WO2011098645A1 - Use of apo j isoforms as tissue lesion biomarkers - Google Patents

Use of apo j isoforms as tissue lesion biomarkers Download PDF

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Publication number
WO2011098645A1
WO2011098645A1 PCT/ES2011/070080 ES2011070080W WO2011098645A1 WO 2011098645 A1 WO2011098645 A1 WO 2011098645A1 ES 2011070080 W ES2011070080 W ES 2011070080W WO 2011098645 A1 WO2011098645 A1 WO 2011098645A1
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Prior art keywords
apo
glycosylated
damage
detected
approximately
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PCT/ES2011/070080
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Spanish (es)
French (fr)
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Lina Badimon Maestro
Judit Cubedo Rafols
Teresa PADRÓ CAPMANY
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Consejo Superior De Investigaciones Científicas (Csic)
Institut Catalá De Ciencies Cardiovasculars (Iccc)
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Publication of WO2011098645A1 publication Critical patent/WO2011098645A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • the present invention is within the field of biomedicine. Specifically, it refers to the use of glycosylated forms of apolipoprotein J (Apo J) as markers of tissue damage and acute inflammation, more specifically produced by acute myocardial infarction (AMI), as well as a method of diagnosing such damage and / or inflammation and a kit comprising the necessary elements to carry out said diagnosis.
  • AMI acute myocardial infarction
  • AMI is one of the major causes of mortality and morbidity worldwide, it can be defined as a disease caused by a stress situation that is accompanied by an acute inflammatory response of the organism.
  • a multitude of current life habits represent risk factors for this disease such as obesity, smoking, high cholesterol levels, etc.
  • This pathology causes damage to the heart tissue which is maintained after the initial event. Due to the impossibility of performing biopsies in said tissue, it is necessary to obtain markers that serve to diagnose the extent of said damage and the acute inflammatory response it generates.
  • proteins have been proposed as markers of this damage, many of which are involved in the formation of atheroma plaques or lipid metabolism such as high density lipoproteins (HDL).
  • proteins have also been proposed that are associated with said lipoproteins such as apolipoprotein A-l (Apo A-l) or Apo J.
  • Apo J Clusterin, TRPM2, CLI, NA1 / NA2, ku70-binding protein 1, Aging-associated gene 4 protein, or SP-40,40
  • Apo Al Clusterin, TRPM2, CLI, NA1 / NA2, ku70-binding protein 1, Aging-associated gene 4 protein, or SP-40,40
  • Apo J was initially described as a heterodimeric glycoprotein, secreted by rat Sertoli cells, grown in vitro.
  • the product of the translation is a simple chain precursor which undergoes a digestion giving rise to two subunits, one of 34 kDa and another of 47 kDa. It has been shown that Apo J has various N-glycosylations that carry complex sugar associates which contribute between 20% and 30% of the total weight of Apo J.
  • Said protein has been associated with a multitude of cellular processes that result in example cytoprotective effects (Schwochau et al. 1998,), anti-inflammatory (McLaughlin et al. 2000), or antiapoptotic (Zhang et al. 2005).
  • Apo J also binds to a multitude of molecules with high affinity including both lipids and peptides or proteins.
  • Apo J has also been related to various pathologies such as, for example, diabetes or different types of cancer. This protein is also abundant in atherosclerotic diseases. The anti-inflammatory properties of Apo J. are also known. In fact, Apo J had already been attributed anti-inflammatory effects through NF- ⁇ (Santilli G. et al. 2003). In addition to its relationship with autoimmune and inflammatory diseases, on the other hand Apo J has been related to coronary heart disease. There is a hypothesis that during the early phase of AMI ischemia, responses are given that try to compensate for the inflammatory state that acute coronary syndromes cause.
  • the present invention relates to the use of the glycosylated forms of Apolipoprotein J, preferably of the sets Apo J-15 and Apo J-29, as markers of tissue damage as well as acute inflammation associated with heart attack, more specifically heart damage caused by acute myocardial infarction. It also refers to a method of diagnosing said damage and / or inflammation and to the kit comprising the elements necessary for performing said method.
  • infarction myocardial infarction
  • AMI acute myocardial infarction
  • heart attack it refers to insufficient blood flow, with associated tissue damage, in a part of the heart, caused by an obstruction in one of the coronary arteries, often by rupture of a plaque of vulnerable atheroma.
  • Ischemia or poor oxygen supply resulting from such obstruction produces angina pectoris, which if recanalized early does not cause death of heart tissue, while if this anoxia is maintained, myocardial injury and finally necrosis occur, that is , the heart attack
  • a first aspect of the invention relates to the use of at least one glycosylated isoform of Apo J as a marker of tissue damage and / or acute inflammation associated with an acute myocardial infarction.
  • the invention relates to the use of at least one glycosylated isoform of Apo J as a marker of acute inflammation both before and after an AMI.
  • tissue damage is understood to mean damage that is generated in a tissue and that implies an alteration in the same of its physiological situation, either in structure or function. This tissue damage for which the use of glycosylated forms of Apo J is useful occurs in tissues that are selected from the list comprising, cardiac tissue, vascular tissue or renal tissue.
