration of MPO in the test sample in step (b) can be determined using any of the previously described methods (e.g., methods that use an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No.
  • the present disclosure relates to a method of monitoring the severity of cardiovascular disease in a subject.
  • the method comprises the steps of:
  • step (b) determining the concentration of MPO in the test sample; and (c) comparing the concentration of MPO in the test sample determined in step (b) with a predetermined level, wherein if the concentration of MPO determined in step (b) is lower than the predetermined level, the subject is determined to have a reduced severity of cardiovascular disease and further wherein if the concentration of MPO in the test sample determined in step (b) is higher than the predetermined level, the subject is determined to have an increased severity of cardiovascular disease.
  • the cardiovascular disease is coronary artery disease, peripheral vascular disease, hypertension, myocardial infarction or heart failure.
  • concentration of MPO in the test sample in step (b) can be determined using any of the previously described methods (e.g., methods that use an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437, an antibody produced by murine hybridoma cell line 1-2169- 715 having AT. C. C. Accession No. PTA-8438 or both of these antibodies).
  • the present disclosure relates to a method of monitoring the progression of cardiovascular disease in a subject. This method comprises the steps of:
  • step (b) determining the concentration of MPO in the test sample; and (c) comparing the concentration of MPO in the test sample determined in step (b) with a predetermined level, wherein if the concentration of MPO determined in step (b) is lower than the predetermined level, the cardiovascular disease in the subject is determined not to have progressed or to that the subject has improved and if the concentration of MPO in the test sample determined in step (b) is higher than the predetermined level, the cardiovascular disease in the subject is determined to have progressed.
  • the concentration of MPO in the test sample in step (b) can be determined using any of the previously described methods (e.g., methods that use an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437, an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438 or both of these antibodies).
  • the present disclosure relates to a method of determining if a subject has suffered a cardiovascular complication as a result of administration to said subject of one or more pharmaceutical compositions.
  • the method comprises the steps of: (a) obtaining a first test sample from the subject before the subject has been administered one or more pharmaceutical compositions and storing said first test sample in a sample collection tube containing a MPO secretion inhibitor;
  • step (d) determining the concentration of MPO in said second test sample; and (e) comparing the concentration of MPO in step (b) with the concentration of MPO in step (d), wherein if the concentration of MPO determined in step (b) is unchanged when compared to the concentration of MPO determined in step (d), then the subject is determined not to have suffered a cardiovascular complication as a result of the administration of one or more pharmaceutical compositions and further wherein if the concentration of MPO determined in step (b) is changed when compared to the concentration of MPO in step (d), then the subject is determined to have suffered a cardiovascular complication as a result of the administration of one or more pharmaceutical compositions.
  • the concentration of MPO in the test sample in step (b), step (d) or in both steps (b) and (d) can be determined using any of the previously described methods (e.g., methods that use an antibody produced by murine hybridoma cell line 1- 1175-509 having A.T.C.C. Accession No. PTA-8437, an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438 or both of these antibodies).
  • the present disclosure relates to a method of monitoring MPO levels in a subject receiving treatment with one or more pharmaceutical compositions. The method comprises the steps of:
  • test sample is stored in a sample collection tube containing a MPO secretion inhibitor
  • step (c) comparing the concentration of MPO determined in step (b) with a predetermined level; (d) treating the subject with one or more pharmaceutical compositions for a period of time if the comparison of the concentration of MPO determined in step (c) is that the concentration of MPO in the first test sample is greater than the predetermined level;
  • test samples are stored in a sample collection tube containing a MPO secretion inhibitor
  • step (g) compare the concentrations of MPO determined in step (f) with the concentration of MPO determined in step (b), wherein if the concentrations of MPO determined in step (f) decrease when compared to the concentration of MPO determined in step (b), then the subject should continue to be administered the one or pharmaceutical compositions of step (d), further wherein, if the concentrations of MPO determined in step (f) are the same or increase when compared to the concentration of MPO determined in step (b), then the subject should be treated with a higher concentration of the one or more pharmaceutical compositions administered to the subject in step (d) or the the subject should be treated with one or more pharmaceutical compositions that are different then the one or more pharmaceutical compositions administered to the subject in step (d).
  • the concentration of MPO in the test sample in step (b), step (f) or in both steps (b) and (f) can be determined using any of the previously described methods (e.g., methods that use an antibody produced by murine hybridoma cell line 1- 1175-509 having A.T.C.C. Accession No. PTA-8437, an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438 or both of these antibodies).
  • Figure 1 shows an antigen titration curve with the antibody concentration held at 0.3125 ⁇ g/mL generated as described in Example 9.
  • -•- represents monoclonal antibody 1-1175-154
  • - represents monoclonal antibody 1-1519-450
  • - ⁇ - represents monoclonal antibody 1-2169-143
  • - ⁇ - represents monoclonal antibody 1- 2244-166.
  • Figure 2 shows an antigen titration curve of the binding of various MPO binding pairs in an immunoassay as described in Example 9.
  • -x- represents the use of monoclonal antibody 1-1175-154 as the capture antibody and monoclonal antibody 1-1519-450 as the conjugate antibody
  • -•- represents the use of monoclonal antibody 1- 1519-450 as the capture antibody and monoclonal antibody 1-1175-154 as the conjugate antibody
  • -*- represents the use of monoclonal antibody 1-1175-154 as the capture antibody and monoclonal antibody 1-2244-166 as the conjugate antibody
  • — represents the use of monoclonal antibody 1-2169-143 as the capture antibody and monoclonal antibody 1-1175-154 as the conjugate antibody.
  • MPO MPO assays
  • immunoassays such as, for example, immunoassays
  • present disclosure also relates to an assay for determining the amount or concentration of MPO in a test sample using one or more of the antibodies of the present disclosure.
  • present disclosure also provides improved test sample handling methods that can be used to preserve original MPO levels in a test sample, thus allowing for a more accurate assessment of MPO levels and thus consequently, more accurate patient stratification and treatment selection.
  • the present disclosure relates to an improved assay which employs a test sample wherein the original MPO levels in the a test sample have been preserved to allow for the accurate detection of MPO levels in said assay.
  • antibody refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies (such as, but not limited to, a bird (for example, a duck or goose), a shark or whale, a mammal, including a non-primate (for example, a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, mouse, etc) or a non-human primate (for example, a monkey, such as a cynomologous monkey, a chimpanzee, etc), recombinant antibodies, chimeric antibodies, single-chain Fvs ("scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs ("sdFv”)
  • scFv single-chain Fvs
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an antigen binding site.
  • Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2 ) or subclass.
  • An antibody whose affinity (namely, K D , kj or ka) has been increased or improved via the screening of a combinatory antibody library that has been prepared using bio-display, is referred to herein as an "affinity maturated antibody”.
  • an antibody against an analyte is frequently referred to herein as being either an "anti-analyte antibody", or merely an “analyte antibody” (e.g., an MPO antibody or an anti-MPO antibody).
  • an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody”, or merely an “analyte antibody” (e.g., an MPO antibody or an anti-MPO antibody).
  • association rate constant refers to the value indicating the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen as shown by the equation below:
  • Ab Antibody + Antigen
  • Ag Antigen
  • dissociation rate constant refers to the value indicating the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free antibody and antigen as shown by the equation below: Ab + Ag ⁇ Ab-Ag.
  • association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho) can also be used.
  • BIAcore® biological interaction analysis
  • KinExA® KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho) can also be used.
  • the association rate, the dissociation rate and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen.
  • blood or blood sample refers to a whole blood sample, serum or a plasma fraction derived therefrom.
  • blood or blood sample is peripheral blood or a plasma fraction derived therefrom.
  • the blood or blood sample is a human peripheral blood or plasma fraction derived therefrom.
  • cardiovascular disease refers to various clinical diseases, disorders or conditions involving the heart, blood vessels or circulation. The diseases, disorders or conditions may be due to atherosclerotic impairment of coronary, cerebral or peripheral arteries.
  • Cardiovascular disease includes, but is not limited to, coronary artery disease, peripheral vascular disease, hypertension, myocardial infarction, heart failure, etc.
  • increased severity refers to the worsening of disease as indicated by increased NYHA classification, to, for example, Class III or Class IV and "reduced severity” of cardiovascular disease refers to an improvement of the disease as indicated by reduced NYHA classification, from, for example, class III or IV to class II or I.
  • reduced severity refers to an improvement of the disease as indicated by reduced NYHA classification, from, for example, class III or IV to class II or I.
  • epitope refers to sites or fragments of a polypeptide or protein having antigenic or immunogenic activity in a subject.
  • An epitope having immunogenic activity is a site or fragment of a polypeptide or protein that elicits an antibody response in an animal.
  • An epitope having antigenic activity is a site or fragment of a polypeptide or protein to which an antibody immunospecifically binds as determined by any method well-known to those skilled in the art, for example by immunoassays. f) Heart Failure
  • heart failure refers to a condition in which the heart cannot pump blood efficiently to the rest of the body.
  • Heart failure may be due to damage to the heart or narrowing of the arteries due to infarction, cardiomyopathy (primary or secondary), hypertension, coronary artery disease, valve disease, birth defects or infection.
  • Heart failure can further be described as chronic, congestive, acute, decompensated, systolic or diastolic.
  • the New York Heart Association (NYHA) classification describes the severity of the disease based on functional capacity of the patient; NYHA class can progress and/or regress based on treatment or lack of response to treatment.
  • humanized antibody refers to an immunoglobulin variant or fragment thereof, which is capable of binding to a predetermined antigen and which comprises framework regions having substantially the amino acid sequence of a human immunoglobulin and CDRs having substantially the amino acid sequence of a non- human immunoglobulin.
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human.
  • the humanized antibody will include substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGi, IgG 2 , IgG 3 and IgG 4 .
  • the humanized antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within those skilled in the art h) MPO Hybridoma
  • MPO hybridoma refers to a particular hybridoma clone or subclone (as specified) that produces an anti-MPO antibody of interest.
  • hybridoma clone or subclone (as specified) that produces an anti-MPO antibody of interest.
  • affinity of antibodies produced by a hybridoma clone as compared to those from a subclone of the same type, e.g., reflecting purity of the clone.
