EP2612150A2 - Elisa für einen haptoglobin-matrix-metalloproteinase-9-komplex als diagnostischer test auf erkrankungen mit akuten entzündungen - Google Patents

Elisa für einen haptoglobin-matrix-metalloproteinase-9-komplex als diagnostischer test auf erkrankungen mit akuten entzündungen

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Publication number
EP2612150A2
EP2612150A2 EP11820374.4A EP11820374A EP2612150A2 EP 2612150 A2 EP2612150 A2 EP 2612150A2 EP 11820374 A EP11820374 A EP 11820374A EP 2612150 A2 EP2612150 A2 EP 2612150A2
Authority
EP
European Patent Office
Prior art keywords
mmp
serum
concentration
sample
inflammatory condition
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Withdrawn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
EP11820374.4A
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English (en)
French (fr)
Other versions
EP2612150A4 (de
Inventor
Jeffrey Lakritz
Grigori Andreevich Bannikov
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Ohio State University
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Ohio State University
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Filing date
Publication date
Priority claimed from US12/861,395 external-priority patent/US20120045778A1/en
Application filed by Ohio State University filed Critical Ohio State University
Publication of EP2612150A2 publication Critical patent/EP2612150A2/de
Publication of EP2612150A4 publication Critical patent/EP2612150A4/de
Withdrawn legal-status Critical Current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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • 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
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96486—Metalloendopeptidases (3.4.24)
    • G01N2333/96491—Metalloendopeptidases (3.4.24) with definite EC number
    • G01N2333/96494—Matrix metalloproteases, e. g. 3.4.24.7

Definitions

  • the present invention relates to methods of diagnosing or predicting acute inflammation and conditions including acute inflammation in a mammal.
  • Systemic inflammatory conditions e.g., sepsis
  • sepsis may result from an interaction between a pathogenic microorganism and the host's defense system that triggers an excessive and dysregulated inflammatory response in the host.
  • the complexity of the host's response during the systemic inflammatory response may complicate efforts towards understanding disease pathogenesis. And, this incomplete understanding of the disease pathogenesis, in turn, contributes to the difficulty in finding diagnostic biomarkers.
  • Early and reliable diagnosis is imperative, however, because of the remarkably rapid progression of sepsis into a life- threatening condition.
  • SIRS systemic inflammatory response syndrome
  • SIRS is often defined by the presence of two or more of the following parameters: a body temperature greater than 38°C or less than 36°C; a heart rate greater than 90 beats per minute; a respiratory rate greater than 20 breaths per minute; P C o2 less than 32 mm Hg; and a white blood cell count either less than 4.0 x 10 9 cells/L or greater than 12.0 x 10 9 cells/L, or having greater than 10% immature band forms.
  • SIRS body temperature greater than 38°C or less than 36°C
  • a heart rate greater than 90 beats per minute
  • a respiratory rate greater than 20 breaths per minute
  • P C o2 less than 32 mm Hg
  • white blood cell count either less than 4.0 x 10 9 cells/L or greater than 12.0 x 10 9 cells/L, or having greater than 10% immature band forms.
  • SIRS is commonly defined as SIRS coupled with a confirmed infectious process.
  • Severe sepsis is associated with MOF, hypotension, disseminated intravascular coagulation ("DIC”), or hypoperfusion abnormalities, including lactic acidosis, oliguria, and changes in mental status.
  • DIC disseminated intravascular coagulation
  • Septic shock is commonly defined as sepsis-induced hypotension that is resistant to fluid resuscitation with the additional presence of hypoperfusion abnormalities.
  • BRD bovine respiratory disease
  • NASHMS National Animal Health Monitoring System
  • APP acute phase proteins
  • haptoglobin (Hp) responses to inflammation in cattle have been evaluated in acute bronchopneumonia, [Eckersall PD, Young FJ, McComb C, Hogarth CJ, Safi S, Weber A, McDonald T, Nolan AM, Fitzpatrick JL (2001 ), Acute phase proteins in serum and milk from dairy cows with clinical mastitis, Vet.Rec. 148: 35-41 ; Humblet MF, Coghe J, Lekeux P, Godeau JM (2004), Acute phase proteins assessment for an early selection of treatments in growing calves suffering from bronchopneumonia under field conditions, Res.Vet.Sci.
  • Most existing sepsis scoring systems or predictive models predict only the risk of late-stage complications, including death, in patients who already are considered septic. Such systems and models, however, do not predict the development of sepsis itself.
  • What is further needed is a method to predict acute inflammation in humans that are not presenting symptoms and other mammals that are not presenting signs.
  • researchers will define a biomarker or biomarkers expressed at a different level in a group of septic patients versus a normal (i.e., non-septic) control group of patients.
  • 7,465,555 discloses a method of indicating early sepsis by analyzing time-dependent changes in the expression level of various biomarkers. Accordingly, optimal methods of diagnosing early sepsis currently require both measuring a plurality of biomarkers and monitoring the expression of these biomarkers over a period of time.
  • diagnosis would be made by a technique that accurately and rapidly measures a single biomarker at a single point in time, thereby allowing for early diagnosis and minimizing disease progression during the time required for diagnosis.
  • diagnosis would be made by a technique that accurately and rapidly measures a single biomarker at a single point in time, thereby allowing for early diagnosis and minimizing disease progression during the time required for diagnosis.
  • MMP 9 have been identified in neutrophil granules and the serum of cattle with acute septic inflammation of the abdomen or thorax [Bannikov GA, Mattoon JS, Abrahamsen EJ,
  • Hp-MMP 9 complexes are released from neutrophils upon degranulation [Bannikov GA, Mattoon JS, Abrahamsen EJ, Premanandan C, Green-Church KB, Marsh AE, Lakritz J (2007), Biochemical and enzymatic characterization of purified covalent complexes of matrix metalloproteinase -9 and haptoglobin released by bovine granulocytes in vitro, Am.J.Vet.Res. 68:995-1004].
  • Hp-MMP 9 matrix metalloproteinase 9
  • Hp-MMP 9 complexes of haptoglobin and matrix metalloproteinase 9
  • neutrophils in vitro and found in acute phase sera have specific functional significance that differs from un-complexed forms of free Hp and MMP 9 alone.
  • serum concentrations of Hp-MMP 9 may serve as an independent indicator of clinically important events occurring during acute inflammation.
  • Hp-MMP 9 complex ELISA in comparison to ELISA for un-complexed Hp or MMP 9 alone as an indicator of acute septic inflammatory disease in mammals, by providing a method to specifically identify systemic neutrophil activation in humans, cattle, and other mammals.
  • one aspect of the present invention provides a method of detecting whether a subject is suffering from an acute inflammatory condition or at risk of same by detecting the presence of a biomarker, or a particular concentration of a biomarker, in a sample from the subject.
  • the sample may be a blood sample, such as the serum fraction.
  • the biomarker may include a protein, such as a serum protein.
  • the biomarker may include the serum protein haptoglobin, or isoforms of the serum protein haptoglobin.
  • the biomarker may include a protein, such as a serum protein, in complex with an enzyme, such as a matrix metalloproteinase, such as matrix metalloproteinase 9 (an Hp-MMP 9 complex).
  • the biomarker may include various isoforms of the Hp-MMP 9 complex.
  • the method may include determining the concentration of the biomarker in samples collected from a subject. The method of this embodiment may also include comparing that concentration to a range of standard concentrations.
  • the method may include comparing the level of the biomarker in samples collected from the subject at different times.
  • the method may include comparing the biomarker with a reference biomarker from the same subject or a different subject.
  • test for performing the method described above.