  • Another aspect of the invention relates to the use of at least one glycosylated isoform of Apo J as a marker of tissue damage and / or acute inflammation associated and / or prior to acute myocardial infarction.
  • acute inflammation is understood to mean inflammation that is associated with changes in vascular flow and caliber, which increase blood flow, structural changes in blood vessels that increase blood flow.
  • vascular permeability and induce the formation of inflammatory exudate and the passage of leukocytes from the vascular space to the extravascular thus reaching the focus of the lesions. The result of all this is the accumulation of a fluid rich in proteins, fibrin and leukocytes.
  • the chronic inflammation differs from chronic inflammation mainly in the duration of the process. While the acute inflammation can last from a few minutes to a few days, the chronic inflammation lasts for weeks or months and is characterized in that the cell infiltrate is mainly composed of macrophages, lymphocytes and plasma cells, and the inflammatory reaction is more productive than exudative, that is, the formation of fibrous tissue prevails over fluid exudate.
  • chronic inflammation means inflammation that is associated with various causes: a) progression of acute inflammation; b) recurrent episodes of acute inflammation and c) chronic inflammation from the beginning frequently associated with intracellular infections (tuberculosis, leprosy, etc.).
  • microscopically, chronic inflammation is characterized by the presence of macrophages and their derivatives (epithelioid and giant cells), lymphocytes, plasma cells, neutrophils, eosinophils and fibroblasts.
  • the Apolipoprotein J or Apo J protein (also called clusterin, TRPM2, CLI, NA1 / NA2, ku70-binding protein 1, Aging-associated gene 4 protein, or SP-40,40), is encoded in Homo sapiens by the gene CLU with identification number in GenBank 1 191.
  • This protein includes both glycosylated and glycosylated forms.
  • Isoform 1 is translated from the mRNA with GenBank accession number NM_001831.
  • Isoform 2 is translated from the mRNA with GenBank accession number NM_203339.
  • total Apo J is understood as the amount of Apo J present including its various isoforms and different glycosylation states.
  • glycosylated isoform of Apo J or "glycosylated form of Apo J” is understood in the present invention as a protein whose amino acid sequence is equal to Apo J, and which has at least one sugar attached to any of its amino acids via a link N-glycosidic or O-glycosidic.
  • the spots of the glycosylated forms that exhibit a greater variation and therefore a greater utility for diagnosis are the forms corresponding to spots 6 and 9 (Apo J-15). which decrease, and spots 3, 7, 8 and 1 1 (Apo J-29), which increase.
  • the spots that make up the Apo J-15 group are spot number 6 that corresponds to a glycosylated form of Apo J with a molecular weight of approximately 43.2 kDa and an isoelectric point (pl) of approximately 4.8 in a two-dimensional electrophoresis, and spot number 9 that corresponds to a glycosylated form of Apo J with a molecular weight of approximately 41.2 kDa and an isoelectric point (pl) of approximately 4.9 in a two-dimensional electrophoresis.
  • spot number 6 that corresponds to a glycosylated form of Apo J with a molecular weight of approximately 43.2 kDa and an isoelectric point (pl) of approximately 4.8 in a two-dimensional electrophoresis
  • spot number 9 that corresponds to a glycosylated form of Apo J with a molecular weight of approximately 41.2 kDa and an isoelectric point (pl) of approximately 4.9 in a two-dimensional electrophores
  • Apo J-29 encompasses four different glycosylated forms of Apo J. These forms correspond to spots 3, 7, 8 and 1 1 of the examples of the present invention.
  • Spot 3 is a glycosylated form of Apo J with a molecular weight of approximately 44.8 kDa and a pl of approximately 4.6;
  • spot 7 is a glycosylated form of Apo J with a molecular weight of approximately 41 kDa and a pl of approximately 4.8;
  • spot 8 is a glycosylated form of Apo J with a molecular weight of approximately 44 kDa and a pl of approximately 4.8;
  • spot 3 is a glycosylated form of Apo J with a molecular weight of approximately 42.2 kDa and a pl of approximately 5.
  • isoelectric points and molecular weights of the Apo J-15 and Apo J-29 assembly forms they refer to an electrophoretic mobility obtained by means of the two-dimensional electrophoresis technique and subjected, therefore, to the variability that this methodology entails.
  • the use of the forms included in the Apo J-15 and Apo J-29 sets either independently or in combination, represents an improvement in the markers for the study of tissue damage as well as the associated and / or previous acute inflammation to the IAM. Being the forms of these sets that best respond, these are the preferred glycosylated forms to identify tissue damage.
  • a preferred embodiment of this aspect of the invention refers to the use of at least one glycosylated form of Apo J as a marker of tissue damage and / or inflammation where the glycosylated form is selected from the Apo J-15 and / or of the Apo J-29 set.
  • the glycosylated forms are all of the Apo J-15 set and / or all of the Apo J-29 set.
  • the present invention demonstrates that total Apo J exhibits variations in damaged and / or inflamed heart tissue versus healthy heart tissue. This is determined by the variations in the various glycosylated forms of the protein.