  • all hybridoma subclones originating from the same clone and further, that produce the anti-MPO antibody of interest produce antibodies of identical sequence and/or identical structure.
  • composition refers to any agent or drug, whether a small molecule (e.g., a drug containing an active agent, typically a non-peptidic) or biologic (e.g., a peptide or protein based drug, including any with modifications, such as, but not limited to pegylation) that can be used to treat a subject suffering from a disease or condition that requires treatment.
  • a small molecule e.g., a drug containing an active agent, typically a non-peptidic
  • biologic e.g., a peptide or protein based drug, including any with modifications, such as, but not limited to pegylation
  • compositions include, but are not limited to, antineoplastics (chemotherapeutics), antidepressants (e.g., tricyclic antidepressants), multiple sclerosis drugs, anesthetics, interferons, hormones, HIV-antiviral drugs, hyperlipidemia drugs (including, but not limited to, niacin, f ⁇ brates (e.g., clof ⁇ brate, fenofibrate, fenofibric acid, simfrate, salts of fenofibric acid and any combinations thereof), ezetimibe, HMG-CoA reductase inhibitors (e.g., statins, such as, but not limited to rosuvastatin, simvastatin, and combinations thereof (including combinations with other hyperlipidemia drugs (e.g., simvastatin and ezetimibe)), anti-inflammatories, etc.
  • chemotherapeutics chemotherapeutics
  • antidepressants e.g.
  • predetermined level refers generally at an assay cutoff value that is used to assess diagnostic results by comparing the assay results against the predetermined level, and where the predetermined level already that has been linked or associated with various clinical parameters (e.g., severity of disease, progression/nonprogression/improvement, etc.).
  • various clinical parameters e.g., severity of disease, progression/nonprogression/improvement, etc.
  • the present disclosure provides exemplary predetermined levels, and describes the initial linkage or association of such levels with clinical parameters for exemplary immunoassays as described herein.
  • cutoff values may vary dependent on the nature of the immunoassay (e.g., antibodies employed, etc.).
  • sample collection tube or “sample tube”
  • sample collection tube refers to any type of container used to collect and store a test sample, such as a blood sample, for shipment to an analytical processing site.
  • Sample collection tubes can be made with any suitable material known in the art (e.g., plastic or glass).
  • the sample collection tube can be made of a suitable plastic material that is non-reactive with the stabilizing agents and does not interfere with the test sample.
  • Preferred plastic material include any type of polyethylene terepththlate (PET) or polypropylene.
  • PET polyethylene terepththlate
  • An example of plastic sample collection tube are the sample collection tubes known as VACUETTE ®, available from Greiner Bio-One GmbH (Kremsmunster, Austria) and BD VACUT AINER® available from BD (Becton, Dickinson and Company, Franklin Lakes, NJ). Particularly preferred are the plastic EDTA-containing VACUT AINER® tubes (BD, Franklin Lakes, N.
  • sample collection tubes can also contain other chelators that function similar to EDTA, such as, but not limited to, ethylene glycol tetraacetic acid (EGTA) or glycol ether diamine tetraacetic acid, nitrilotriacetic acid (NTA) or diethylenetriaminepentaacetic acid (DTPA).
  • EGTA ethylene glycol tetraacetic acid
  • NTA nitrilotriacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • the sample collection tube can be made of glass or siliconized glass.
  • Sample collection tubes can be made by any process known in the art, such as, for example, injection molding.
  • the sample tube can be of any design, including nested designs, such as described in U.S. 6,910,597, M. Iskra, "Collection Container Assembly".
  • the sample collection tubes may be coated with a clot activator (such as, for example, silicon and/or micronized silica particles). In addition to the clot activator, these tubes may also contain a gel. Alternatively, the sample collection tube may contain a gel and other materials (such as lithium heparin) to facilitate the processing of the test sample.
  • sample collection tubes are the BD VACUT AINER® Serum Tubes, the BD VACUT AINER® SSTTM tubes (which are plastic tubes that contain a gel and a clot activator ) and BD VACUT AINER® PSTTM tubes (which are plastic tubes that contain a gel and lithium heparin).
  • the terms “subject” and “patient” are used interchangeably.
  • the terms “subject” and “subjects” refer to an animal, in one aspect, a bird (for example, a duck or goose), in another aspect, a shark or whale, or in a further aspect, a mammal including, a non-primate (for example, a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse) and a primate (for example, a monkey, such as a cynomolgous monkey, chimpanzee, and a human).
  • a non-primate for example, a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse
  • a primate for example, a monkey, such as a cynomolgous monkey, chimpanze
  • test sample generally refers to a biological material being tested for and/or suspected of containing an analyte of interest.
  • the test sample may be derived from any biological source, such as, a physiological fluid, including, but not limited to, whole blood (hemolyzed or unhemolyzed), serum, plasma, red blood cells (erythrocytes), white blood cells (leukocytes including granulocytes such as neutrophils, eosinophils and basophils, and including lymphoid cells such as lymphocytes and monocytes), and other blood cells or forms of blood (e.g., platelets, lymph), interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen and so forth.
  • a physiological fluid including, but not limited to, whole blood (hemolyzed or unhemolyze
  • the test sample may be tested immediately following its collection (i.e., fresh) or following some period of storage under appropriate storage conditions.
  • the test sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample.
  • pretreatment may include preparing plasma from blood, diluting viscous fluids and so forth.
  • Methods of pretreatment may also involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, etc.
  • MPO Antibodies The present disclosure provides antibodies that specifically bind to MPO. More specifically, the inventors have discovered antibodies that bind to distinct epitope groupings on MPO. In fact, the inventors of the present disclosure have discovered that human MPO contains at least eight (8) distinct epitope groups.
  • the present disclosure provides for isolated antibodies that bind to one distinct epitope group on MPO, referred to herein as epitope group number 3.
  • the present disclosure relates to murine hybridoma cell line 1- 1175-509 (also referred to as "MPO 1-1175-509") having A.T.C.C. Accession No. PTA- 8437, deposited on May 16, 2007.
  • the present disclosure relates to an antibody made from DNA extracted from murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437.
  • the present disclosure relates to an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437, deposited on May.
  • the antibody produced by murine hybridoma cell line 1-1175-509 can bind to epitope group number 3 on MPO.
  • the present disclosure provides for isolated antibodies that bind to one distinct epitope group on MPO, referred to herein as epitope group number 5.
  • the present disclosure relates to murine hybridoma cell line 1- 2169-715 (also referred to as "MPO 1-2169-715”) having A.T.C.C. Accession No. PTA- 8438, deposited on May 16, 2007.
  • the present disclosure relates to antibody made from DNA extracted from murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No.
  • the present disclosure relates to an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438, deposited on May.
  • the antibody produced by murine hybridoma cell line 1-2169-715 can bind to epitope group number 5 on MPO.
  • polyclonal and monoclonal antibodies against MPO can be raised by immunizing a suitable subject (such as, but not limited to, a rabbit, goat, mouse or other mammal) with an immunogenic preparation which contains a suitable immunogen, such as purified MPO antigen.
  • a suitable immuogen can be MPO purified from human neutrophils, which is commercially available from Athens Research & Technology (Athens, GA).
  • the antibodies raised in the subject can then be screened to determine if the antibodies bind to MPO.
  • Such antibodies can be further screened using the methods described herein (See, Example 9). For example, these antibodies can be assayed to determine if they bind to epitope group number 3 or epitope group number 5. Suitable methods to identify an antibody with the desired characteristics are described herein (See, Example 10).
  • the unit dose of immunogen namely, the purified protein, or recombinantly produced human MPO protein
  • the immunization regimen will depend upon the subject to be immunized, its immune status, and the body weight of the subject.
  • an immunogen can be administered with an adjuvant, such as Freund's complete or incomplete adjuvant, Ribi's adjuvant or any combinations thereof.
  • Immunization of a subject with an immunogen as described above induces a polyclonal antibody response.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques such as an ELISA using an immobilized antigen, namely, MPO.
  • Human monoclonal antibodies can be produced by introducing an antigen into immune deficient mice that have been engrafted with human antibody-producing cells or tissues (for example, human bone marrow cells, peripheral blood lymphocytes (PBL), human fetal lymph node tissue, or hematopoietic stem cells).
  • human antibody-producing cells or tissues for example, human bone marrow cells, peripheral blood lymphocytes (PBL), human fetal lymph node tissue, or hematopoietic stem cells.
  • Such methods include raising antibodies in SCID-hu mice (See, for example, WO 93/05796, U.S. Patent No. 5,411,749; or McCune et al, Science, 241 :1632-1639 (1988)) or Rag-l/Rag-2 deficient mice.
  • Human antibody-immune deficient mice are also commercially available. For example, Rag-2 deficient mice are available from Taconic Farms (Germantown, NY).
  • Monoclonal antibodies can be generated by immunizing a subject with an immunogen.
  • antibody producing cells can be harvested from an immunized animal and used to prepare monoclonal antibodies using standard techniques.
  • the antibody producing cells can be fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • Such techniques are well known in the art, and include, for example, the hybridoma technique as originally developed by Kohler and Milstein, Nature, 256:495-497 (1975)), the human B cell hybridoma technique (Kozbar et al, Immunology Today, 4:72 (1983)), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal
  • Monoclonal antibodies can also be made by harvesting antibody producing cells, for example, splenocytes, from transgenic mice expressing human immunoglobulin genes and which have been immunized with MPO.
  • the splenocytes can be immortalized through fusion with human myelomas or through transformation with Epstein-Barr virus (EBV).
  • EBV Epstein-Barr virus
  • hybridomas can be made using human B cell-or EBV-hybridoma techniques described in the art (See, for example, Boyle et al., European Patent Publication No. 0 614 984).
  • MPO are detected by screening the hybridoma culture supernatants by, for example, screening to select antibodies that specifically bind to the immobilized MPO, or by testing the antibodies as described herein to determine if the antibodies have the desired characteristics, namely, the ability to bind to MPO at the unique epitope groups (namely, MPO epitope group 3 or 5) described herein.
  • the clones may be subcloned, e.g., by limiting dilution procedures, for example the procedure described by Wands et al. (Gastroenterology 80:225-232 (1981)), and grown by standard methods.