  • the test may be an enzyme-linked immunosorbent assay
  • One such ELISA test embodiment is directed to a method for detecting one or more isoforms of an Hp-MMP 9 complex in a biological sample.
  • the method includes: (1 ) incubating a biological sample with a capture reagent immobilized on a solid support to bind multiple isoforms of Hp-MMP 9 to the capture reagent, wherein the capture reagent includes an antibody, such as a monoclonal antibody, that binds MMP9; and (2) detecting Hp-MMP 9 bound to the immobilized capture reagent by contacting the bound Hp-MMP 9 with a detectable antibody that binds to Hp.
  • Another ELISA test embodiment is directed to a process for identifying a patient with an acute inflammatory condition or at risk of an acute inflammatory condition by determining concentration of Hp-MMP 9 in a bodily fluid sample.
  • the process includes (a) obtaining monoclonal antibodies specific for MMP9 and attaching the monoclonal antibodies to a solid support; (b) obtaining a sample of a bodily fluid from a patient wherein the sample is suspected of containing Hp-MMP 9 and/or immunogenic fragments of Hp-MMP 9; (c) adding the sample to the monoclonal antibodies of step (a) wherein the Hp-MMP 9 and/or immunogenic fragments of Hp-MMP 9 contained in the sample is captured by the monoclonal antibodies; (d) providing second antibodies specific for Hp; (e) labeling the second antibodies with a detector; (f) adding the second antibodies of step (e) to the Hp-MMP 9 and/or immunogenic fragments of Hp-MMP 9 captured in step (c
  • Another ELISA test embodiment is directed to a method for detecting Hp-MMP 9 in plasma or serum to screen for an acute inflammatory condition or risk of same.
  • This test includes the steps of: (a) providing polyclonal or monoclonal antibodies against MMP9; (b) providing a microtiter plate coated with the antibodies; (c) adding the serum or plasma to the microtiter plate; (d) providing horseradish peroxidase-anti-Hp conjugates reactive with Hp to the microtiter plate; (e) providing hydrogen peroxide as a reactor to the microtiter plate; and (f) comparing the reaction which occurs as a result of steps (a) to (e) with a standard curve to determine the level of Hp-MMP 9 compared to a normal individual.
  • Fig. 1 is a graphical representation of ELISA data demonstrating the specificity of the MMP 9 and Hp-MMP 9 ELISA assays for free MMP 9 and Hp-MMP 9 complexes.
  • the graph is representative of results of 3 independent experiments.
  • the line with solid black circles (— ⁇ — ) represents absorbance for increasing concentrations of Hp-MMP 9 captured on MMP 9 monoclonal Ab and detected with anti-Hp HRP conjugate (Hp-MMP 9 standard curve).
  • the line with open circles (— o— ) represents wells where affinity purified Hp was added to the anti-MMP 9 coated wells followed by anti-Hp HRP conjugate.
  • the line having open, downward triangles represents wells where affinity purified MMP 9 was added to wells followed by anti-Hp HRP conjugate.
  • the line with open, upward triangles represents wells where Hp-MMP 9 complexes were added to wells, and HRP-conjugated anti-MMP 9 (clone 10.1 ) was added to wells (simulation of MMP 9 ELISA).
  • Fig. 2A is a plot of data for serum haptoglobin in cattle by disease classification
  • Fig. 2B is a plot of data for serum Hp-MMP 9 in cattle by disease classification
  • Open upward triangles ( ⁇ ) represent acute septic animals
  • downward triangles represent chronic/metabolic disease
  • diamonds (0) represent normal animals.
  • Box plots depict the median (solid line), mean (dashed green line), 5th percentile, 25th percentile, 75th percentile and 95th percentile. Filled triangles or diamonds depict outliers.
  • Fig. 2D shows the box plot data from Fig. 2A.
  • the box plot depicts the median
  • Fig. 2E shows the box plot data from Fig. 2B.
  • the box plot depicts the median
  • Fig. 2F shows the box plot data from Fig. 2C.
  • the box plot depicts the median
  • Fig. 3 is a graph showing the effect of lipopolysaccharide (LPS) infusion on Hp-
  • Fig. 4 is a graph showing the effect of LPS infusion on Hp-MMP 9 and serum
  • Fig. 5 is a graph showing the effect of LPS infusion on Hp-MMP 9 and band PMN
  • Fig. 6 is a graph showing the effect of LPS infusion on serum Hp-MMP 9 and Hp in cattle.
  • Fig. 7 is a graph showing the effect of LPS infusion on Hp-MMP 9 and serum a1- acid glycoprotein (AGP) in cattle.
  • Fig. 8 is a graph showing serum Hp-MMP 9 in experimental Mycoplasma bovis infection.
  • Fig. 9 is a graph showing the presence of serum Hp-MMP 9 in human patients having systemic lupus erythematosis, rheumatoid arthritis, or osteoarthritis.
  • Fig. 10 is a graph showing various white blood cell counts post injection of LPS for a first calf (calf 101 ).
  • Fig. 1 1 is a graph showing various white blood cell counts post injection of LPS for a second calf (calf 102).
  • Fig. 12 is a graph showing various white blood cell counts post injection of LPS for a third calf (calf 104).
  • Fig. 13 is a graph showing concentrations of neutrophils and Hp-MMP 9 in the calf of Fig. 10 (calf 101 ) post injection of LPS.
  • Fig. 14 is a graph showing concentrations of neutrophils and Hp-MMP 9 in the calf of Fig. 1 1 (calf 102) post injection of LPS.
  • Fig. 15 is a graph showing concentrations of neutrophils and Hp-MMP 9 in the calf of Fig. 12 (calf 104) post injection of LPS.
  • Hp-MMP 9 refers to a complex of haptoglobin and matrix metalloproteinase 9.
  • a complex is a group of two or more associated polypeptide chains - and herein the Hp-MMP 9 complex is a group of two associated polypeptide chains: the chain for haptoglobin and the chain for matrix metalloproteinase 9.
  • haptoglobin is a protein that in humans is encoded by the Hp gene. In blood plasma, haptoglobin binds free hemoglobin (Hb) released from erythrocytes with high affinity and thereby inhibits its oxidative activity.
  • MMPs matrix metalloproteinases
  • apoptotic ligands such as the FAS ligand
  • chemokine/cytokine in/activation MMPs are also thought to play a major role on cell behaviors such as cell proliferation, migration (adhesion/dispersion), differentiation, angiogenesis, apoptosis and host defense.
  • the MMPs share a common domain structure.
  • the three common domains are the pro-peptide, the catalytic domain and the haemopexin-like C-terminal domain which is linked to the catalytic domain by a flexible hinge region.
  • two - MMP2 and MMP9 - are gelatinases.
  • the main substrates of the gelatinases are type IV collagen and gelatin, and these enzymes are distinguished by the presence of an additional gelatin-binding domain inserted into the catalytic domain. This gelatin- binding region is positioned immediately before the zinc binding motif, and forms a separate folding unit which does not disrupt the structure of the catalytic domain.
  • detecting is used in the broadest sense to include both qualitative and quantitative measurements of a target molecule.
  • the detecting method as described herein is used to identify the mere presence of Hp-MMP 9 in a biological sample.
  • the method is used to test whether Hp-MMP 9 in a sample is at a particular level.
  • the method can be used to quantify the amount of Hp-MMP 9 in a sample and further to compare the Hp-MMP 9 levels from different samples or compare the amount of Hp-MMP 9 in a sample to reference standards.
  • biological sample refers to a body sample from any animal, such as any mammal, such as a human.
  • biological fluids such as serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid, saliva, sputum, lung lavage, tears, perspiration, mucus, and tissue culture medium, as well as tissue extracts such as homogenized tissue, and cellular extracts.