  • One of the most important events that generate damage to heart tissue is the acute myocardial infarction, which is one of the diseases with the highest incidence in the world.
  • this myocardial infarction causes variations in the amount of Apo J forms in various states of glycosylation with respect to individuals who do not suffer from this infarction.
  • this aspect of the invention refers to the use of at least one glycosylated form of Apo J, preferably selected from the Apo J-15 assembly and / or Apo J-29 assembly, more preferably all of the Apo J-15 and / or Apo J-29, as a marker of cardiac damage and / or inflammation, preferably caused by acute myocardial infarction.
  • Apo J can also be found altered in renal tissue. Therefore, another preferred embodiment of this aspect of the invention relates to the use of at least one glycosylated form of Apo J, preferably selected from the Apo J-15 set and / or the Apo J-29 set, more preferably all of the Apo J-15 set and / or Apo J-29 set, as a marker of renal damage and / or acute inflammation associated and / or prior to infarction.
  • Another preferred embodiment relates to the use of glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8 pl, and / or approximately 41.2 kDa molecular weight and approximately 4.9 4.9 in an electrophoresis two-dimensional, as markers of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably of heart damage, more preferably caused by myocardial infarction.
  • Another preferred embodiment relates to the use of glycosylated forms of Apo J of approximately 44.8 kDa molecular weight and approximately 4.6 pl, approximately 41 kDa molecular weight and approximately 4.8 pl, approximately 44 kDa molecular weight and pl Approximately 4.8, and / or molecular weight approximately 42.2 kDa and approximately 5 in a two-dimensional electrophoresis as markers of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably heart damage, more preferably caused by myocardial infarction.
  • Another preferred embodiment relates to the use of glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8 pl, of approximately 41.2 kDa molecular weight and approximately 4.9 pl, of 44.8 molecular weight about kDa and about 4.6 pl, about 41 kDa molecular weight and about 4.8 pl, about 44 kDa molecular weight and about 4.8 pl, and / or about 42.2 kDa molecular weight and about 5 pl in a two-dimensional electrophoresis as markers of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably of heart damage, more preferably caused by myocardial infarction.
  • Another aspect of the present invention relates to a method of obtaining useful data for the diagnosis, prognosis or monitoring of the treatment of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably heart damage, more preferably caused by acute myocardial infarction comprising the following steps:
  • step (b) detect the amount of at least one glycosylated form of Apo J in the sample obtained in step (a).
  • the method of obtaining useful data for the diagnosis, prognosis or monitoring of the treatment of tissue damage and / or acute inflammation associated and / or prior to the infarction, preferably heart damage, more preferably produced by acute myocardial infarction which comprises in addition to steps (a) and (b) described above, an additional step:
  • step (b) compare the amount detected in step (b) with a reference amount obtained from a control sample.
  • the glycosylated forms detected in step (b) are selected from the Apo J-15 assembly and / or the Apo J-29 assembly.
  • the glycosylated forms detected in step (b) are all of the Apo J-15 set and / or all of the Apo J-29 set.
  • the glycosylated forms detected in step (b) are the glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8, and / or of weight molecular 41, approximately 2 kDa and approximately 4.9 in a two-dimensional electrophoresis.
  • the glycosylated forms detected in step (b) are the glycosylated forms of Apo J of approximately 44.8 kDa molecular weight and approximately 4.6 pl, of 41 kDa molecular weight approximately and pl approximately 4.8, molecular weight approximately 44 kDa and approximately 4.8, and molecular weight approximately 42.2 kDa and approximately 5 in a two-dimensional electrophoresis.
  • the glycosylated forms detected in step (b) are the glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8, of molecular weight 41, About 2 kDa and about 4.9 pl, about 44.8 kDa molecular weight and about 4.6 pl, about 41 kDa molecular weight and about 4.8 pl, about 44 kDa molecular weight and about 4.8 pl , and molecular weight 42.2 kDa approximately and pl 5 approximately in a two-dimensional electrophoresis.
  • Another aspect of the present invention relates to a diagnostic method comprising the steps (a) - (c) mentioned above, which further comprises a step (d) where an amount of at least one of the glycosylated forms of the Apo J assembly -15 detected in step (b) less than the reference amount of step (c), is indicative of tissue damage and / or acute inflammation associated and / or prior to AMI, preferably heart damage, more preferably caused by acute heart attack.
  • myocardium In a preferred embodiment of this aspect of the invention the isoforms of step (b) are all glycosylated forms of the Apo J-15 assembly.
  • Another aspect of the present invention relates to a diagnostic method comprising the steps (a) - (c) mentioned above, which further comprises a step (d) where a quantity of the glycosylated forms of Apo J of molecular weight 43, 2 kDa and pl 4.8 approximately, and / or molecular weight 41.2 kDa approximately and pl 4.9 approximately in a two-dimensional electrophoresis, detected in step (b) less than the reference amount of step (c), It is indicative of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably heart damage, more preferably caused by acute myocardial infarction.