  • Hybridoma cells that produce monoclonal antibodies that test positive in the screening assays described herein can be cultured in a nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium, to thereby produce whole antibodies.
  • Tissue culture techniques and culture media suitable for hybridoma cells are generally described in the art (See, for example, R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980)).
  • Conditioned hybridoma culture supernatant containing the antibody can then be collected.
  • the monoclonal antibodies secreted by the subclones optionally can be isolated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Monoclonal antibodies can be engineered by constructing a recombinant combinatorial immunoglobulin library and screening the library with the MPO.
  • Kits for generating and screening phage display libraries are commercially available (See, for example, the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612).
  • yeast display vectors are known in the art and are commercially available (for example, pYDl available from Invitrogen Corp., Carlsbad, CA).
  • the antibody library is screened to identify and isolate phages or yeast cells that express an antibody that specifically binds to MPO.
  • the primary screening of the library involves screening with an immobilized MPO.
  • the display phage or yeast is isolated and the polynucleotide encoding the selected antibody can be recovered from the display phage or yeast (for example, from the phage or yeast genome) and subcloned into other expression vectors (e.g., into Saccharomyces cerevesiae cells, for example EBYlOO cells (Invitrogen Corporation, Carlsbad, CA)) by well known recombinant DNA techniques.
  • the polynucleotide can be further manipulated (for example, linked to nucleic acid encoding additional immunoglobulin domains, such as additional constant regions) and/or expressed in a host cell.
  • recombinant forms of antibodies can also be prepared to minimize the response by a human patient to the antibody.
  • antibodies produced in non-human subjects or derived from expression of non-human antibody genes are used therapeutically in humans, they are recognized to varying degrees as foreign, and an immune response may be generated in the patient.
  • One approach to minimize or eliminate this immune reaction is to produce chimeric antibody derivatives, namely, antibody molecules that combine a non-human animal variable region and a human constant region.
  • Such antibodies retain the epitope binding specificity of the original monoclonal antibody, but may be less immunogenic when administered to humans, and therefore more likely to be tolerated by the patient.
  • Chimeric monoclonal antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the constant region of a non-human antibody molecule is substituted with a gene encoding a human constant region (See, for example, PCT Patent Publication PCT/US86/02269, European Patent Application 184,187 or European Patent Application 171 ,496).
  • a chimeric antibody can be further "humanized” by replacing portions of the variable region not involved in antigen binding with equivalent portions from human variable regions.
  • General reviews of "humanized” chimeric antibodies can be found in Morrison, S. L., Science, 229:1202-1207 (1985) and in Oi et al, BioTechniques, 4-214 (1986). Such methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of an immunoglobulin variable region from at least one of a heavy or light chain.
  • the cDNA encoding the humanized chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector.
  • Suitable "humanized” antibodies can be alternatively produced by complementarity determining region (CDR) substitution (See, for example, U.S. Patent No. 5,225,539; Jones et al., Nature, 321 :552- 525 (1986); Verhoeyan et al., Science 239:1.534 (1988); and Beidler et al., J. Immunol,. 141 :4053-4060 (1988)).
  • CDR complementarity determining region
  • Epitope imprinting can also be used to produce a "human" antibody polypeptide dimer that retains the binding specificity of the antibodies (for example, hamster antibodies) specific for MPO.
  • a gene encoding a non-human variable region (VH) with specific binding to an antigen and a human constant region (CHl) is expressed in E. coli and infected with a phage library of human V ⁇ .C ⁇ genes. Phage displaying antibody fragments are then screened for binding to MPO. Selected human V ⁇ genes are recloned for expression of V ⁇ .C ⁇ . chains and E. coli harboring these chains are infected with a phage library of human VHCHl genes and the library is subject to rounds of screening with antigen coated tubes (See, WO 93/06213).
  • the present disclosure contemplates that the antibody is an antibody fragment.
  • the antibody fragment can include, but is not limited to, a Fab, a Fab', a Fab'-SH fragment, a di-sulfide linked Fv, a single chain Fv (scFv) and a
  • F(ab')2 fragment Various techniques are known to those skilled in the art for the production of antibody fragments. For example, such fragments can be derived via proteolytic digestion of intact antibodies (See, for example, Morimoto et al, J. Biochem. Biophys. Methods, 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)) or produced directly by recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (See, Carter et al., Bio /Technology, 10:163-167 (1992)).
  • the F(ab') 2 is formed using the leucine zipper GCN4 to promote assembly of the F(ab')2 molecule.
  • Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell culture.
  • Single chain variable region fragments are made by linking light and/or heavy chain variable regions by using a short linking peptide (See, Bird et al. Science, 242:423-426 (1998)).
  • An example of a linking peptide is GPAKELTPLKEAKVS (SEQ ID NO: 1).
  • Linkers can in turn be modified for additional functions, such as attachment of drugs or attachment to solid supports.
  • linker sequences examples include Bird et al., Science, 2AlAlT)-AIe (1988), Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988) and McCafferty et al., Nature, 348:552-554 (1990).
  • the single chain variants can be produced either recombinantly or synthetically.
  • an automated synthesizer can be used for synthetic production of scFv.
  • a suitable plasmid containing polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli.
  • a suitable host cell either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli.
  • Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides.
  • the resultant scFv can be isolated using standard protein purification techniques known in the art.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (See, for example, Holliger, P., et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); Poljak, R. J., et al., Structure, 2:1121-1123 (1994)).
  • the antibodies of the present disclosure have a variety of uses. More specifically, the antibodies of the present disclosure can be used as one or more capture antibodies, one or more conjugate antibodies or as both one or more capture antibodies and one or more conjugate antibodies in immunoassays to detect the presence of MPO in a test sample.
  • D. MPO Test Sample Collection and Improved Test Sample Handling Methods The present disclosure also provides improved test sample handling methods that can be used to preserve original MPO levels obtained from a collected test sample. Specifically, these improved test sampling methods involve storing a test sample obtained from a subject in a sample collection tube containing a leukocyte MPO secretion inhibitor.
  • the methods involve storing a blood sample (e.g., a peripheral blood sample) obtained from a subject in a sample tube containing a leukocyte MPO secretion inhibitor.
  • a blood sample e.g., a peripheral blood sample
  • a sample tube containing a leukocyte MPO secretion inhibitor.
  • test samples that have had their original MPO levels preserved according to the methods described herein can be used in assays to determine the concentration of MPO in a test sample.
  • determining the concentration of MPO in a test sample involves: (a) providing a test sample obtained from a subject, preferably, a peripheral blood sample, stored in a sample collection tube containing a MPO secretion inhibitor; and (b) determining the concentration of MPO in the test sample.
  • Methods for collecting, handling and processing test samples such as whole blood, serum and plasma, and other body fluids that can be used in the practice of the methods of the present disclosure are well known in the art.
  • a leukocyte MPO secretion inhibitor can be any reagent added to the test sample on or shortly after collection into the sample collection tube (e.g., a plasma collection tube), that inhibits release of MPO from leukocytes present in the test sample (e.g., serum or plasma) particularly, release at room temperature.
  • leukocyte MPO secretion inhibitors include, but are not limited to, salts of EDTA (including, but not limited to sodium or potassium salts) or salts of citrate (including, but not limited to sodium citrate).
  • EDTA salts include, but are not limited to, dipotassium and tripotassium salts.
  • citrate salts are sodium citrate.
  • the amount of leukocyte MPO secretion inhibitor to be added can be determined by one skilled in the art. Specifically, the amounts used in commercially available sample collection tubes for whole blood are acceptable. Examples of such sample collection tubes that can be used include, but are not limited to, EDTA-containing tubes, e.g., plastic EDTA-containing
  • VACUT AINER® tubes such as sold by BD, Franklin Lakes, N. J.
  • lavendar- top tubes tube spray-coated with EDTA (namely, VACUT AINER® K 2 EDTA or K 3 EDTA tubes) and plasma preparation tubes (PPT) with EDTA.
  • PPT plasma preparation tubes
  • sample collection tube After collection of the test sample into a sample collection tube (referred to herein as a "collected test sample") with a leukocyte MPO secretion inhibitor, the sample collection tube may be stored for a period of time not longer than about 8 hours at room temperature.
  • the inventors of the present disclosure have found that the levels of MPO in a collected test sample can be preserved (e.g., maintained) at room temperature for up to about 8 hours in sample collection tubes containing a MPO secretion inhibitor prior to processing. Particularly good results were obtained using sample collection tubes that contained salts of EDTA.
  • test samples in sample collection tubes containing salts of citrate can be stored at other than room temperature, namely at a temperature of from about 2°C to about 8 0 C, for a period of from up to about eight (8) hours prior to processing.
  • the collected test sample can be centrifuged and then placed on ice or stored at a temperature in a range of about 2°C to about 8 0 C, until the collected test sample is processed for MPO determination.
  • a temperature range lower than about 2°C to about 8 0 C this is not preferred and should be avoided to lessen the likelihood the sample will freeze.
  • sample collection tube can be further stored at a temperature in a range of about 2°C to about 8 0 C for a period of up to about seven (7) days after processing (e.g., such as after centrifugation and/or plasma separation).
  • sample collection tubes contain salts of EDTA or salts of citrate.
  • the preservation status of the test sample can be checked, reviewed or verified.
  • the preservation status (namely, the MPO preservation conditions) of the test sample can be checked, reviewed or verified in a number of different ways.
  • the temperature and time at which the test sample has been stored can be assessed to determinine whether or not the preservation conditions are unacceptable for further processing and use of the test sample in an assay.
  • unacceptable preservation conditions would be storage of the test sample in a sample collection tube that does not contain a MPO secretion inhibitor or storage of the test sample in a sample collection tube containing a leukocyte MPO secretion inhibitor at room temperature for a period of over about 8 hours prior to processing.
  • the test sample can be analyzed for the presence of lysis of leukocytes in the test sample.
  • MPO preservation status of a test sample can be automated in an analytical instrument, such as an automated immunoassay analyzer.
  • an automated immunoassay analyzer can be programmed to check on the MPO preservation conditions used for the test sample, and where potentially unacceptable preservation conditions are determined as present, the analyzer can be programmed to send an error message that it is unable to perform the MPO assay, because of the likelihood of inaccurately high results.