  • capture reagent refers to a reagent capable of binding and capturing a target molecule in a sample such that under suitable condition, the capture reagent-target molecule complex can be separated from the rest of the sample.
  • the capture reagent is immobilized or immobilizable.
  • the capture reagent may be an antibody or a mixture of different antibodies against a target antigen.
  • detectable antibody refers to an antibody that is capable of being detected either directly through a label amplified by a detection agent, or indirectly through, e.g., another antibody that is labeled.
  • the antibody is typically conjugated to a moiety that is detectable by some means.
  • One such antibody is an antibody conjugated to horse radish peroxidase.
  • detection agent refers to a moiety or technique used to detect the presence of the detectable antibody, and includes detection agents that amplify the immobilized label such as a label captured onto a microtiter plate. On such detection agent is hydrogen peroxide.
  • antibody is used in the broadest sense and includes monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired binding specificity.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. Nature 352:624- 628 (1991 ) and Marks et al. J. Mol. Biol. 222:581-597 (1991 ), for example.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic, and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc. In various aspects of the present invention, the mammal may be human or cattle.
  • Systemic inflammatory response syndrome refers to a clinical response to a variety of severe clinical insults, as manifested by two or more of the following conditions within a 24-hour period in a human: body temperature greater than 38°C. (100.4°F.) or less than 36°C. (96.8°F.); heart rate (HR) greater than 90 beats/minute; respiratory rate (RR) greater than 20 breaths/minute, or P C o2 less than 32 mm Hg, or requiring mechanical ventilation; and white blood cell count (WBC) either greater than 12.0x10 9 /L or less than 4.0x10 9 /L or having greater than 10% immature forms (bands).
  • body temperature greater than 38°C. (100.4°F.) or less than 36°C. (96.8°F.)
  • HR heart rate
  • RR respiratory rate
  • WBC white blood cell count
  • a subject with SIRS has a clinical presentation that is classified as SIRS, as defined above, but is not clinically deemed to be septic.
  • Sepsis refers to a SIRS-positive condition that is associated with a confirmed infectious process. Clinical suspicion of sepsis arises from the suspicion that the SIRS-positive condition of a SIRS patient is a result of an infectious process. As used herein, “sepsis” includes all stages of sepsis including, but not limited to, the onset of sepsis, severe sepsis and MOF associated with the end stages of sepsis.
  • the "onset of sepsis” refers to an early stage of sepsis, i.e., prior to a stage when the clinical manifestations are sufficient to support a clinical suspicion of sepsis. Because the methods of the present invention are used to detect sepsis prior to a time that sepsis would be suspected using conventional techniques, the patient's disease status at early sepsis can only be confirmed retrospectively, when the manifestation of sepsis is more clinically obvious. The exact mechanism by which a patient becomes septic is not a critical aspect of the invention. The methods of the present invention can detect changes in the biomarker profile independent of the origin of the infectious process. Regardless of how sepsis arises, the methods of the present invention allow for determining the status of a patient having, or suspected of having, sepsis or SIRS, as classified by previously used criteria.
  • hypoperfusion abnormalities include, but are not limited to, lactic acidosis, oliguria, or an acute alteration in mental status.
  • Steptic shock refers to sepsis-induced hypotension that is not responsive to adequate intravenous fluid challenge and with manifestations of peripheral hypoperfusion.
  • a “biomarker” is virtually any biological compound, such as a protein and a fragment thereof, a peptide, a polypeptide, a proteoglycan, a glycoprotein, a lipoprotein, a carbohydrate, a lipid, a nucleic acid, an organic on inorganic chemical, a natural polymer, and a small molecule, that is present in the biological sample and that may be isolated from, or measured in, the biological sample.
  • a biomarker can be the entire intact molecule, or it can be a portion thereof that may be partially functional or recognized, for example, by an antibody or other specific binding protein.
  • a biomarker is considered to be informative if a measurable aspect of the biomarker is associated with a given state of the subject, such as a particular stage of sepsis.
  • a measurable aspect may include, for example, the presence, absence, or particular concentration of the biomarker in the biological sample from the subject.
  • a "phenotypic change” is a detectable change in a parameter associated with a given state of the subject.
  • a phenotypic change may include an increase or decrease of a biomarker in a bodily fluid, where the change is associated with sepsis or the onset of sepsis.
  • a phenotypic change may further include a change in a detectable aspect of a given state of the patient that is not a change in a measurable aspect of a biomarker.
  • a change in phenotype may include a detectable change in body temperature, respiration rate, pulse, blood pressure, or other physiological parameter.
  • Such changes can be determined via clinical observation and measurement using conventional techniques that are well-known to the skilled artisan.
  • "conventional techniques” are those techniques that classify an individual based on phenotypic changes without obtaining a biomarker profile according to the present invention.
  • Isoform is understood to mean proteins that have undergone alternative splicing or post-translational modifications. For example, a cell will generally transcribe and translate one gene into many related proteins. The differences in the proteins, called isoforms, are a result of alterations in the originally translated protein. There are over 800 known post- translational modifications which include, but are not limited to, protein cleavage fragments, phosphorylation, glycosylation, lipidation, cross-linking, protein folding, hydrogen bond interactions with other molecules, Van der Waals force interactions with other molecules, and covalent interactions with other molecules, etc.
  • Hp-MMP 9 serum concentrations of Hp-MMP 9 may serve as an independent indicator of clinically important events occurring during acute inflammation.
  • the utility of Hp-MMP 9 complex ELISA in comparison to ELISA for un-complexed Hp or MMP 9 alone as an indicator of acute septic inflammatory disease in cattle is evaluated herein.
  • Serum Hp-MMP 9 complexes are present in animals with acute, septic inflammation and may therefore provide a means to specifically identify systemic neutrophil activation in humans, cattle, and other mammals.
  • one aspect of the present invention provides a method of detecting whether a subject is suffering from an acute inflammatory condition or at risk of same by detecting the presence of a biomarker, or a particular concentration of a biomarker, in a sample from the subject.
  • the sample may be a blood sample, such as the serum fraction.
  • the biomarker may include a protein, such as a serum protein.
  • the biomarker may include the serum protein haptoglobin, or isoforms of the serum protein haptoglobin.
  • the biomarker may include a protein, such as a serum protein, in complex with an enzyme, such as a matrix metalloproteinase, such as matrix metalloproteinase 9 (an Hp-MMP 9 complex).
  • the biomarker may include isoforms of the Hp-MMP 9 complex.
  • the method may include determining the concentration of the biomarker in samples collected from a subject. The method of this embodiment may also include comparing that concentration to a range of standard concentrations.
  • the method may include comparing the level of the biomarker in samples collected from the subject at different times.
  • the method may include comparing the biomarker with a reference biomarker from the same subject or a different subject.
  • biomarkers may include a protein, such as a serum protein, a nucleic acid or nucleotide, a carbohydrate, a lipid, a glycoprotein, a glycolipid, a protein fragment, a hormone, a steroid, a cytokine, a lymphokine, a chemokine, an immune modulator, a cell, a hematologic parameter, genomic expression from a cell in the sample, and combinations thereof.
  • a protein such as a serum protein, a nucleic acid or nucleotide, a carbohydrate, a lipid, a glycoprotein, a glycolipid, a protein fragment, a hormone, a steroid, a cytokine, a lymphokine, a chemokine, an immune modulator, a cell, a hematologic parameter, genomic expression from a cell in the sample, and combinations thereof.
  • the biomarker is an isoform of a serum protein, such as haptoglobin, transferrin, hemopexin, albumin, and combinations thereof.
  • the biomarker includes haptoglobin.
  • the biomarker may include a matrix metalloproteinase.