  • Another aspect of the present invention relates to a diagnostic method comprising the steps (a) - (c) mentioned above, which further comprises a step (d) where an amount of at least one of the glycosylated forms of the Apo J assembly -29 detected in step (b) greater than the reference amount of step (c) is indicative of tissue damage and / or acute inflammation associated and / or prior to AMI, preferably heart damage, more preferably caused by acute myocardial infarction .
  • the isoforms of step (b) are all glycosylated forms of the Apo J-29 assembly.
  • Another aspect of the present invention relates to a diagnostic method comprising the steps (a) - (c) mentioned above, which further comprises a step (d) where a quantity of the glycosylated forms of Apo J of molecular weight 44, About 8 kDa and about 4.6 pl, about 41 kDa molecular weight and about 4.8 pl, about 44 kDa molecular weight and about 4.8 pl, and / or about 42.2 kDa molecular weight and pl 5 approximately in a two-dimensional electrophoresis, detected in step (b) greater than the reference amount of step (c) is indicative of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably heart damage, more preferably caused by acute myocardial infarction.
  • Another aspect of the present invention relates to a diagnostic method comprising steps (a) - (c) mentioned above, which further comprises a step (d) where an amount of at least at least the glycosylated forms of the Apo set J-15 detected in step (b) less than the reference amount of step (c), and / or an amount of at least all of the glycosylated forms of the Apo J-29 set detected in step (b) greater than
  • the reference amount of step (c) are indicative of tissue damage and / or acute inflammation associated and / or prior to AMI, preferably heart damage, more preferably caused by acute myocardial infarction.
  • the glycosylated forms detected in step (b) are all glycosylated forms of the Apo J-15 assembly and / or all glycosylated forms of the Apo J-29 assembly.
  • Another aspect of the present invention relates to a diagnostic method comprising the steps (a) - (c) mentioned above, which further comprises a step (d) where a quantity of the glycosylated forms of Apo J of molecular weight 43, Approximately 2 kDa and approximately 4.8 and / or molecular weight 41.2 approximately 2 kDa and approximately 4.9 in a two-dimensional electrophoresis, detected in step (b) less than the reference amount of step (c), and an amount of the glycosylated forms of Apo J of approximately 44.8 kDa molecular weight and approximately 4.6 pl, approximately 41 kDa molecular weight and approximately 4.8 pl, approximately 44 kDa molecular weight and approximately 4.8 pl , and / or molecular weight 42.2 kDa approximately and pl 5 in a two-dimensional electrophoresis, detected in step (b) greater than the reference amount of step (c) are indicative of tissue damage and / or associated acute inflammation and / or before the infarction,
  • steps (b) and / or (c) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the quantity in step (b) or the computerized comparison in step (c).
  • diagnosis refers to the ability to discriminate between samples from patients presenting with tissue damage and / or acute inflammation associated and / or prior to infarction and samples from individuals healthy who have not suffered this damage and / or inflammation, when a sample classification method is applied based on the analysis of the amount of glycosylated forms of Apo J and the comparison of said detected amount with respect to a reference amount .
  • This detection as understood by one skilled in the art, is not intended to be correct in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly.
  • the amount that is significantly statistical can be established by one skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the p-value, Student's test or discriminant functions of Fisher
  • the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%.
  • the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001.
  • the present invention allows to correctly detect the damage and / or inflammation in at least 60%, in at least 70%, in at least 80%, or in at least 90% of the subjects of a certain group or population analyzed.
  • detect or detect the amount of a protein refers to the measure of the quantity or the concentration, preferably semi-quantitatively or quantitatively.
  • the measure can be carried out directly or indirectly.
  • Direct measurement refers to the measure of the amount or concentration of the protein, based on a signal that is obtained directly from the protein, and that is directly correlated with the number of protein molecules present in the sample. That signal -to the that we can also refer to as an intensity signal - it can be obtained, for example, by measuring an intensity value of a chemical or physical property of the protein.
  • the indirect measurement includes the measurement obtained from a secondary component (for example, a component other than the protein) or a biological measurement system (for example the measurement of cellular responses, ligands, "tags” or enzymatic reaction products).
  • Quantity refers to, but is not limited to, the absolute or relative amount of the protein, as well as any other value or parameter related to or derived from them.
  • Said values or parameters comprise values of signal intensity obtained from any of the physical or chemical properties of the protein by direct measurement, such as, for example, intensity values of mass spectroscopy or nuclear magnetic resonance. Additionally, said values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.
  • comparison refers to, but is not limited to, the comparison of the amount of Apo J glycosylated forms of the biological sample to be analyzed, also called the biological sample. problem, with a quantity of the glycosylated forms of Apo J of a desirable reference sample described elsewhere in the present description.
  • the reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample.
  • reference amount refers to the amount of the absolute or relative amount of glycosylated forms of the Apo J protein that allows discriminating tissue damage and / or associated acute inflammation and / or before the infarction, of a non-pathological situation. Suitable reference amounts can be determined by the method of the present invention from a reference sample that can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample.
  • the reference sample may be, for example, an extract of proteins obtained from a biological sample isolated from a healthy individual, or a mixture of protein extracts obtained from biological samples isolated from one or several different healthy individuals.