  • the present disclosure also relates to assays for determining MPO concentration in a test sample obtained from a subject.
  • Assays contemplated include immunoassays (such as sandwich and competitive immunoassays), clinical chemistry assays and enzymatic assays.
  • the MPO measurement is done using an immunoassay, and more preferably, a sandwich immunoassay, which will be discussed in more detail herein.
  • Assays for determining MPO concentration in a test sample obtained from a subject can comprise the steps of: (a) providing a test sample obtained from a subject; and (b) determining the concentration of MPO in the test sample.
  • the test sample obtained from a subject is a peripheral blood sample.
  • the peripheral blood sample has been stored in a sample collection tube containing a MPO secretion inhibitor.
  • the assay can also comprise an intermediate or additional step. This intermediate or additional step involves checking the preservation conditions in the test sample.
  • This intermediate or additional step of checking, reviewing or verifying the preservation conditions in the test sample must be performed prior to determining the concentration of MPO in the test sample.
  • this intermediate or additional step is particularly advantageous for use with an automated analytical instrument, such as an automated immunoassay analyzer.
  • the analyzer can be programmed to check, review or verify the preservation conditions in the test sample as well as identify unacceptable MPO storage conditions, such as storage of the collected test sample at room temperature for longer than about 8 hours or further storage of the collected test sample at a temperature of or 2°C to 8 0 C for over about 7 days before test sample centrifugation and plasma separation.
  • the analyzer can be programmed to send an error message that it is unable to perform the MPO assay, because of the likelihood of inaccurately high results.
  • the analyzer would proceed to process a test sample stored in a collection tube containing a MPO secretion inhibitor and stored at room temperature for no longer than about 8 hours.
  • Immunoassays can be conducted using any format known in the art, such as, but not limited to, a sandwich format, a competitive inhibition format (including both forward or reverse competitive inhibition assays) or in a fluorescence polarization format. As mentioned above, preferably, the immunoassay is in a sandwich format.
  • At least two antibodies are employed to separate and quantify human MPO in a test sample. More specifically, the at least two antibodies bind to certain epitopes of MPO forming an immune complex which is referred to as a "sandwich". Generally, in the immunoassays one or more antibodies can be used to capture the MPO in the test sample (these antibodies are frequently referred to as a
  • capture antibody or capture antibodies and one or more antibodies can be used to bind a detectable (namely, quantifiable) label to the sandwich (these antibodies are frequently referred to as the “detection antibody”, “detection antibodies”, a “conjugate” or “conjugates”).
  • detection antibody namely, quantifiable
  • detection antibodies a detectable label label to the sandwich
  • conjugates conjugates
  • antibodies should be selected so that the one or more first antibodies brought into contact with a test sample or test sample extract suspected of containing MPO do not bind to all or part of the binding site recognized by the second or subsequent antibodies, thereby interfering with the ability of the one or more second detection antibodies to bind to MPO.
  • Excellent immunoassays can be performed using the antibodies of the present disclosure as the capture antibodies, detection antibodies or as capture and detection antibodies.
  • the antibodies of the present disclosure can be used as a first capture antibody and other commercially available antibodies can be used as the detection antibodies.
  • the antibodies of the present disclosure can be used as a second or subsequent capture antibody.
  • a different antibody other than an antibody of the present disclosure, namely, other commercially available antibodies can be used as a second capture antibody.
  • the antibodies of the present disclosure can be used only as detection antibodies and not as capture antibodies with other commercially available antibodies being used as the capture antibodies. Still in another alternative, the antibodies of the present disclosure can be used as both capture and detection antibodies.
  • an antibody (or an antibody fragment thereof) produced by murine hybridoma cell line 1- 1175-509 can be used as a capture antibody and an antibody (or a antibody fragment thereof) produced by murine hybridoma cell line 1-2169-715 can be used as a detection antibody.
  • an antibody (or an antibody fragment thereof) produced by murine hybridoma cell line 1-2169-715 can be used as a capture antibody and an antibody (or an antibody fragment thereof) produced by hybridoma cell line 1-1175-509 can be used as a detection antibody.
  • the test sample being tested for (for example, suspected of containing) MPO can be contacted with at least one capture antibody (or antibodies) and at least one detection antibody (which is either a second detection antibody or a third detection antibody) either simultaneously or sequentially and in any order.
  • the test sample can be first contacted with at least one capture antibody and then (sequentially) with at least one detection antibody.
  • the test sample can be first contacted with at least one detection antibody and then (sequentially) with at least one capture antibody.
  • the test sample can be contacted simultaneously with a capture antibody and a detection antibody.
  • the test sample being tested can be stored is a sample collection tube containing a leukocyte MPO secretion inhibitor as described previously herein.
  • the test sample does not need to have been stored in a sample collection tube containing a leukocyte MPO secretion inhibitor .
  • a test sample suspected of containing MPO is first brought into contact with an at least one first capture antibody under conditions which allow the formation of a first antibody-MPO complex. If more than one capture antibody is used, a first multiple capture antibody-MPOcomplex is formed.
  • the antibodies, preferably, the at least one capture antibody are used in molar excess amounts of the maximum amount of MPO expected in the test sample. For example, from about 5 ⁇ g/mL to about 1 mg/mL of antibody per mL of buffer (e.g., microparticle coating buffer) can be used.
  • the at least one capture antibody prior to contacting the test sample with the at least one capture antibody (for example, the first capture antibody), the at least one capture antibody can be bound to a solid support or solid phase which facilitates the separation the first antibody-MPO complex from the test sample.
  • a solid support known in the art can be used, including but not limited to, solid supports made out of polymeric materials in the forms of wells of a reaction tray, test tubes or beads (for example, polystyrene beads, magnetic beads), nitrocellulose strips, membranes, microparticles (for example, latex particles, sheep and DURACYTES® (Abbott Laboratories, Abbott Park, IL; DURACYTES® are red blood cells that have been "fixed” by pyruvic aldehyde and formaldehyde)).
  • the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens.
  • Microporous structures generally are preferred, but materials with gel structure in the hydrated state may be used as well.
  • Such useful solid supports include, but are not limited to, nitrocellulose and nylon.
  • Such porous solid supports are preferably in the form of sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm.
  • the pore size may vary within wide limits, and preferably is from about 0.025 to about 15 microns, especially from about 0.15 to about 15 microns.
  • the surface of such supports may be activated by chemical processes which cause covalent linkage of the antigen or antibody to the support.
  • the irreversible binding of the antigen or antibody is obtained, however, in general, by adsorption on the porous material by poorly understood hydrophobic forces.
  • the antibody (or antibodies) can be bound to the solid support or solid phase by adsorption, by covalent bonding using a chemical coupling agent or by other means known in the art, provided that such binding does not interfere with the ability of the antibody to bind MPO.
  • the antibody (or antibodies) can be bound with microp articles that have previously coated with streptavidin or biotin (for example, using Power-BindTM- SA-MP streptavidin coated microparticles, available from Seradyn, Indianapolis, Indiana).
  • the antibody can be bound using microparticles that have been previously coated with anti-species specific monoclonal antibodies.
  • the solid support can be derivatized to allow reactivity with various functional groups on the antibody. Such derivatization requires the use of certain coupling agents such as, but not limited to, maleic anhydride, N-hydroxysuccinimide and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide.
  • the mixture is incubated in order to allow for the formation of a first antibody (or multiple antibody)-MPO complex.
  • the incubation can be carried out at a pH of from about 4.5 to about 10.0, at a temperature of from about 2°C to about 45°C, and for a period from at least about one (1) minute to about eighteen (18) hours, preferably from about 1 to 20 minutes, most preferably from about 2-6 minutes.
  • the immunoassay described herein can be conducted in one step (meaning the test sample, at least one capture antibody and at least one detection antibody are all added sequentially or simultaneously to a reaction vessel) or in more than one step, such as two steps, three steps, etc.
  • the complex is then contacted with at least one detection antibody (under conditions which allow for the formation of a (first or multiple) capture antibody-MPO-second antibody detection complex).
  • the at least one detection antibody can be the second, third, fourth, etc. antibodies used in the immunoassay. If the capture antibody-MPO complex is contacted with more than one detection antibody, then a (first or multiple) capture antibody-MPO-(multiple) detection antibody complex is formed.
  • At least one detection antibody contains a detectable label.
  • the detectable label can be bound to the at least one detection antibody (e.g., the second detection antibody) prior to, simultaneously with or after the formation of the (first or multiple) capture antibody-MPO-(second or multiple) detection antibody complex. Any detectable label known in the art can be used.
  • the detectable label can be a radioactive label, such as, 3 H, 125 1, 35 S, 14 C, 32 P, 33 P, an enzymatic label, such as horseradish peroxidase, alkaline phosphatase, glucose 6-phosphate dehydrogenase, etc., a chemiluminescent label, such as, acridinium esters, luminol, isoluminol, thioesters, sulfonamides, phenanthridinium esters, etc.
  • a radioactive label such as, 3 H, 125 1, 35 S, 14 C, 32 P, 33 P
  • an enzymatic label such as horseradish peroxidase, alkaline phosphatase, glucose 6-phosphate dehydrogenase, etc.
  • a chemiluminescent label such as, acridinium esters, luminol, isoluminol, thioesters, sulfonamides, phenanthrid
  • a fluorescence label such as, fluorescein (5- fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6- hexachloro -fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (zinc sulfide-capped cadmium selenide), a thermometric label or an immuno -polymerase chain reaction label.
  • fluorescein 5- fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6- hexachloro -fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.
  • fluorescein 5- fluorescein, 6-carbox
  • the detectable label can be bound to the antibodies either directly or through a coupling agent.