  • the biomarker may include complexes of proteins as described above.
  • the biomarker may include a complex of haptoglobin and matrix metalloproteinase 9 (Hp-MMP 9 complex).
  • the biomarker is an isoform of a protein.
  • samples used for detecting biomarkers include whole blood, fractions of blood, such as the serum fraction, the plasma fraction, or the cellular fraction, such as white blood cells or red blood cells, other fluids (i.e., saliva, urine, cerebral spinal fluid, bile, extracellular fluid, cytosolic fluid, etc.), cells, tissues (such as skin, bone, muscle, hair, etc.), and combinations thereof.
  • fractions of blood such as the serum fraction, the plasma fraction, or the cellular fraction
  • white blood cells or red blood cells such as white blood cells or red blood cells
  • other fluids i.e., saliva, urine, cerebral spinal fluid, bile, extracellular fluid, cytosolic fluid, etc.
  • cells such as skin, bone, muscle, hair, etc.
  • the biomarker and levels of biomarker may be detected with a compound that selectively binds the biomarker.
  • the selective compound can include a polyclonal antibody, a monoclonal antibody, an antibody fragment, a receptor, a phage display, a peptide, a protein fragment, a nucleic acid, a sequence of nucleic acids, a ribonucleic acid, a deoxyribonucleic acid, a small molecule, and combinations thereof.
  • the selective compound may be coupled to or associated with a detection system to indicate the level of the detected biomarker.
  • Exemplary detection systems may have elements that include, but are not limited to a test strip, chip, slide, microarray, titer plate, membrane, electrode, probe, bead, column, matrix, gel, and liquids.
  • detection may occur other than by binding the biomarker.
  • biomarker levels may alternatively be detected by analyzing a chemical parameter such as the size, charge, structure, and/or sequence of the biomarker.
  • the biomarker or levels of the biomarker may be detected using enzyme-linked immunosorbent assay (ELISA), gel electrophoresis, immunoprecipitation, radio-immunoassay, protein blotting, a test strip, chromatography, liquid chromatography, gas chromatography, binding assays, mass spectrometry, microarray, genomic microarray, polymerase chain reaction (PC ), Reverse transcription PCR (RT-PCR), real time RT-PCR, and combinations thereof.
  • ELISA enzyme-linked immunosorbent assay
  • gel electrophoresis immunoprecipitation
  • radio-immunoassay protein blotting
  • a test strip chromatography, liquid chromatography, gas chromatography, binding assays
  • mass spectrometry mass spectrometry
  • microarray genomic microarray
  • PC polymerase chain reaction
  • RT-PCR Reverse transcription PCR
  • real time RT-PCR real time RT-PCR
  • a comparison of the level of the biomarker may be detected by comparing the level of the biomarker in at least two samples collected from the subject.
  • the two samples can be collected at different times or from different sources.
  • the level of the biomarker from the first sample is compared with the level of the biomarker in the second sample to determine the presence of increased Hp-MMP 9 in the subject when the amount in the second taken sample is greater than in the first taken sample.
  • the level of the biomarker may be detected by comparing the level of the biomarker with the level of a reference biomarker. This could indicate the presence or absence of a condition, such as an acute inflammatory condition or risk of same.
  • the reference biomarker is from another subject.
  • Another subject in this context is understood to mean one or more other subjects and can represent the levels of reference biomarkers found in a population of subjects that are found to be indicative of either the presence or absence of an acute inflammatory condition or risk of same.
  • the invention also includes a kit for use in the methods described above for detecting the presence of Hp-MMP 9 in a subject.
  • Exemplary uses for the kit include determining the presence of an inflammatory condition or risk of same in the tested subject.
  • the kit includes devices and reagents configured to detect the presence of and levels of a biomarker (e.g., Hp-MMP 9) in a sample from the subject.
  • a biomarker e.g., Hp-MMP 9
  • Another aspect of the invention provides a test for performing the method described above.
  • the test may be an ELISA.
  • One ELISA test embodiment is directed to a method for detecting multiple isoforms of a haptoglobin-matrix m eta No proteinase 9 (Hp-MMP 9) complex in a biological sample.
  • the method includes: (1 ) incubating a biological sample with a capture reagent immobilized on a solid support to bind multiple isoforms of Hp-MMP 9 to the capture reagent, wherein the capture reagent includes an antibody, such as a monoclonal antibody, that binds MMP9; and (2) detecting Hp-MMP 9 bound to the immobilized capture reagent by contacting the bound Hp-MMP 9 with a detectable antibody that binds to Hp.
  • Another ELISA test embodiment is directed to a process for identifying a patient with an acute inflammatory condition or at risk of an acute inflammatory condition by determining concentration of Hp-MMP 9 in a bodily fluid sample.
  • the process includes (a) obtaining monoclonal antibodies specific for MMP9 and attaching the monoclonal antibodies to a solid support; (b) obtaining a sample of a bodily fluid from a patient wherein the sample is suspected of containing Hp-MMP 9 and/or immunogenic fragments of Hp-MMP 9; (c) adding the sample to the monoclonal antibodies of step (a) wherein the Hp-MMP 9 and/or immunogenic fragments of Hp-MMP 9 contained in the sample is captured by the monoclonal antibodies; (d) providing second antibodies specific for Hp; (e) labeling the second antibodies with a detector; (f) adding the second antibodies of step (e) to the Hp-MMP 9 and/or immunogenic fragments of Hp-MMP 9 captured in step (c
  • Another ELISA test embodiment is directed to a method for detecting Hp-MMP 9 in plasma or serum.
  • This test includes the steps of: (a) providing polyclonal or monoclonal antibodies against MMP9; (b) providing a microtiter plate coated with the antibodies; (c) adding the serum or plasma to the microtiter plate; (d) providing horseradish peroxidase-anti-Hp conjugates reactive with Hp to the microtiter plate; (e) providing hydrogen peroxide as a reactor to the microtiter plate; and (f) comparing the reaction which occurs as a result of steps (a) to (e) with a standard curve to determine the level of Hp-MMP 9 compared to a normal individual.
  • the assay described herein is a multi-site immunoassay.
  • the biological sample is contacted and incubated with an immobilized capture (or coat) reagent or reagents, which may be an anti-MMP9 monoclonal antibody.
  • an immobilized capture (or coat) reagent or reagents which may be an anti-MMP9 monoclonal antibody.
  • These antibodies may be from any species, but the monoclonal antibody is often a murine monoclonal antibody.
  • the monoclonal antibodies may be prepared by methods well known to those of ordinary skill in the art. For example, a mixture of affinity purified MMP 9 monomer and dimer produced by bovine neutrophils were used for immunization of BALB/C mice.
  • mice were immunized twice with ⁇ ⁇ of purified MMP 9, over a 7 day interval. Four weeks later, mice were boosted with 10 ⁇ of MMP 9 daily for 4 days and 4 days later, splenocytes were fused to the B cell myeloma SP2/0 and plated in 96 well plates.
  • Hybridomas were selected in the presence of 100 ⁇ hypoxanthine, 0.4 ⁇ aminopterin, and 16 ⁇ thymidine following established methods [as described in Kohler G, Milstein C (1975), Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256: 495- 497, incorporated by reference herein in its entirety], well known to those of ordinary skill in the art. Clones that secreted antibodies with specificity for MMP 9 as tested by ELISA were further cloned by limiting dilution twice in order to ensure clonality [as described in Kohler G, Milstein C (1975), Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256:495-497, incorporated by reference herein]. Clone 10.1 was selected for positive reactivity and cellular amplification in a bioreactor [Celline, CL1000, Sartorius Stedim, North America, Inc. Bohemia NY 1 1716], which was performed as follows.