  • the reference amount is obtained from a reference sample from a healthy individual, that is, that there is no tissue damage and / or acute inflammation associated and / or prior to AMI.
  • the reference amount can also be obtained from a reference sample from the same individual that suffers the damage and / or inflammation, but prior to suffering the damage and / or inflammation.
  • isolated biological sample refers to, but is not limited to, tissues and / or biological fluids of a subject, obtained by any method known to a person skilled in the art that serves for such finish.
  • the isolated biological sample comprises: heart tissue, blood, serum, plasma or urine.
  • subject or “individual”, as used in the description, refer to animals, preferably mammals, and more preferably, humans.
  • healthy subject or “healthy individual” in the present invention refer to that individual who has not altered the standard biochemical parameters for measuring cardiac damage and / or acute inflammation associated and / or prior to infarction [troponin, creatine kinase (CK), creatine kinase MB (CK-MB) and natriuretic peptide type B (BNP)] or signs of pathology when performing diagnostic tests to measure heart damage and / or inflammation (ECG and cardiovascular magnetic resonance).
  • standard biochemical parameters for measuring cardiac damage and / or acute inflammation associated and / or prior to infarction troponin, creatine kinase (CK), creatine kinase MB (CK-MB) and natriuretic peptide type B (BNP)
  • ECG and cardiovascular magnetic resonance signs of pathology when performing diagnostic tests to measure heart damage and / or inflammation
  • the detection of the variation in the amount of glycosylated forms of Apo J is performed by a two-dimensional electrophoresis (2-DE).
  • Two-dimensional electrophoresis is an analytical technique of separation of kinetic foundation based on the movement or migration of macromolecules dissolved in a certain medium (electrophoresis buffer solution), through a matrix or cross-linked support as a result of the action of a field electric.
  • the separation of proteins is based on two characteristics: first, the proteins are separated according to their isoelectric point (pl) by isoelectric focusing (IEF) and, secondly, the separation is carried out according to their molecular mass, by electrophoresis under denaturing conditions (SDS-PAGE).
  • IEF isoelectric point
  • SDS-PAGE electrophoresis under denaturing conditions
  • these gels are stained with a specific protein stain, such as, but not limited to Coomassie blue or silver staining, and the spots corresponding to the protein of interest between the different gels are compared.
  • a specific protein stain such as, but not limited to Coomassie blue or silver staining
  • Another way of detecting and comparing the amount of proteins is by means of the DIGE (Differential In Gel Electrophoresis) technique, which is based on the protein mapping of the study samples with one of the three fluorochromes (Cy2, Cy3 or Cy5) before separation. The samples are then mixed and separated in a single two-dimensional gel, minimizing experimental variability. Due to the specific marking of each sample they can be observed individually and perform a comparative analysis of the differential expression of proteins, allowing precise quantification.
  • DIGE Different In Gel Electrophoresis
  • the detection of the variation in the amount of glycosylated forms of Apo J is performed by incubation with a specific antibody in an immunoassay.
  • immunoassay refers to any analytical technique that is based on the reaction of conjugation of an antibody with the sample obtained. Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry or protein chips.
  • the immunoassay is an immunoblot or Western blot.
  • a protein extract is obtained from an isolated biological sample of a subject and the proteins are separated in a support medium capable of retaining them by electrophoresis. Once the proteins are separated, they are transferred to a different support where they can be detected through the use of specific antibodies that recognize the glycosylated forms of the Apo J. protein.
  • Electrophoresis is an analytical technique of kinetic basis separation based on the movement or migration of the macromolecules dissolved in a certain medium (electrophoresis buffer solution), through a matrix or cross-linked support as a result of the action of an electric field.
  • the behavior of the molecule is given by its electrophoretic mobility and this by the charge, size and shape thereof. The higher the charge / size ratio, the faster an ion migrates within the electric field.
  • this technique depending on the equipment used, support and physical-chemical conditions in which the separation will be carried out.
  • the proteins obtained from an isolated biological sample of a subject are separated by the two-dimensional electrophoresis described above.
  • the proteins are transferred to a support or to a membrane, for example, but not limited to, PDVF, nitrocellulose or cellulose acetate.
  • a membrane for example, but not limited to, PDVF, nitrocellulose or cellulose acetate.
  • This membrane is hybridized with a specific antibody (also called primary antibody) that recognizes the glycosylated forms of the Apo J. protein.
  • an antibody also called secondary antibody
  • it is the antibody that recognizes the glycosylated forms of the Apo J protein that is conjugated or bound to a marker compound, and the use of a secondary antibody is not necessary.
  • the protein Once the protein is detected, its relative molecular size can be determined by comparing its migration with the migration of a control protein that is detected simultaneously, preferably on the same support, which has a known size. Therefore, in an even more preferred embodiment, the detection of the variation in the amount of glycosylated forms of Apo J is performed by a two-dimensional electrophoresis followed by an immunoblot.
  • the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay).
  • the ELISA is based on the premise that an immunoreactive (biological sample antigen or antibody) can be immobilized on a solid support, then putting that system in contact with a fluid phase containing the complementary reagent that can bind to a marker compound .