  • a coupling agent that can be used is EDAC (l-ethyl-3-(3- dimethylaminopropyl) carbodiimide, hydrochloride) that is commercially available from EDAC (l-ethyl-3-(3- dimethylaminopropyl) carbodiimide, hydrochloride) that is commercially available from EDAC (l-ethyl-3-(3- dimethylaminopropyl) carbodiimide, hydrochloride) that is commercially available from EDAC (l-ethyl-3-(3- dimethylaminopropyl) carbodiimide, hydrochloride) that is commercially available from EDAC (l-ethyl-3-(3- dimethylaminopropyl) carbodiimide, hydrochloride) that is commercially available from EDAC (l-ethyl-3-(3- dimethylaminopropyl
  • detectable labels can be purchased or synthesized that already contain end groups that facilitate the coupling of the detectable label to the antibody, such as, NlO- (3-sulfopropyl)-N-(3-carboxypropyl)-acridinium-9-carboxamide, otherwise known as CPSP- Acridinium Ester or N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9- carboxamide, otherwise known as SPSP- Acridinium Ester.
  • the (first or multiple) capture antibody-MPO-(second or multiple) detection antibody complex can be, but does not have to be, separated from the remainder of the test sample prior to quantification of the label.
  • the at least one capture antibody e.g., the first capture antibody
  • a solid support or solid phase such as, but not limited to a well of a reaction tray, a bead or a microparticle
  • separation can be accomplished by removing the fluid (of the test sample) from contact with the solid support.
  • the at least first capture antibody is bound to a solid support it can be simultaneously contacted with the MPO-containing sample and the at least one second detection antibody to form a first (multiple) antibody- MPO-second (multiple) antibody complex, followed by removal of the fluid (test sample) from contact with the solid support. If the at least one first capture antibody is not bound to a solid support, then the (first or multiple) capture antibody-MPO-(second or multiple) detection antibody complex does not have to be removed from the test sample for quantification of the amount of the label.
  • the amount of label in the complex is quantified using techniques known in the art. For example, if an enzymatic label is used, the labeled complex is reacted with a substrate for the label that gives a quantifiable reaction such as the development of color. If the label is a radioactive label, the label is quantified using a scintillation counter. If the label is a fluorescent label, the label is quantified by stimulating the label with a light of one color (which is known as the "excitation wavelength”) and detecting another color (which is known as the "emission wavelength”) that is emitted by the label in response to the stimulation.
  • the labeled capture antibody-MPO-detection antibody complex e.g., the first capture antibody-MPO-second detection antibody complex
  • the amount of label in the complex is quantified using techniques known in the art. For example, if an enzymatic label is used, the labeled complex is reacted with a substrate for the label that gives a quantifiable reaction such as the development of color
  • the label is quantified detecting the light emitted either visually or by using luminometers, x-ray film, high speed photographic film, a CCD camera, etc.
  • concentration of MPO in the test sample is determined by use of a standard curve that has been generated using serial dilutions of MPO of known concentration.
  • the standard curve can be generated gravimetrically, by mass spectroscopy and by other techniques known in the art.
  • control compositions can be used in the MPO immunoassays.
  • the control compositions generally comprise the MPO antigen to be assayed for along with any desirable additives.
  • a preferred control MPO antigen is available commercially from Athens Research and Technology Inc. (Athens, Georgia).
  • the MPO assays described herein can be used for diagnosing cardiovascular disease in a subject. Specifically, such assays involve providing a test sample obtained from a subject (which may or may not have been stored in a sample collection tube containing a MPO secretion inhibitor as described previously herein in Section D). The concentration of MPO in the test sample can then determined using any of the MPO assays described herein (e.g., using the methods described in this Section E, namely, methods that use an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437, an antibody produced by murine hybridoma cell line 1-2169- 715 having A.T.C.C. Accession No.
  • concentration of MPO in the test sample can be compared to a predetermined level to determine whether or not the subject is suffering from cardiovascular disease. Specifically, if the concentration of MPO in the test sample is lower or the same as a predetermined level, then the subject would be considered not to have cardiovascular disease. However, if the concentration of MPO in the test sample were higher then a predetermined level, then the subject would be considered to have cardiovascular disease.
  • concentration of MPO in a test sample determined using the method described herein can also be used to determine whether or not a subject is at risk of developing cardiovascular disease. Specifically, such a method can comprise the steps of:
  • test sample which can optionally be stored in a sample collection tube containing a MPO secretion inhibitor such as that described in Section D
  • concentration of MPO in the test sample according to any of the assays described herein (e.g., using the methods described in this Section E, namely, methods that use an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437, an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438 or both of these antibodies); and (c) comparing the concentration of MPO in the test sample determined in step (b) with a predetermined level.
  • step (b) when making such a comparision, if the concentration of MPO determined in step (b) is lower than the predetermined level, then the subject is considered not to be at risk of developing cardiovascular disease. However, when making such a comparision, if the concentration of MPO in the test sample determined in step (b) is higher then the predetermined level, then the subject is considered to be at risk of developing cardiovascular disease.
  • concentration of MPO in a test sample determined using the methods described herein can also be useful to provide an indicator of the clinical status (i.e., severity or progression of disease) of a subject.
  • concentration of MPO determined using the methods of the present disclosure can be used to determine whether or not subject is suffering from a disease such as heart failure.
  • the concentration of MPO determined as described herein can be used to determine whether a subject suffering from heart failure should be classified in any of New York Heart Association (NYHA) Classifications I, II, III or IV or whether a subject classified as certain New York Heart Association Classification has progressed to a different New York Heart Association Classification (e.g., the subject was initially classified as New York Heart Association Classification II and then the subject progress to New York Heart Association Classification III).
  • NYHA New York Heart Association
  • the severity or progression of disease, such as cardiovascular disease, in a subject can be determined using a method comprising the steps of:
  • test sample from a subject which can optionally be stored in a sample collection tube containing a MPO secretion inhibitor such as that described in Section D
  • concentration of MPO in the test sample according to any of the assays described herein (e.g., using the methods described in this Section E, namely, methods that use an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437, an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No.
  • step (c) comparing the concentration of MPO in the test sample determined in step (b) with a predetermined level. Specifically, when making such a comparison with respect to severity of cardiovascular disease in a subject, if the concentration of MPO determined in step (b) is lower than the predetermined level, the subject is determined to have a reduced severity of cardiovascular disease. If the concentration of MPO determined in step (b) is higher than the predetermined level, the subject is determined to have an increased severity of cardiovascular disease.
  • the concentration of MPO in the test sample determined in step (b) When comparing the concentration of MPO in the test sample determined in step (b) with respect to progression of cardiovascular disease, if the concentration of MPO determined in step (b) is lower or unchanged to a predetermined level, the subject is determined not to have progressed or to have improved with respect to cardiovascular disease. If the concentration of MPO in the test sample determined in step (b) is lower or unchanged to a predetermined level, the subject is determined not to have progressed or to have improved with respect to cardiovascular disease. If the concentration of MPO in the test sample determined in step (b) is lower or unchanged to a predetermined level, the subject is determined not to have progressed or to have improved with respect to cardiovascular disease. If the concentration of MPO in the test sample determined in step
  • (b) is higher when compared to a predetermined level, the subject is determined to have progressed with respect to cardiovascular disease.
  • the progression of disease such as cardiovascular disease, can be monitored either before treatment is commenced in a subject or after commencement of treatment in a subject.
  • the concentration of MPO determined using the methods described herein can be used to determine if a subject has suffered a cardiovascular complication as a result of administration to said subject of one or more pharmaceutical compositions.
  • such a method can comprise the steps of: (a) obtaining a first test sample from the subject before the subject has been administered one or more pharmaceutical compositions (which can optionally be stored in a sample collection tube containing a MPO secretion inhibitor such as that described in Section D);
  • step (e) comparing the concentration of MPO in step (b) with the concentration of MPO in step (d). Specifically, if the concentration of MPO determined in step (b) is unchanged when compared to the concentration of MPO determined in step (d), then the subject is determined not to have suffered a cardiovascular complication as a result of the administration of one or more pharmaceutical compositions. Moreover, if the concentration of MPO determined in step (b) is changed when compared to the concentration of MPO in step (d), then the subject is determined to have suffered a cardiovascular complication as a result of the administration of one or more pharmaceutical compositions.
  • the concentration of MPO determined using the methods described herein can be used in methods for monitoring MPO levels in a subject receiving treatment with one or more pharmaceutical compositions. Specifically, such methods involve providing a first test sample from a subject before the subject has been administered one or more pharmaceutical compositions (which can optionally be stored in a sample collection tube containing a MPO secretion inhibitor such as that described in Section D). Next, the concentration of MPO (e.g., the level of MPO) in the test sample is determined according to any of the assays described herein (e.g., using the methods described in this Section E, namely, methods that use an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No.
  • PTA-8437 an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438 or both of these antibodies).
  • concentration of MPO in the test sample is determined, the concentration of MPO is then compared with a predetermined level. If the concentration of MPO determined in the first test sample is lower then the predetermined level, then the subject is not treated with one or more pharmaceutical compositions. However, if the concentration of MPO determined in the first test sample is higher then the predetermined level, then the subject is treated with one or more pharmaceutical compositions for a period of time.
  • the period of time that the subject is treated with the one or more pharmaceutical compositions can be determined by one skilled in the art (for example, the period of time can be from about seven (7) days to about two years, preferably from about fourteen (14) days to about one (1) year).
  • second and subsequent test samples are then obtained from the subject (any or all of these test samples can optionally be stored in a sample collection tube containing a MPO secretion inhibitor such as that described in Section D).
  • the number of test samples and the time in which said test samples are obtained from the subject are not critical.
  • a second test sample could be obtained seven (7) days after the subject is first administered the one or more pharmaceutical compositions
  • a third test sample could be obtained two (2) weeks after the subject is first administered the one or more pharmaceutical compositions
  • a fourth test sample could be obtained three (3) weeks after the subject is first administered the one or more pharmaceutical compositions
  • a fifth test sample could be obtained four (4) weeks after the subject is first administered the one or more pharmaceutical compositions, etc.
  • the concentration of MPO in the second and subsequent test samples is determined according to any of the assays described herein (e.g., using the methods described in this Section E, namely, methods that use an antibody produced by murine hybridoma cell line 1-1175-509 having A.T.C.C. Accession No. PTA-8437, an antibody produced by murine hybridoma cell line 1-2169-715 having A.T.C.C. Accession No. PTA-8438 or both of these antibodies).
  • the concentrations of MPO determined in each of these second and subsequent test samples is then compared with the concentration of MPO determined in the first test sample (e.g., the test sample that was originally compared to the predetermined level).