  • the hybridoma cells were maintained in the cell compartment of the CL 1000 and harvested at approximately 7 day intervals. During harvest, cells and supernatant were collected from the cell compartment, a fraction of the harvest was reinoculated into the cell compartment with fresh medium. Nutrient medium was removed and replaced with fresh medium on day of harvest.
  • Murine hybridoma cell lines were thawed from frozen stocks and expanded in static culture (RPMI-1640, 10-15% FBS, 2X L-Glutamine, Pen- Strep). After demonstration of consistent cell doubling in static culture, cells were inoculated into the CL 1000 devices.
  • Basal medium was supplemented with 10% FBS (commercially available from Hyclone, Logan Utah). Additional supplementation of medium with a hybridoma growth supplement (0.1 % Vitacyte, J. Brooks Irvine, CA)) was done to remain consistent with prior batch production runs of the same cell lines in traditional flasks.
  • Cells were inoculated from static culture at Day 0 in a 20 ml volume into the cell compartment of the CL 1000 devices. Inoculation density was maintained above 3.0 x 10 6 cells/ml. Cells were removed from frozen stock initiated cultures and resuspended in fresh cell compartment medium prior to inoculation. Nutrient medium (1000 ml) was supplied to the nutrient medium compartment and the devices placed into a 5% C02, 37°C humidified tissue culture incubator.
  • the total cell compartment volume was removed from the CL 1000 units by pipette.
  • Cell numbers were determined by diluting and counting samples using a standard hemacytometer. Viable cells were discriminated from non-viable cells by trypan blue staining and phase contrast microscopy. Cell compartment contents were split back between 3- 5 fold determined by cell numbers in the cell compartment at time of harvest.
  • Fresh cell compartment medium (17-15 ml) was added to the cell fraction (3-5 ml) to achieve a 20 ml volume and the cell suspension returned to the cell compartment.
  • the harvested cell containing supernatant fraction was kept sterile and stored at 4°C until purification by affinity
  • Culture supernatant was processed by eluting antibody from protein A affinity chromatography columns following manufacturers protocol. Eluted antibody fractions were collected, pooled and antibody quantified by spectrophotometer and ELISA. Sandwich ELISA was performed with polyclonal goat anti-mouse IgG or IgM capture antibody and polyclonal anti- mouse IgG or IgM antibody labeled with peroxidase. Color was developed with ABTS. Antibody purity was assessed by SDSPAGE and Coomassie blue staining.
  • Immobilization conventionally is accomplished by insolubilizing the capture reagents either before the assay procedure, as by adsorption to a water-insoluble matrix or surface (as described in U.S. Pat. No. 3,720,760) or non-covalent or covalent coupling [for example, using glutaraldehyde or carbodiimide cross-linking, with or without prior activation of the support with, e.g., nitric acid and a reducing agent as described in U.S. Pat. No. 3,645,852 or in Rotmans et al. J. Immunol. Methods 57:87-98 (1983)], or afterward, e.g., by
  • the solid phase used for immobilization may be any inert support or carrier that is essentially water insoluble and useful in immunometric assays, including supports in the form of, e.g., surfaces, particles, porous matrices, etc.
  • supports in the form of, e.g., surfaces, particles, porous matrices, etc.
  • commonly used supports include small sheets, Sephadex®, polyvinyl chloride, plastic beads, and assay plates or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like including 96-well microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S.
  • Pat. Nos. 3,969,287; 3,691 ,016; 4,195, 128; 4,247,642; 4,229,537; and 4,330,440 are suitably employed for capture reagent immobilization.
  • the immobilized capture reagents are coated on a microtiter plate, and in particular one exemplary solid phase used is a multi-well microtiter plate that can be used to analyze several samples at one time.
  • One such ELISA plate is that sold as Dynatech Immobilon II (commercially available from Dynatech Laboratories, Chantily, VA).
  • the solid phase is coated with the capture reagent(s), such as those described above, which may be linked by a non-covalent or covalent interaction or physical linkage as desired.
  • Techniques for attachment include those described in U.S. Pat. No. 4,376,1 10 and the references cited therein. If covalent, the plate or other solid phase is incubated with a cross- linking agent together with the capture reagent under conditions well known in the art (such as for 1 hour at room temperature).
  • cross-linking agents for attaching the pre-mixed capture reagents to the solid phase substrate include, e.g., 1 ,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis
  • succinimidylpropionate and bifunctional maleimides such as bis-N-maleimido-1 ,8-octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates capable of forming cross-links in the presence of light.
  • 96-well plates are utilized, they are generally coated with the mixture of capture reagents (typically diluted in a buffer such as T IS buffered saline by incubation for at least about 12 hours, at temperatures of about 3°C to 4°C, and at a pH of about 7.5.
  • a buffer such as T IS buffered saline
  • the plates may be stacked and coated long in advance of the assay itself, and then the assay can be carried out simultaneously on several samples in a manual, semiautomatic, or automatic fashion, such as by using robotics.
  • the coated plates are then typically treated with a blocking agent that binds non- specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate.
  • a blocking agent that binds non- specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate.
  • appropriate blocking agents include, e.g., gelatin, bovine serum albumin (BSA), egg albumin, casein, and non-fat milk.
  • BSA bovine serum albumin
  • TBS or SuperBlock Blocking Buffer are used as blocking agents.
  • the blocking treatment typically takes place under conditions of ambient temperatures for about 1-4 hours.
  • the biological sample to be analyzed is added to the immobilized phase.
  • the dilution rate is about 2.5% (1 :40) to 20% (1 :5 dilution), such as about 10%, by volume.
  • Buffers that may be used for dilution for this purpose include TBS + 1 mg/mL BSA.
  • the amount of capture reagents employed is sufficiently large to give a good signal in comparison with the Hp-MMP 9 standards.
  • the amount of biological sample added is such that the immobilized capture reagents are in molar excess of the maximum molar concentration of free Hp-MMP 9 anticipated in the biological sample after appropriate dilution of the sample. This anticipated level depends mainly on any known correlation between the concentration levels of the free Hp-MMP 9 in the particular biological sample being analyzed with the clinical condition of the patient. In general, the concentration of all reagents should be tittered to obtain optimal results of the ELISA.
  • the concentration of the capture reagents will generally be determined by the concentration range of interest of the Hp-MMP 9 taking any necessary dilution of the biological sample into account, the final concentration of the capture reagents is determined empirically to maximize the sensitivity of the assay over the range of interest.
  • the conditions for incubation of sample and immobilized capture reagent are selected to maximize sensitivity of the assay and to minimize dissociation.
  • the incubation is generally accomplished at fairly constant temperatures, ranging from about 3°C to about 5°C, to obtain a less variable, lower coefficient of variant (CV) than at, e.g., room temperature.
  • the time for incubation depends primarily on the temperature, being generally no greater than about 2 hours to avoid reduction of sensitivity of the assay. Generally, the incubation time is from about 2 hours at 3°C to 5°C to maximize binding of free Hp-MMP 9 to capture reagents.
  • the pH of the incubation mixture will ordinarily be in the range of about 7.2 - 7.5.
  • the pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagents to the Hp-MMP 9 being captured.
  • Various buffers may be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, Tris-HCI or Tris-phosphate, acetate, barbital, and the like.
  • the particular buffer employed is not critical to the invention.
  • the biological sample is separated (by washing) from the immobilized capture reagents to remove uncaptured Hp-MMP 9.
  • the solution used for washing is generally a buffer ("washing buffer") with a pH determined using the considerations and buffers described above for the incubation step, with a pH range of about 7.2-7.5.
  • the washing may be done three or more times.