  • ELISA There are different types of ELISA.
  • the solid support In the direct ELISA or simple two-layer ELISA assay, the solid support is coated with the biological sample and incubated with an antibody that recognizes glycosylated forms of Apo protein, conjugated or bound to a marker compound.
  • the solid support In the indirect ELISA, the solid support is coated with the biological sample and incubated with a primary antibody, which recognizes the glycosylated forms of the Apo J protein and then a secondary antibody, which recognizes the primary antibody, conjugated or bound to a marker compound.
  • the well is coated with a first antibody that recognizes the glycosylated forms of Apo J protein so that glycosylated forms of Apo J will be retained in the well upon recognition. by the first antibody, and then a second antibody is applied that recognizes the glycosylated forms of the Apo J protein, conjugated or bound to a marker compound.
  • the immunoassay is an immunohistochemistry (IHQ).
  • IHQ immunohistochemistry
  • Immunohistochemical techniques allow the identification, on tissue or cytological samples of characteristic antigenic determinants. Immunohistochemical analysis is performed on tissue sections, either frozen or included in paraffin, from an isolated biological sample of a subject. These sections hybridize with a specific antibody or primary antibody that recognizes the glycosylated forms of the Apo J. protein. The membrane is then hybridized with a secondary antibody capable of recognizing specifically the primary antibody and that is conjugated or bound with a marker compound. In an alternative embodiment, it is the antibody that recognizes the glycosylated forms of Apo J proteins that is conjugated or bound to a marker compound, and the use of a secondary antibody is not necessary.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with the forms Apo J. protein glycosylates.
  • portions of immunologically active immunoglobulin molecules include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin.
  • the antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen).
  • the monoclonal antibody may be biochemically altered, by genetic manipulation, or it may be synthetic, possibly lacking the antibody in whole or in part, from portions that are not necessary for the recognition of the glycosylated forms of Apo J and being replaced by others that give the antibody additional advantageous properties.
  • the antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing.
  • a "recombinant antibody or polypeptide” is an antibody that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination.
  • Antibodies that recognize the glycosylated forms of Apo J known in the state of the art, such as those described, but not limited to, in the examples of the present description.
  • the antibodies used to carry out the methods of the present invention can be polyclonal or monoclonal antibodies capable of recognizing only each of the glycosylated forms of the Apo J protein. These antibodies can be used to carry out the methods of the present invention, for example, but not limited, by immunoblot, ELISA or immunohistochemistry.
  • marker compound refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and quantification of the amount of Apo protein. J or its truncated forms.
  • the marker compound is selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and / or quantified directly. This marker compound can bind to the antibody directly, or through another compound.
  • directly binding marker compounds are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as 33P or 35S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, auto-radiography, fluorimetry , or metallography respectively.
  • enzymes such as alkaline phosphatase or peroxidase
  • radioactive isotopes such as 33P or 35S
  • fluorochromes such as fluorescein or metal particles
  • kit or device comprising the elements necessary to carry out the methods of the present invention.
  • the kit of the present invention comprises the elements necessary for:
  • step (b) compare the quantity detected in step (a) with a reference quantity.
  • Said kit may contain all those reagents necessary to detect the amount of glycosylated forms of the Apo J protein by any of the methods described hereinbefore such as, but not limited to, antibodies specific to the glycosylated forms of the protein. Apo J, secondary antibodies or positive and / or negative controls.
  • the kit can also include, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc.
  • the kit can include all the supports and containers necessary for commissioning and optimization.
  • the kit further comprises instructions for carrying out the method of the invention.
  • the glycosylated forms to be detected in step (a) are selected from the Apo J-15 assembly and / or the Apo J-29 assembly. In a more preferred embodiment of this aspect of the invention the glycosylated forms to be detected in step (a) are all of the Apo J-15 assembly and / or all of the Apo J-29 assembly. In another preferred embodiment of this aspect of the invention the glycosylated forms to be detected in step (a) are the glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8 pl, and / or approximately 41.2 kDa molecular weight and pl 4, 9 approximately in a two-dimensional electrophoresis.
  • the glycosylated forms to be detected in step (a) are the glycosylated forms of Apo J of approximately 44.8 kDa molecular weight and approximately 4.6 pl, of approximately 41 kDa molecular weight and pl 4.8 approximately, molecular weight 44 kDa approximately and pl 4.8 approximately, and / or molecular weight 42.2 kDa approximately and isoelectric point 5 approximately in a two-dimensional electrophoresis.
  • the glycosylated forms to be detected in step (a) are the glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8 pl, of 41.2 kDa molecular weight approximately and pl 4.9 approximately, of molecular weight approximately 44.8 kDa and pl 4.6 approximately, of molecular weight approximately 41 kDa and pl 4.8 approximately, of molecular weight approximately 44 kDa and approximately 4.8, and / or molecular weight 42.2 kDa approximately and pl 5 approximately in a two-dimensional electrophoresis.
  • the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps.
  • other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention.
  • the following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.
  • the present invention comprises a method of detection / diagnosis / prevention / treatment of both tissue damage and acute inflammation associated with acute myocardial infarction by using at least one glycosylated form of Apo J as a marker.