  • concentrations of MPO determined in the second and subsequent test samples are lower or have decreased when compared to the concentration of MPO determined in the first test sample, then treatment with the one or more pharmaceutical compositions can be continued. However, if the concentrations of MPO determined in the second and subsequent test samples are the same or have increased when compared to the concentration of MPO determined in the first test sample, then a determination is made that the one or more pharmaceutical composition are not efficacious for reducing MPO levels in that subject.
  • the subject can then is either: (a) treated with a higher dose of the one or more pharmaceutical compositions when compared to the dose of the one or more pharmaceutical compositions previously given to said subject; or (b) switched to one or more alternate or different pharmaceutical compositions. Specifically, the subject can be treated with one or more pharmaceutical compositions that are different then the one or more pharmaceutical compositions that the subject had previously received to decrease or lower said subject's MPO levels.
  • kits for detecting the presence of MPO in a test sample can comprise one or more of the antibodies described herein. More specifically, if the kit is a kit for performing an immunoassay, the kit optionally can contain (1) at least one capture antibody that specifically binds to MPO; (2) at least one conjugate; and (3) one or more instructions for performing the immunoassay.
  • the antibodies of the present disclosure can be included in such a test kit as a capture antibody, as a detection antibody or both as a capture antibody and a detection antibody.
  • an antibody produced by murine hybridoma cell line 1-1175-509 can be included in the kit as capture antibody and an antibody produced by murine hybridoma cell line 1- 2169-715 can be included in the kit as a detection antibody.
  • an antibody produced by murine hybridoma cell line 1-2169-715 can be included in the kit as a capture antibody and an antibody produced by hybridoma cell line 1-1175-509 can be included in the kit as a detection antibody.
  • an antibody produced by murine hybridoma cell line 1-1175-509 or an antibody produced by murine hybridoma cell line 1-2169-715 can be included in the kit as a capture antibody and a different antibody included in the kit as a detection antibody.
  • an antibody produced by murine hybridoma cell line 1-1175-509 or an antibody produced by murine hybridoma cell line 1-2169-715 can be included in the kit as a detection antibody and a different antibody included in the kit as a capture antibody.
  • the kit can also contain at least one calibrator or control. Any calibrator or control can be included in the kit. Preferably, however, the calibrator or control is a purified MPO.
  • the kit can also contain at least one sample collection tube.
  • the kit can also contain at least one leukocyte MPO secretion inhibitor.
  • An example of at least one leukocyte MPO secretion inhibitor that can be included in the kit is a salt of any salt of EDTA or sodium citrate.
  • the kit can also containing at least one sample collection tube that contains at least one leukocyte MPO secretion inhibitor.
  • the present disclosure further provides for diagnostic and quality control kits comprising one or more antibodies of the present disclosure.
  • the assays, kits and kit components of the invention are optimized for use on commercial platforms (e.g., immunoassays on the PRISM®, AxSYM®, ARCHITECT® and EIA (Bead) platforms of Abbott Laboratories, Abbott Park, IL, as well as other commercial and/or in vitro diagnostic assays).
  • the assays, kits and kit components can be employed in other formats, for example, on electrochemical or other hand-held or point-of-care assay systems.
  • the present disclosure is, for example, applicable to the commercial Abbott Point of Care (i-STAT®, Abbott Laboratories, Abbott Park, IL) electrochemical immunoassay system that performs sandwich immunoassays for several cardiac markers, including TnI, CKMB and BNP.
  • Immunosensors and methods of operating them in single- use test devices are described, for example, in U.S. Patent Applications 20030170881, 20040018577, 20050054078 and 20060160164. Additional background on the manufacture of electrochemical and other types of immunosensors is found in U.S. Patent 5,063,081.
  • kits include quality control reagents (for example, sensitivity panels, calibrators, and positive controls). Preparation of quality control reagents is well known in the art, and is described, e.g., on a variety of immunodiagnostic product insert sheets.
  • the present disclosure provides for a quality control kit comprising one or more antibodies of the present disclosure for use as a sensitivity panel to evaluate assay performance characteristics and/or to quantitate and monitor the integrity of the antigen(s) used in the assay.
  • kits can optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co- factors, substrates, detection reagents, and the like.
  • Other components such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), may also be included in the kit.
  • the kit may additionally include one or more other controls.
  • One or more of the components of the kit may be lyophilized and the kit may further comprise reagents suitable for the reconstitution of the lyophilized components.
  • the various components of the kit optionally are provided in suitable containers. As indicated above, one or more of the containers may be a microtiter plate.
  • the kit further can include containers for holding or storing a sample (e.g., a container or cartridge for a blood or urine sample). Where appropriate, the kit may also optionally contain reaction vessels, mixing vessels and other components that facilitate the preparation of reagents or the test sample. The kit may also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.
  • a sample e.g., a container or cartridge for a blood or urine sample.
  • the kit may also optionally contain reaction vessels, mixing vessels and other components that facilitate the preparation of reagents or the test sample.
  • the kit may also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.
  • the kit further can optionally include instructions for use, which may be provided in paper form or in computer-readable form, such as a disc, CD, DVD or the like.
  • the disclosure herein also can be adapted for use in a variety of automated and semi-automated systems (including those wherein the solid phase comprises a microparticle), as described, e.g., in U.S. Patent Nos. 5,089,424 and 5,006,309, and as, e.g., commercially marketed by Abbott Laboratories (Abbott Park, IL) including but not limited to Abbott's ARCHITECT®, AxS YM®, IMx®, PRISM®, and QuantumTM II instruments, as well as other platforms.
  • the disclosure optionally is adaptable for the Abbott Laboratories commercial Point of Care (i-STATTM) electrochemical immunoassay system for performing sandwich immunoassays.
  • i-STATTM Point of Care
  • Example 1 A.T.C.C. Deposit Information
  • Murine hybridoma cell line for monoclonal antibody 1-1175-509 was deposited with the American Type Culture Collection (hereinafter referred to as "A.T.C.C"), 10801 University Boulevard., Manassas, VA 20110-2209, on May 16, 2007 and assigned A.T.C.C. Accession No. PT A-8437.
  • A.T.C.C American Type Culture Collection
  • Murine hybridoma cell line for monoclonal antibody 1-2169-715 was deposited with the A.T.C.C., 10801 University Boulevard., Manassas, VA 20110-2209, on May 16, 2007 and assigned A. T. C. C. Accession No. PTA-8438.
  • the analytical range of the assay is from 5 to 10,000 pmol/L with precision ranging from 2.4 to 6.0% CV over the range of 250 to 5000 pmol/L.
  • the two monoclonal antibodies used in the experimental ARCHITECT® assay are 1-1175-509 and 1-2169-715.
  • sample tube types and measured mean MPO values are in the following Table 1 :
  • Table 4 The results shown in Table 4 are the mean MPO values of the 5 donors at each timepoint and storage condition. Citrate and lithium heparin storage tubes were able to maintain MPO levels relatively stable with storage at 2-8°C for 2 hours, note however the MPO concentration in citrate was similar to the concentration measured in EDTA, whereas the concentration in lithium heparin was increased over the concentration in EDTA or citrate at baseline. Storage at room temperature for both citrate and lithium heparin showed marked increase in MPO levels over time. In contrast, use of the storage tubes with the leukocyte MPO secretion inhibitor EDTA was able to maintain MPO levels essentially constant at both room temperature and 2-8°C. Table 4
  • BD367964 SST (8.5 mL, BD367988) and EDTA PPT (5 mL, BD362788) tube types.
  • PST and PPT tubes were centrifuged immediately; SST tubes were allowed to clot for 30 minutes at room temperature prior to centrifugation.
  • the isolated plasma or serum were tested neat and spiked with MPO. Samples were then placed at room temperature and tested at time 0, 3.5 hours, 24 hours and 48 hours. Samples were also placed at 2-8°C and tested using the ARCHITECT® assay of Example 2 at time 0, 24 hours, 48 hours, 5 days and 8 days. As seen in Tables 5 and 6 the isolated plasma or serum was stable under all conditions tested.
  • Example 7 Testing Naturally Elevated Samples In Various Tube Types Fifty matched specimens were collected from patients presenting to the Emergency
  • the collection tubes evaluated were plastic serum (SST), K 2 EDTA plasma, lithium heparin plasma (PST) and citrate tubes. All samples were stored at room temperature for no more than 60 minutes prior to centrifugation except the EDTA samples that were stored at 2-8°C as whole blood samples for 24 hours prior to centrifugation. Separated serum and plasma were stored at 2-8°C until tested by the ARCHITECT® MPO assay of Example 2. The mean, median, minimum and maximum concentration for each tube type are summarized in the Table 8: Table 8
  • RBF/DnJ female mice (The Jackson Laboratory, Bar Harbor, Maine) were immunized three times with purified MPO antigen (Advanced Immunochemical, Long Beach, CA), using either the Freund's Adjuvant (Difco, Detroit, Michigan) or Ribi
  • MPL+TDM (Corixa, Hamilton, MT) Adjuvant System.
  • the inoculum was prepared by diluting the MPO antigen in 0.9% sodium chloride (Abbott Laboratories, Abbott Park, IL) and emulsifying with one of the two adjuvants.
  • a 10 ⁇ g boost of MPO was administered to the mice.
  • Freund's Complete Adjuvant was used for the primary boost and delivered subcutaneously.
  • Freund's Incomplete Adjuvant was used for the next 2 boosts delivered through intradermal injection.
  • Ribi Adjuvant was used for all three immunizations on the Ribi mice, and each was delivered by intradermal injection.
  • the mice were administered a pre-fusion boost of 20 ⁇ g of MPO.
  • mice On the day of fusion, the mice were euthanized and their spleens containing anti-
  • MPO splenocytes were harvested and placed into Iscove's Modified Dulbecco's Medium (IMDM) (Invitrogen Corporation, Grand Island NY). A cell fusion was performed as described by Kohler and Milstein ⁇ Nature, 256:495-7 (1975)). Each mouse spleen was placed into a separate petri dish containing IMDM. The splenocytes were perfused out of each spleen using a syringe containing IMDM and cell scraper, then counted using a hemocytometer. Approximately 5.O x 10 6 splenocytes were pooled from each mouse and washed by centrifugation into a cell pellet and re-suspended in IMDM.