  • the temperature of washing is generally from refrigerator to moderate temperatures, with a constant temperature maintained during the assay period, typically from about 22°C to 25°C.
  • any immobilized complexes are contacted with detectable antibodies, such as at a temperature of about 22°C to 25°C, with the temperature and time for contacting the two being dependent primarily on the detection agent employed.
  • detectable antibodies such as at a temperature of about 22°C to 25°C, with the temperature and time for contacting the two being dependent primarily on the detection agent employed.
  • H P horseradish peroxidase
  • the contacting is generally carried out for 1 hour to 2 hours to amplify the signal to the maximum.
  • the detectable antibody may be a polyclonal or monoclonal antibody.
  • the detectable antibody may be directly detectable, and may have a fluorimetric or colorimetric label.
  • a molar excess of an antibody with respect to the maximum concentration of captured Hp-MMP 9 expected is added to the plate after it is washed.
  • This antibody (which is directly or indirectly detectable) may be a polyclonal antibody, although any antibody can be employed.
  • the affinity of the antibody is generally sufficiently high that small amounts of the free Hp-MMP 9 can be detected, but not so high that it causes nonspecific background binding of the detectable antibodies.
  • the amount of Hp-MMP 9 that is now bound to the capture reagents is measured using a detection agent for the detectable antibody.
  • the antibody added to the immobilized capture reagents will be either directly labeled, or detected indirectly by addition, after washing off of excess first unlabeled detectable antibody, of a molar excess of a second, labeled antibody directed against the first antibody.
  • the label used for either the first or second antibody may be any detectable functionality that does not interfere with the binding of free Hp-MMP 9 to the antibody.
  • suitable labels are those numerous labels known for use in immunoassay, including moieties that may be detected directly, such as fluorochrome, chemiluminscent, chromogenic, and radioactive labels, as well as moieties, such as enzymes, that can be reacted or derivatized to be detected.
  • labels include horseradish peroxidase (H P), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, biotin/streptavidin, biotin/Streptavidin-3-galactosidase with MUG, spin labels, bacteriophage labels, stable free radicals, the radioisotopes 32 P, 14 C, 125 l, 3 H, and 131 1, fluorophores such as rare earth cheats or fluorescein and
  • the amount of bound antibody is determined by removing excess unbound labeled antibody through washing and then measuring the amount of the attached label using a detection method appropriate to the label, and correlating the measured amount with the amount of free Hp-MMP 9 in the biological sample.
  • a detection method appropriate to the label for example, in the case of enzymes, the amount of color developed and measured will be a direct measurement of the amount of Hp-MMP 9 present.
  • the assay method of this invention can be provided in the form of a kit.
  • a kit is a packaged combination including the basic elements of: (1 ) capture reagents comprised of polyclonal and monoclonal antibodies against human Hp-MMP 9 molecule; and (2) detection reagents comprised of detectable (labeled or unlabeled) antibodies that bind to Hp-MMP 9.
  • the kit further comprises a solid support for the capture reagents, which may be provided as a separate element or on which the capture reagents are already immobilized.
  • the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such support that is included with the kit or provided separately from the kit.
  • the capture reagents may be coated on a microtiter plate.
  • the detection reagent may be labeled antibodies detected directly or unlabeled antibodies that are detected by labeled antibodies directed against the unlabeled antibodies raised in a different species.
  • the kit will ordinarily include substrates and cofactors required by the enzyme, and where the label is a fluorophore, a dye precursor that provides the detectable chromophore.
  • the detection reagent is unlabeled, the kit may further comprise a detection agent for the detectable antibodies, such as the labeled antibodies directed to the unlabeled antibodies, such as in a fluorimetric-detected format.
  • the kit also typically contains instructions for carrying out the assay, and/or Hp- MMP 9 as an antigen standard (e.g., purified Hp-MMP 9), as well as other additives such as stabilizers, washing and incubation buffers, and the like.
  • Hp- MMP 9 as an antigen standard (e.g., purified Hp-MMP 9), as well as other additives such as stabilizers, washing and incubation buffers, and the like.
  • the components of the kit may be provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for combining with the sample to be tested.
  • the present example describes an ELISA that can be used as a test for Hp-MMP 9 complexes, as well as ranges of Hp-MMP 9 complex concentration that can be used as an indicator of an acute inflammatory condition, or risk of same.
  • 35 cattle were tested in this Example.
  • the animals were those admitted to The Ohio State University VTH for evaluation of a variety of disorders (22 animals) or sampled from the Ohio State University Waterman Dairy (13 animals). Animals were either bled for routine clinico-pathologic work-up or as part of a herd infectious disease survey. Serum was harvested in routine fashion by centrifugation after clotting and frozen at -20°C until analyzed.
  • the individual analyzing the samples by ELISA was not aware of the individual animal's identity or classification and was only given frozen serum samples with the animal's laboratory designation.
  • the animals selected for inclusion were done so under the guidance of The Ohio State University VTH Hospital Executive Committee clinical investigation approval (Jan 1 , 2006 to Jan 1 , 2008) and an approval of laboratory studies obtained from The Ohio State University Institutional Animal Care and Use committee.
  • [00135] A mixture of affinity purified MMP 9 monomer and dimer produced by bovine neutrophils were used for immunization of BALB/C mice. Mice were immunized twice with 100 ⁇ g of purified MMP 9, over a 7 day interval. Four weeks later, mice were boosted with 100 ⁇ g of MMP 9, sacrificed after 4 days, and splenocytes were fused to the B cell myeloma SP2/0 and plated in 96 well plates.
  • Hybridomas were selected in the presence of 100 ⁇ hypoxanthine, 0.4 ⁇ aminopterin, and 16 ⁇ thymidine following established methods [as described in Kohler G, Milstein C (1975), Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256:495-497, incorporated by reference herein in its entirety], well known to those of ordinary skill in the art. Clones that secreted antibodies with specificity for MMP 9 as tested by ELISA were further cloned by limiting dilution twice in order to ensure clonality [as described in Kohler G, Milstein C (1975), Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256:495-497].
  • Clone 10.1 was selected for positive reactivity and cellular amplification in a bioreactor [Celline, CL1000, Sartorius Stedim, North America, Inc. Bohemia NY 1 1716] was performed as previously described. The reactivity of the hybridoma supernatant was tested using western immunoblot of purified monomer and dimer under reducing and non-reducing conditions and with enzymatically activated MMP 9 monomer and dimer. In addition, whole cell lysates from bovine neutrophils, and from peripheral blood leukocytes, were immunoblotted with the MAb.
  • Immunoglobulin class (lgG1 ) of the MAb was determined using a commercial MAb isotyping kit [Pierce Rapid ELISA Mouse mAb isotyping kit, commercially available from Thermo Fisher Scientific, Rockford, IL 61 105]. IgG from the hybridoma media was purified with Protein G agarose [commercially available from Thermo Fisher Scientific, Rockford IL 61 105] and stored at concentration of 1 mg/ml at 4°C.
  • the resulting antibody-HRP conjugate was purified by gel-filtration on Ultragel AcA 34 (Sigma, U8878) [commercially available from Sigma-Aldrich Co., St. Louis, MO 63178] and stored at 4°C at a concentration 60 ⁇ g ml.
  • Serum Hp ELISA - Serum Hp concentrations were determined using commercial Bovine haptoglobin ELISA test kits [Bovine haptoglobin 96-well ELISA, commercially available from Life Diagnostics, West Chester PA 19380], according to manufacturers instructions.
  • Standard curves were prepared using purified bovine haptoglobin standard (2.5 ⁇ g/mL) included with the kit at a concentration range from 7.8 - 500 ng/mL. Serum samples were diluted according to the kit instructions (1 :2,000 dilution) or used at lower dilution for samples containing low concentration of Hp and were run in duplicate. Controls included were normal bovine serum, 5% BSA in TBS, and blank wells.