  • the present invention comprises the use of at least one glycosylated form of Apo J for the manufacture of a medicament for detection / diagnosis / prevention / treatment of both tissue damage and acute inflammation associated with acute myocardial infarction, wherein said form Apo J glycosylated is used as a marker.
  • Figure 1 Two-dimensional electrophoresis gel.
  • Figure 2 Increased images of Apo J areas in controls and patients with acute myocardial infarction.
  • Apo J was detected as a series of 12-13 points with an isoelectric point range between 4.5 and 5 and a molecular mass between 37.1 and 47.3 kDa.
  • the spots identified as Apo J they were numbered from the most acidic to the most basic pH. Point number 2 was only detected in AMI patients. Points 1, 3, 7, 8, 10, 1 1 and 13 showed an increase in detection levels in AMI patients while points 6 and 9 showed a reduced intensity in AMI when compared to the control group.
  • the spots 6 and 9 correspond to Apo J-15, and the spots 3, 7, 8 and 1 1 correspond to Apo J-29
  • Serum Apo J levels (pg / ml) in AMI patients and healthy individuals. Apo J levels were quantified by a commercial ELISA.
  • B Graph of the monitoring of serum Apo J levels in AMI patients from the moment of admission (time 0) to 96 hours. The values of each moment are expressed as mean ⁇ ES ( ⁇ p ⁇ 0.0001 vs. control; * p ⁇ 0.02 vs. time 0; ANOVA and Fisher PSLD as Post hoc test).
  • Apo J is located around the capillaries in the ischemic myocardia. Intense brown signals are detected around the capillaries in myocardial sections of an ischemic heart, while no signals are detected in the capillaries of a non-ischemic myocardial tissue.
  • Short-term myocardial ischemia induces cardiac modified C-reactive protein expression and proinflammatory gene (cyclo-oxygenase-2, monocyte chemoattractant protein-1, and tissue factor) upregulation in peripheral blood mononuclear cells. J Thromb Haemost. 2009; 7: 485-93.
  • cardiac modified C-reactive protein expression and proinflammatory gene cyclo-oxygenase-2, monocyte chemoattractant protein-1, and tissue factor
  • Serum samples (after the extraction of the six most abundant proteins) from 27 patients with AMI and healthy donors were analyzed individually in duplicate by two-dimensional electrophoresis (2-DE) and the proteins were identified by MALDI type mass spectrometry. -ToF.
  • 2-DE two-dimensional electrophoresis
  • HDL-associated proteins were identified, including Apo J, PON1 and Apo A-1 ( Figures 1A and 1 B; Table 1).
  • NS Not significant. A change was detected towards the most electrophoretic mobility forms in the AMI group (see table 2). When comparing the AMI patients with the controls, it was found that the relative volume of spots 6 and 9 (PM 43.2-41, 2 kDa) decreased more than double (p ⁇ 0.001), while spots 7, 10 and 13 (PM 41, 0 - 37.1 kDa) presented an increase of more than double (p ⁇ 0.001).
  • Necrosis (%): Percentage of necrotic myocardial mass (calculated as the percentage of the total mass of the heart measured by cardiovascular magnetic resonance).
  • TnTmax Maximum value of troponin levels T from the moment of admission
  • t PCR t 0: Levels of C-reactive protein at the time of admission.
  • ⁇ Max PCR Maximum value of C-reactive protein levels from the moment of admission.
  • PROTEOMIC PATTERN OF THE GLOSSYLED FRACTION OF APO J SERICA The proteomic pattern of the glycosylated fraction of Apo J serum was studied after the purification of glycosylated proteins through their binding to lectins.
  • the serum glycoprotein subproteome of the group of AMI patients and the control group (pools of 10 individuals) was analyzed by 2-DE ( Figure 5). Patients with diabetes were excluded from this analysis.
  • MALDI-ToF 6 spots with a molecular mass between 40 and 45 kDa and a range of pl between 4.5 and 5.0 were identified as Apo J, both in the control group and in the patients.
  • spots are numbered as 1, 2, 4, 5, 8 and 1 1 in the 2-SD analysis of the total serum fraction (compare Figure 5 with Figure 2A).
  • AMI vs. control mean ⁇ ES: 9072 ⁇ 166 vs. 12351 ⁇ 1717
  • the values indicate the total intensity of Apo J forms normalized by the intensity of the volume of all spots in the gel.
  • HDL cholesterol (mg / dL) 44.2 ⁇ 1 1.8 43 ⁇ 13.5
  • LDL cholesterol (mg / dL) 127.8 ⁇ 40.1 139.5 ⁇ 38.7
  • PCR levels Levels of C-reactive protein at the time of admission.
  • TnTmax Maximum value of troponin T levels from the time of admission,
  • ASA Acetylsalicylic acid
  • ACEI angiotensin conversion enzyme inhibitors
  • ARA2 acetylsalicylic acid
  • ACEI angiotensin conversion enzyme inhibitors
  • ARA2 acetylsalicylic acid
  • NIT Nitroglycerin
  • ADO oral antidiabetics.