  • IMDM Iscove's Modified Dulbecco's Medium
  • splenocytes were mixed with an equal number of SP 2/0 myeloma cells and centrifuged into a pellet.
  • the fusion was accomplished by exposing the splenocytes and SP 2/0 cells to 50% polyethylene glycol (PEG) (A.T.C.C. Molecular Weight 1300-1600, Manassas VA) in IMDM. Two mL of the PEG solution was added to the cell pellet followed by a one- minute incubation. The PEG and cell pellet was diluted by slowly adding thirty mL of IMDM over 30 seconds. The fused cells were then removed from suspension by centrifugation and decanting the supernatant.
  • PEG polyethylene glycol
  • the cell pellet was re-suspended into 502 mL containing an approximate 50% mixture of spent medium from the SP 2/0 myeloma cell culture and fresh IMDM supplemented with FBS (Hyclone Laboratories, Logan UT), HAT (Hypoxanthine, Aminopterin, Thymidine) (Sigma Laboratories, St. Louis, MO), Hybridoma Cloning Factor (Bioveris Corporation, Gaithersburg MD), and L-Glutamine
  • hybridomas were allowed to grow for 7-10 days prior to supernatant screening for antibody production.
  • MPO antigen was added to the plates at 500 ng/mL and incubated for approximately 30 minutes. Following this incubation, the antigen was washed from the plates using distilled water. Rabbit anti-MPO antibody was added to the plates at 250 ng/mL and incubated for approximately 30 minutes. Following this incubation, the antibody was washed from the plates using distilled water. Goat anti- rabbit-HRPO (Kirkegaard & Perry) diluted to approximately 250 ng/mL in block solution was added to the plates and allowed to incubate for 30 minutes.
  • the plates were washed with distilled water to remove the Goat anti-rabbit-HRPO and o-phenylenediamine substrate (OPD; Abbott Laboratories, Abbott Park, IL) was used as the chromogen to generate signal. Plates were read at 492 nm and the results were analyzed. Hybrids were considered positive if they had an EIA signal at least 3 times greater than background (See Table 9, below).
  • Hybrid MPO binding epitope groups were determined by measuring the ability of each MPO hybrid mAb to complete a mAb-antigen-mAb sandwich with five existing outside vendor monoclonal antibodies (See, Table 11, below) that are commercially available and were biotin labeled and believed to have different and distinct binding epitopes on MPO. Briefly, rabbit anti-mouse IgG Fc (Jackson Immunoresearch, West Grove, PA) was coated on 96 well microtiter EIA plates at 10 ug/mL. After the capture reagent has been coated on the solid phase, it was removed and any open binding sites on the plates were blocked using BSA or Fish Gelatin block solution.
  • the antibody was washed from the plates using distilled water. Streptavidin-HRPO diluted to approximately 100 ng/mL in block solution was added to the plates and allowed to incubate for 30 minutes. The plates were washed and o-phenylenediamine substrate was used as the chromogen to generate signal. Plates were read at 492 nm and the results were analyzed. The supernatant dilution that generates approximately 50% of maximal binding was compared for each of the hybrids. Table 11, below, summarizes the absorbance values for each hybrid supernatant with each of the biotin labeled mAbs. Based on their ability to form sandwiches with each of these antibodies, the hybrids were divided into groups. Hybrids 1-1175 and 1-2169 were believed to bind to distinct epitopes on MPO and were therefore selected for cloning to stabilize the cell line and ensure there that there was no mixed cell population.
  • Hybrids 1-1175 and 1-2169 were cloned using a Fluorescent Activated Cell Sorter (FACS). Hybrid cultures were stained with Propidium Iodide to stain non-viable cells so they could be deselected from the population. The stained cultures were analyzed on the FACSAria (BD Biosciences). Single cells were deposited into each well of 96 well culture dishes containing Hybridoma Serum Free Medium (HSFM) supplemented with L- Glutamine and 10% FBS. The cultures were allowed to incubate for approximately 7 days and screened by EIA, as previously described in this example. Clones 1-1175-154 and 1-
  • HSFM Hybridoma Serum Free Medium
  • 2169-143 were isolated and purified antibody was generated from each cell line.
  • the purified antibody was tested for it's relative affinity ranking with the other antibodies generated (See, Table 11).
  • Rabbit anti-mouse IgG Fc (Jackson Immunoresearch) was coated on 96 well microtiter EIA plates at 5 ug/mL. After the capture reagent has been coated on the solid phase, it was removed and any open binding sites on the plates were blocked using BSA or Fish Gelatin block solution.
  • Purified antibody was added to the plates at six different dilutions and allowed to incubate for approximately 60 minutes. The antibody was washed off and biotin labeled MPO antigen was added in concentrations from 0 to 100 ng/mL and allowed to incubate for 60 minutes.
  • the antigen was washed off and streptavidin-HRPO diluted to approximately 100 ng/mL in block solution was added to the plates and allowed to incubate for 30 minutes. The plates were washed and o-phenylenediamine substrate was used as the chromogen to generate signal. Plates were read at 492 nm and the results were analyzed. The antibody concentration generating approximately half of the maximum binding signal was used to generate an antigen titration curve shown in Figure 1.
  • Purified antibody from each of these clones was labeled with biotin. Epitope group confirmation was completed using a competitive inhibition micro-titer assay, using a number of different proprietary and commercial available monoclonal antibodies.
  • MPO antigen was coated on 96 well microtiter EIA plates at 0.5 ug/mL. After the capture reagent had been coated on the solid phase, it was removed and any open binding sites on the plates were blocked using BSA or Fish Gelatin block solution. Purified antibody (100 uL/well) from each clone at 50 ug/mL was then added to the blocked plates and allowed to incubate at room temperature for one hour.
  • Biotin labeled antibody 50 uL/well was then added to the wells, without washing out the unlabeled antibody, and allowed to incubate for approximately 10-15 minutes. After that incubation, the antibody solution was washed from the plate using distilled water. Streptavidin-HRPO diluted to approximately 200 ng/mL in block solution was added to the plates and allowed to incubate for 30 minutes. The plates were washed and o-phenylenediamine substrate was used as the chromogen to generate signal. Plates were read at 492 nm and the results were analyzed.
  • Antibody pairing studies confirmed the above data. Briefly, the unlabeled mAb is coated on to 96 well micro-titer plates at 1 ug/mL. After the capture reagent had been coated on the solid phase, it was removed and any open binding sites on the plates were blocked using BSA or Fish Gelatin block solution. MPO antigen from 0 to 50 ng/mL was then added to the blocked plates and allowed to incubate at room temperature for 30 minutes. Following this incubation, the antigen was washed from the plates using distilled water. The biotin labeled anti-MPO antibodies were added to the plates and incubated for approximately 30 minutes. Following this incubation, the antibody was washed from the plates using distilled water.
  • Streptavidin-HRPO diluted to approximately 200 ng/mL in block solution was added to the plates and allowed to incubate for 30 minutes. The plates were washed and o-phenylenediamine substrate was used as the chromogen to generate signal. Plates were read at 492 nm and the results were analyzed. As shown in Figure 2, these results confirmed that clones 1-1175-154 and 1-2169-143 were good MPO binding partners.
  • Clone 1-1175-154 was weaned to HSFM without FBS and subcloned using the FACS cell sorting method previously described.
  • Cell line 1-1175-509 was selected for scale up and cell banking purposes. Liquid nitrogen freezers are used for long-term storage of the cell bank.
  • Anti-MPO mAb 1-1175-154 is the parental clone from which subclone 1-1175-509 was derived.
  • Clone 1-2169-143 was weaned to HSFM without FBS and subcloned by counting the viable cells in culture and seeding 1 cell per well in 96 well tissue culture plates. The plates were allowed to incubate for 7-10 days and the subclone supernatant was tested for anti-MPO antibody using a micro-titer EIA.
  • Cell line 1-2169-715 was selected for scale up and cell banking purposes. Liquid nitrogen freezers are used for long-term storage of the cell bank.
  • Anti-MPO mAb 1-2169-143 is the parental clone from which subclone 1-2169- 715 was derived.
  • the 1-1175-509 and 1-2169-715 cell lines were expanded in HSFM and seeded into roller bottles at approximately 0.5 xlO "5 cells/mL.
  • the cultures were incubated at 37°C while rotating at approximately 1 revolution per minute for 10-14 days, or until a terminal end culture was obtained.
  • the terminal roller bottle supernatant was harvested and clarified with a 0.45 ⁇ M filter.
  • the clarified supernatant was concentrated using a Pellicon system and filtered with a 0.45 ⁇ M filter.
  • the mAb concentrate was diluted with an equal volume of 1.5 M glycine / 3 N NaCl buffer at pH 8.9, then loaded onto a pre- equilibrated 5 ml Protein A column using the AKTA automated purification system (Amersham/Pharmacia). The column was then washed with 5 column volumes of binding buffer and when a stable baseline was achieved, the mAb was eluted with a pH 3.0 citrate buffer. The mAb was then transferred to a 70 mL G25 column for an exchange into PBS. The antibody was aliquoted and stored at -70 0 C.
  • the affinity of MPO monoclonal antibodies 1-1175-509 and 1-2169-143 for MPO was determined using a BIAcore 2000 instrument (BIAcore International AB, Uppsala, Sweden).
  • a -5,000 RU goat anti-mouse IgG Fc Capture Biosensor was created by amine- coup ling polyclonal goat anti-mouse IgG Fc antibody (Jackson Immunoresearch Laboratories) to a CM4 biosensor chip (BIAcore) via EDC/NHS/Ethanolamine chemistry provided in a Amine Coupling Kit (BIAcore).
  • MPO antibody and MPO antigen were diluted into a running buffer (hereinafter "Running Buffer") composed of HBS-EP buffer spiked with 0.1% BSA, 0.1% CM-Dextran, and 10 mM CaCl 2 .
  • Each MPO antibody was diluted to 0.6 ⁇ g/mL and MPO antigen (Athens Research & Technology, Athens, GA) was diluted to concentrations ranging from 0.0105 to 207 nM using a 3 -fold dilution series.