  • Serum MMP 9 ELISA The ELISA for bovine MMP 9 was developed in the laboratory, exploiting un-conjugated bovine MMP 9 MAb 10.1 as a capture antibody (100 ⁇ , containing ⁇ g per well) and HRP conjugated 10.1 antibody (0.3 ⁇ g/ml in TBS, 1 mg/ml BSA) for detection of bound MMP 9. These concentrations were chosen on basis of preliminary experiments.
  • the plates were washed 3 x 5 minutes with TBS and the wells blocked with bovine serum albumin [Bovine Serum Albumin, Fraction V, commercially available from Fisher Scientific, Thermo Fisher Scientific, Pittsburg PA 15275], at a concentration of 20 mg/ml in TBS or with Super Block Blocking Buffer [Pierce Super Block Blocking buffer, commercially available from Thermo Fisher Scientific, Rockford IL 61 105].
  • bovine serum albumin Bovine Serum Albumin, Fraction V, commercially available from Fisher Scientific, Thermo Fisher Scientific, Pittsburg PA 15275
  • Super Block Blocking Buffer commercially available from Thermo Fisher Scientific, Rockford IL 61 105.
  • Serum samples from sick and healthy animals were analyzed by serially diluting each sample with TBS containing 1 mg/ml BSA in duplicate. Once each set of samples was analyzed, and the absorbance value from the dilution which lay within the linear range of the standard curve was used to quantitate MMP 9 concentrations.
  • sera were serially diluted with PBS, containing 1 mg/ml BSA and run in duplicate. Controls included were normal bovine serum, 5% BSA in TBS and blank wells.
  • Serum Hp-MMP 9 complex ELISA - Purified MAb 10.1 was used as a capture antibody as described above (100 ⁇ , containing ⁇ g per well).
  • the detection antibody chosen was an affinity purified HRP-conjugated rabbit anti-bovine haptoglobin [obtained from Immunology Consultants Laboratory, RHPT-10P, Newberg, OR 97132] used at a final concentration of 0.033 ⁇ g/ml in TBS, 1 mg/ml BSA. These concentrations of both capture and detection antibodies were chosen on basis of preliminary experiments.
  • Purified bovine Hp-MMP 9 complexes were used to create standard curves using concentrations of 40 - 5000 ng/ml. Samples registering lower than 40 ng/mL were considered to be zero ng/mL. Once each set of samples were analyzed, the absorbance value from the dilution which lay within the linear range of the standard curve was used to quantitate Hp-MMP 9 concentrations. For analysis sera were serially diluted with PBS, containing 1 mg/ml BSA and run in duplicate. Controls included were normal bovine serum, 5% BSA in TBS and blank wells.
  • Eluates were diluted to 6 ml with the same buffer, without DMSO and concentrated to 300 ⁇ in iCon concentrators [Pierce Concentrators, 9K MWCO, 7 ml_, commercially available from Thermo Fisher Scientific. Rockford, IL 61 105]. After dilution to 2 ml samples were again concentrated to 200 ⁇ in iCon concentrators. Samples were diluted with 4X LDS sample buffer and electrophoresed through 4- 20% Tris-Glycine gels
  • Hp and MMP 9 bands were visualized by labeling with rabbit anti-bovine haptoglobin [Rabbit anti-bovine haptoglobin polyclonal antibody, commercially available from ICL, Inc., Newburg, OR 97132] and goat anti-human MMP 9 [Goat anti-human MMP 9 affinity purified polyclonal AB, commercially available from R & D Systems Inc., Minneapolis, MN 55413].
  • HRP-conjugated anti-Rabbit and ant-Goat antibodies were used as secondary antibody followed by ECL visualization [20X Lumiglo reagent and 20x peroxide, commercially available from Cell Signaling Technology, Danvers, MA 01923]. [00147] Statistical analysis
  • Animals were categorized by either (1 ) clinical diagnosis based upon the findings of clinical examination, laboratory, or other diagnostics, or (2) postmortem diagnosis based upon postmortem examination. Based on the results of those diagnoses, the animals were designated as acute septic disease; chronic or metabolic disease; or normal. Acute septic disease animals were desingated with the number "1 ,” chronic or metabolic disease animals were designated with the numeral "2,” and normal animals were designated with the numeral "3.”
  • ELISA assays were performed by an individual without prior knowledge of the disease classification. Data from each animal, for each of the 3 ELISA assays were then placed into the spreadsheet by animal ID (Numbers 1-35).
  • the anti-bovine MMP 9 monoclonal antibody (Clone 10.1 ) reacted positively in Western blot with bovine MMP 9 monomer and dimer whether reduced or not and after enzymatic activation. Clone 10.1 antibody also reacted positively in Western blot with recombinant MMP 9 of human origin. Analytical sensitivity of both MMP 9 ELISA and Hp-MMP 9 complex ELISA used in this study was 39 ng/ml.
  • Fig. 2B all normal and 8 of 10 disease classification 2 animals had no measureable serum Hp - MMP 9 concentrations. Animals grouped in disease classification 1 had measureable concentrations which were significantly greater than animals in Disease classification 2 and 3. Two disease classification 2 animals had elevated serum Hp-MMP 9 concentrations (Fig. 2B). One animal was diagnosed with thymic lymphoma and the other possibly had an acute disease that was not detected clinically. In acute septic animals, 14/15 animals possessed high serum concentrations of Hp-MMP 9 complexes (Fig. 2B). And as will be discussed, in Fig. 1 C, animals in disease classification 1 and 2 had significantly greater serum MMP 9 than animals in disease classification 3. The serum concentrations of MMP 9 in disease classification 1 and 2 animals were not significantly different.
  • inflammation/metabolic disease animals (median 0 ng/mL; 25th, 75th percentiles:0,0 ng/mL; p ⁇ 0.0031 ) and in normal animals (median 0 ng/mL; 0,0 25th, 75th percentiles: 0,0 ng/mL;
  • Table 1 C Disease classification 3 - Normal animal population of dairy cattle analyzed for serum haptoglobin, MMP 9 and Hp-MMP
  • MMP 9 matrix metalloproteinase 9
  • Hp haptoglobin
  • Hp- MMP 9 haptoglobin-matrix metalloproteinase 9
  • Hp-MMP 9 in conditioned media from phorbol ester stimulated bovine neutrophils in vitro and the observation of the presence of Hp-MMP 9 in serum of septic cattle but not in serum chronically inflamed or healthy cows [Bannikov GA, Mattoon JS, Abrahamsen EJ, Premanandan C, Green-Church KB, Marsh AE, Lakritz J (2007), Biochemical and enzymatic characterization of purified covalent complexes of matrix metalloproteinase -9 and haptoglobin released by bovine granulocytes in vitro, Am. J. Vet. Res. 68:995-1004].
  • the design of this study also included measurement of serum concentrations of two individual components of Hp-MMP 9 complex: Hp, which is a major acute phase protein in cattle and MMP 9 which concentrates in serum of cattle undergoing inflammation.
  • serum Hp-MMP 9 complexes in cattle with acute septic disease do not distort data for Hp-ELISA, because the serum concentrations of Hp-MMP 9 complexes are present in concentrations that are 3 orders of magnitude lower than the concentrations of Hp.
  • MMP 9 is stored pre-formed in granules in peripheral blood neutrophils [Bannikov GA, Mattoon JS, Abrahamsen EJ, Premanandan C, Green-Church KB, Marsh AE, Lakritz J (2007), Biochemical and enzymatic characterization of purified covalent complexes of matrix metalloproteinase-9 and haptoglobin released by bovine granulocytes in vitro, Am. J. Vet. Res.
  • Hp is recognized as an indicator of acute inflammation in cattle
  • moderate increases of serum Hp have been described for cows with hepatic lipidosis, despite having no clinically apparent signs of inflammation
  • concentration in sera of healthy cows is negligible (approximately 20 ng/mL or lower) but can increase in acute inflammation to values as high as 950 ⁇ g/mL.
  • Hp-MMP 9 ELISA Data from the current study demonstrated a significant diagnostic advantage of the Hp-MMP 9 ELISA over the Hp and MMP 9 ELISA assays. There are significant differences in serum Hp-MMP 9 concentrations observed in cattle with acute septic disease compared to those animals with chronic inflammatory/metabolic disease or healthy animals. The data suggest that the Hp-MMP 9 assay is specific for acute, septic diseases. Of the 15 animals identified clinically as having acute inflammation, 14 animals had high serum concentrations of Hp-MMP 9. Only 1 of 15 animals had serum concentrations of Hp-MMP 9 complexes ⁇ 39 ng/mL (interpreted as 0). This cow (# 8; Table 1A), had recently calved and developed
  • Hp and MMP 9 Serum concentrations of Hp and MMP 9 were within the ranges of those observed in the serum of both disease category 2 and 3 animals. However, this animal had been treated with Predef 2XTM (isoflupredone acetate) for 3 days.
  • Predef 2XTM isoflupredone acetate
  • the lack of serum Hp- MMP 9 complexes may be related to prior corticosteroid treatment since corticosteroids have profound effects on inflammation in vitro and in vivo [Cohn.L.A. (2003), The influence of corticosteroids on host defense mechanisms, J. Vet. Intern. Med.. 5:95-104].
  • Hp-MMP 9 complex ELISA for acute septic condition is further corroborated by its presence in only 2 of 12 chronically ill animals.
  • One of these two cases (Table 1 B, #16) was diagnosed with thymic lymphoma, without any pathologically described necrosis or bacterial infection.
  • the presence of the Hp-MMP 9 complexes in cattle with neoplasia should be investigated.
  • a second Hp-MMP 9 positive animal was placed into disease classification 2 based upon clinical findings (Table 1 B, # 19). Although it did possess serum Hp- MMP 9 complexes, the lack of diagnostic testing performed on this particular animal prior to discharge, precludes an explanation of the presence of these serum Hp-MMP 9 complexes.
  • Hp-MMP 9 ELISA Another favorable feature of the Hp-MMP 9 ELISA was the narrow range of concentrations in the sera of acutely septic animals: median concentrations of Hp-MMP 9 in animals classified clinical as acute septic were 1 ,014 ng/mL (with 25th, 75th percentiles ranging from 586 - 1373 ng/mL). Median values for the chronic inflammation/metabolic disease cases were 0 ng/mL. The relatively narrow range of serum Hp-MMP 9 complex concentrations in acutely septic animals enhances its diagnostic utility.
  • Hp-MMP 9 ELISA may be beneficial for the diagnosis of early events in acute septic conditions in the bovine.
  • Intravenous LPS infusion induces changes in serum Cortisol, white blood cell activation and Hp-MMP 9.
  • Serum Hp-MMP 9 complexes were absent in 26/28 animals prior to the start of the study.
  • One animal with detectable serum Hp-MMP 9 also had elevated serum cardiac troponin I at the start of the study suggesting thoracic inflammation.
  • the other animal had elevated serum haptoglobin on day 14 suggestive of chronic, non-specific inflammatory abnormalities.
  • Serum Hp-MMP 9 complexes were detected in 4/28 animals on day 7 and 18/28 animals on day 14 (See Figure 8).
  • This Example researches serum Hp-MMP 9 complexes in human patients with auto-immune diseases in comparison to osteoarthritis.
  • the serum Hp-MMP 9 ELISA assay was performed on these samples blinded and the data were returned to Cedar Sinai Medical Center. The ELISA test performed on the serum samples is as described above in Example 1 , though those of ordinary skill in the art will recognize that parameters can be modified (and validated), such as is described below in Example 5. After return of the data, Dr. Wiseman then provided the disease process associated with the tube number so that the disease processes were matched to serum Hp-MMP 9 concentrations.
  • This Example is directed to further methods for modifying and validating of the Hp-MMP 9 ELISA described herein.
  • Serum with known Hp-MMP 9 concentrations is currently being used to optimize ELISA conditions.
  • Serum samples (serial dilutions) used as calibration curve were prepared and checkerboard titrations conducted to evaluate coating antibody concentration (2 ug/well to 64 ug/well) and detection antibody (1 :5,000 to 1 : 160,000 fold dilutions). This demonstrated that coating antibody concentrations >2 ug/well are sufficient and that the standard curves are linear from 1 :5K (0.2 ng/mL) to 1 :160K (0.0063ng/mL) detection antibody. However, as the concentration of secondary antibody declines, the slope of the line and dynamic range of calibration curve decreases from 1 :5K to 1 :160K.
  • Hp-MMP 9 complex ELISA is potentially useful in the diagnosis of other conditions, differing from acute sepsis, but associated with neutrophil degranulation. These conditions may include various types of arthritis, atherosclerosis, coronary plaque formation, etc. Notwithstanding the above, certain variations and modifications, while producing less than optimal results, may still produce satisfactory results. All such variations and modifications are intended to be within the scope of the present invention.

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EP11820374.4A 2010-08-23 2011-08-11 Elisa für einen haptoglobin-matrix-metalloproteinase-9-komplex als diagnostischer test auf erkrankungen mit akuten entzündungen Withdrawn EP2612150A4 (de)

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US12/861,395 US20120045778A1 (en) 2010-08-23 2010-08-23 Elisa for haptoglobin-matrix metalloproteinase 9 complex as a diagnostic test for conditions including acute inflammation
US201161472815P 2011-04-07 2011-04-07
PCT/US2011/047425 WO2012027127A2 (en) 2010-08-23 2011-08-11 Elisa for haptoglobin-matrix metalloproteinase 9 complex as a diagnostic test for conditions including acute inflammation

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* Cited by examiner, † Cited by third party
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Bannikov et al: "Dissecting serum Haptoglobin: A subset of serum haptoglobin is produced by neutrophils and is present in cattle with sepsis.", Ohio Dairy Veterinarians Meeting. , 1 January 2007 (2007-01-01), XP002721954, Retrieved from the Internet: URL:http://www.ohiodairyvets.org/wp-content/uploads/2007/09/abstracts-january-2007.pdf *
BANNIKOV G A ET AL: "Serum haptoglobin-matrix metalloproteinase 9 (Hp-MMP 9) complex as a biomarker of systemic inflammation in cattle", VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY, ELSEVIER BV, AMSTERDAM, NL, vol. 139, no. 1, 1 January 2011 (2011-01-01), pages 41-49, XP027561045, ISSN: 0165-2427, DOI: 10.1016/J.VETIMM.2010.08.004 [retrieved on 2010-08-14] *
HINDS C A ET AL: "SERUM HAPTOGLOBIN (HP)-MATRIX METALLOPROTEINASE 9 (MMP 9) COMPLEX ELISA AS A MARKER OF SEPTIC INFLAMMATION IN CATTLE: A COMPARISON TO HP OR MMP 9 ELISA", JOURNAL OF VETERINARY INTERNAL MEDICINE, vol. 24, no. 3, May 2010 (2010-05), page 713, XP002721953, & ANNUAL ACVIM FORUM; ANAHEIM, CA, USA; JUNE 09 -12, 2010 ISSN: 0891-6640 *
See also references of WO2012027127A2 *

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