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Abstract

La présente invention concerne des anticorps isolés qui peuvent être utilisés dans un dosage visant à déterminer les niveaux de concentration de myélopéroxidase (MPO) dans un échantillon test. En outre, la présente invention concerne l'utilisation de procédés améliorés de manipulation d'un échantillon test dans des dosages visant à préserver les niveaux originaux de MPO dans ledit échantillon test.
EP08827464A 2007-05-18 2008-05-16 Anticorps et procedes ameliores de manipulation d'un echantillon test destines a etre utilises dans des dosages pour myeloperoxidase Withdrawn EP2158491A4 (fr)

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US11/750,507 US20080286818A1 (en) 2007-05-18 2007-05-18 Blood sample handling methods for improved assays for myeloperoxidase
US5006108A 2008-03-17 2008-03-17
PCT/US2008/063930 WO2009023331A2 (fr) 2007-05-18 2008-05-16 Anticorps et procédés améliorés de manipulation d'un échantillon test destinés à être utilisés dans des dosages pour myélopéroxidase

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US11185677B2 (en) 2017-06-07 2021-11-30 Shifamed Holdings, Llc Intravascular fluid movement devices, systems, and methods of use
US11511103B2 (en) 2017-11-13 2022-11-29 Shifamed Holdings, Llc Intravascular fluid movement devices, systems, and methods of use
US11654275B2 (en) 2019-07-22 2023-05-23 Shifamed Holdings, Llc Intravascular blood pumps with struts and methods of use and manufacture
US11724089B2 (en) 2019-09-25 2023-08-15 Shifamed Holdings, Llc Intravascular blood pump systems and methods of use and control thereof
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Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015530560A (ja) * 2012-06-15 2015-10-15 エルビズ，エクレム Ｅｌｉｓａ法におけるゲルを含むｅｄｔａチューブ及び該チューブを使用する分析器
CN111879758A (zh) * 2020-07-28 2020-11-03 江苏扬新生物医药有限公司 一种吖啶酯抗体标记方法及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006117688A2 (fr) * 2005-02-11 2006-11-09 University Of Leicester Diagnostic

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4724213A (en) * 1985-05-24 1988-02-09 Northwestern University Murine hybridoma Lym-1 and diagnostic antibody produced thereby
US5948893A (en) * 1996-01-17 1999-09-07 The United States Of America As Represented By The Secretary Of The Navy Murine hybridoma and antibody binding to CD28 receptor secreted by the hybridoma and method of using the antibody
DK1353666T3 (da) * 2001-01-02 2013-10-14 Cleveland Clinic Foundation Myeloperoxidase, en risikoindikator for cardiovasculær sygdom

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006117688A2 (fr) * 2005-02-11 2006-11-09 University Of Leicester Diagnostic

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
A. Niskanen ET AL: "Myeloperoxidase (MPO) and hs-CRP as predictive factors for myocardial infarction", Clinical Laboratory International, 1 November 2006 (2006-11-01), XP55009479, Retrieved from the Internet: URL:http://www.cli-online.com/uploads/tx_ttproducts/datasheet/myeloperoxidase-%28mpo%29-and-hs-crp-as-predictive-factors-for-myocardial-infarction.pdf [retrieved on 2011-10-13] *
CAVUSOGLU ET AL: "Usefulness of Baseline Plasma Myeloperoxidase Levels as an Independent Predictor of Myocardial Infarction at Two Years in Patients Presenting With Acute Coronary Syndrome", AMERICAN JOURNAL OF CARDIOLOGY, CAHNERS PUBLISHING CO., NEWTON, MA, US, vol. 99, no. 10, 9 May 2007 (2007-05-09), pages 1364-1368, XP022067507, ISSN: 0002-9149, DOI: 10.1016/J.AMJCARD.2006.12.060 *
J. A. VITA: "Serum Myeloperoxidase Levels Independently Predict Endothelial Dysfunction in Humans", CIRCULATION, vol. 110, no. 9, 23 August 2004 (2004-08-23), pages 1134-1139, XP55009474, ISSN: 0009-7322, DOI: 10.1161/01.CIR.0000140262.20831.8F *
KAMYAR KALANTAR-ZADEH ET AL: "Serum Myeloperoxidase and Mortality in Maintenance Hemodialysis Patients", AMERICAN JOURNAL OF KIDNEY DISEASES, vol. 48, no. 1, 1 July 2006 (2006-07-01), pages 59-68, XP55009477, ISSN: 0272-6386, DOI: 10.1053/j.ajkd.2006.03.047 *
MARIE-LUISE BRENNAN ET AL: "Prognostic Value of Myeloperoxidase in Patients with Chest Pain", NEW ENGLAND JOURNAL OF MEDICINE, vol. 349, no. 17, 23 October 2003 (2003-10-23), pages 1595-1604, XP55009475, ISSN: 0028-4793, DOI: 10.1056/NEJMoa035003 *
MOCATTA ET AL: "Plasma Concentrations of Myeloperoxidase Predict Mortality After Myocardial Infarction", JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, ELSEVIER, NEW YORK, NY, US, vol. 49, no. 20, 4 May 2007 (2007-05-04), pages 1993-2000, XP022079403, ISSN: 0735-1097, DOI: 10.1016/J.JACC.2007.02.040 *
NG ET AL: "Myeloperoxidase and C-reactive protein augment the specificity of B-type natriuretic peptide in community screening for systolic heart failure", AMERICAN HEART JOURNAL, MOSBY- YEAR BOOK INC, US, vol. 152, no. 1, 1 July 2006 (2006-07-01), pages 94-101, XP005686371, ISSN: 0002-8703 *
PINCEMAIL J ET AL: "Fast double antibody radioimmunoassay of human granolocyte myeloperoxidase and its application to plasma", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 137, no. 2, 21 March 1991 (1991-03-21), pages 181-191, XP023974059, ISSN: 0022-1759, DOI: 10.1016/0022-1759(91)90023-9 [retrieved on 1991-03-21] *
PUBMEDDEV: 'Plasma concentrations of myeloperoxidase p... [J Am Coll Cardiol. 2007] - PubMed - NCBI', [Online] 04 May 2007, XP055098968 Retrieved from the Internet: <URL:http://www.ncbi.nlm.nih.gov/pubmed/17512353> [retrieved on 2014-01-28] *
S. BALDUS: "Myeloperoxidase Serum Levels Predict Risk in Patients With Acute Coronary Syndromes", CIRCULATION, vol. 108, no. 12, 23 September 2003 (2003-09-23), pages 1440-1445, XP55009476, ISSN: 0009-7322, DOI: 10.1161/01.CIR.0000090690.67322.51 *
S. J. NICHOLLS: "Myeloperoxidase and Cardiovascular Disease", ARTERIOSCLEROSIS, THROMBOSIS, AND VASCULAR BIOLOGY, vol. 25, no. 6, 1 June 2005 (2005-06-01), pages 1102-1111, XP55009482, ISSN: 1079-5642, DOI: 10.1161/01.ATV.0000163262.83456.6d *
See also references of WO2009023331A2 *
T. FRANCK ET AL: "Development of an Enzyme-Linked Immunosorbent Assay for Specific Equine Neutrophil Myeloperoxidase Measurement in Blood", JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION, vol. 17, no. 5, 1 September 2005 (2005-09-01), pages 412-419, XP55009472, ISSN: 1040-6387, DOI: 10.1177/104063870501700502 *
TANG W H W ET AL: "Plasma Myeloperoxidase Levels in Patients With Chronic Heart Failure", AMERICAN JOURNAL OF CARDIOLOGY, CAHNERS PUBLISHING CO., NEWTON, MA, US, vol. 98, no. 6, 15 September 2006 (2006-09-15), pages 796-799, XP025044842, ISSN: 0002-9149, DOI: 10.1016/J.AMJCARD.2006.04.018 [retrieved on 2006-09-15] *
TSIMIKAS ET AL: "Oxidative Biomarkers in the Diagnosis and Prognosis of Cardiovascular Disease", AMERICAN JOURNAL OF CARDIOLOGY, CAHNERS PUBLISHING CO., NEWTON, MA, US, vol. 98, no. 11, 4 December 2006 (2006-12-04), pages S9-S17, XP025045056, ISSN: 0002-9149, DOI: 10.1016/J.AMJCARD.2006.09.015 [retrieved on 2006-12-04] *
ZAKI S R ET AL: "Production and characterization of monoclonal antibodies to human myeloperoxidase", CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY, SAN DIEGO, CA, US, vol. 50, no. 3, 1 March 1989 (1989-03-01), pages 283-297, XP022959084, ISSN: 0090-1229, DOI: 10.1016/0090-1229(89)90137-2 [retrieved on 1989-03-01] *
ZHANG RENLIANG ET AL: "ASSOCIATION BETWEEN MYELOPEROXIDASE LEVELS AND RISK OF CORONARY ARTERY DISEASE", JAMA THE JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION, AMERICAN MEDICAL ASSOCIATION, US, vol. 286, no. 17, 7 November 2001 (2001-11-07), pages 2136-2142, XP009084422, ISSN: 0098-7484, DOI: 10.1001/JAMA.286.17.2136 *

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US11717670B2 (en) 2017-06-07 2023-08-08 Shifamed Holdings, LLP Intravascular fluid movement devices, systems, and methods of use
US11511103B2 (en) 2017-11-13 2022-11-29 Shifamed Holdings, Llc Intravascular fluid movement devices, systems, and methods of use
US10722631B2 (en) 2018-02-01 2020-07-28 Shifamed Holdings, Llc Intravascular blood pumps and methods of use and manufacture
US11229784B2 (en) 2018-02-01 2022-01-25 Shifamed Holdings, Llc Intravascular blood pumps and methods of use and manufacture
US11964145B2 (en) 2019-07-12 2024-04-23 Shifamed Holdings, Llc Intravascular blood pumps and methods of manufacture and use
US11654275B2 (en) 2019-07-22 2023-05-23 Shifamed Holdings, Llc Intravascular blood pumps with struts and methods of use and manufacture
US11724089B2 (en) 2019-09-25 2023-08-15 Shifamed Holdings, Llc Intravascular blood pump systems and methods of use and control thereof

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