  • the control group consisted of fifty-one individuals (34 men and 17 women) with an average age of 45 years ⁇ 1 1, who underwent a routine medical review.
  • the ethical committee of the Hospital de la Santa Creu i Sant Pau approved the project and the studies were carried out in accordance with the principles of the Declaration of Helsinki. All participants gave written informed consent to be part of the study.
  • Contrast cardiac magnetic resonance studies were performed in the chronic phase after AMI in all those patients who presented AMI with ST segment elevation using a Philips Intera 1 .5 Teslas scanner (Philips Medical Systems, Best, The Netherlands).
  • the long axes were acquired in cinema mode and high precision stationary state by means of multiple longitudinal cuts (short axis) at a cutting thickness of 10mm. These cuts were made from the atrioventricular ring to the apex of the left ventricle. A total of sixteen cardiac cycle phases were acquired for each cut.
  • Gadobutrol Gadobutrol (Gadovista, Schering, Berlin, Germany) was administered intravenously at a rate of 0.1 mmol / kg.
  • the inversion-recovery sequences with gradient echo were acquired in the same planes as the cinema mode sequences in order to evaluate the late enhancement.
  • the inversion time was adjusted individually to suppress the healthy myocardium (200 to 300 ms).
  • An assessment of the segmental motility of the left ventricle was performed according to the American Image Societies, which divide the left ventricle into two zones (anteroseptal and inferolateral), 4 walls (anterior, septal, inferior and lateral) and 17 segments.
  • the double contrast enhancement sequences were analyzed double blind by three expert interpreters and each segment was evaluated.
  • Venous blood samples from AMI patients were collected before the start of the medication. Serum was obtained, aliquoted and stored at -80 ° C.
  • HDLs Human HDLs were obtained from normocholesterolemic serum by differential ultracentrifugation. They were determined the concentrations of lipoproteins and cholesterol of the HDL. The protein fraction was obtained by precipitation of 2mg of HDL with pure acetone at -20 ° C for two hours. Serum preparation: For proteomic studies, the serum samples were sonicated (six cycles of 15 seconds each) on ice and filtered (0.22 ⁇ ) by centrifugation to avoid the presence of impurities.
  • the six most abundant whey proteins were removed using a specific affinity column capable of binding albumin, immunoglobulin G, immunoglobulin A, transferrin, a1-antitrypsin and haptoglobin (Multiple Affinity Removal Spin Cartridge, Agilent Technologies).
  • the serum fraction without the six major proteins was concentrated and centrifuged with 5kDa filters for the removal of salts and the change to a urea buffer (8M Urea, 2% CHAPS (3 - [(3 -Cholamidopropyl) dimethylammonium] -1-propanesulfonate)).
  • glycosylated whey protein fraction was purified by the use of a commercial kit (Qproteome Total Glycoprotein Kit, QIAGEN). The protein concentration present in the extracts was quantified with the 2D-Quant Kit (GE Healthcare). All samples were stored at -80 ° C until use.
  • Two-dimensional electrophoresis 2-DE: 120 g (in analytical gels), or 300 g (in preparations) of Urea / CHAPS soluble extract protein were loaded into 18 cm isoelectric focusing strips and a linear pH range of 4 to 7 (GE Healthcare). The proteins in the gels were visualized by staining fluorescent (analytical gels) or with Coomassie blue (preparative gels). For each independent experiment, the 2-ED of the protein extracts from controls and patients were processed in parallel to ensure that they were comparable. Each batch of 2-DE was repeated at least twice. The analysis of the differences between the protein patterns of the control and infarction samples was carried out with the PD-Quest program (BioRad) using a single master that included all the gels of each independent experiment. Each protein spot, after the extraction of the background signal and the normalization between gels, was assigned a relative value that corresponded to the value of each individual spot compared to the volume of all the spots present in the gel.
  • Protein spots of interest were trimmed from the gels, washed, dehydrated, dried and enzymatically digested with porcine trypsin (Promega).
  • the peptides resulting from trypsin digestion were mixed in a 1: 1 ratio with 5mg / ml of a-cyano-4-hydroxy-cinnamic and loaded onto a stainless steel strip for mass spectrometry.
  • Protein identification was carried out by means of their peptide fingerprint using an Ettan MALDI-ToF Pro (matrix-assisted laser desorption / ionisation time-of-flight mass spectrometer, GE-Healthcare) working in the reflector mode.
  • Ettan MALDI-ToF Pro matrix-assisted laser desorption / ionisation time-of-flight mass spectrometer, GE-Healthcare
  • the spectra generated by MALDI were internally calibrated using the peaks of the trypsin autolysis products, Ang III (angiotensin III) and ACTH (adrenocorticotropic hormone). Peptide masses were searched through the non-redundant mammalian database of the National Center for Biotechnology Information using ProFound TM and confirmed using a Matrixscience Mascot search by selecting the SwissProt database. For this study, protein identification was based on the measurement of at least 10 peptides with a minimum of 50% coincidence and coverage of more than 35%. The minimum expectation for a valid identification was 0.0001 and p ⁇ 0.05.
  • the serum Apo J concentration was determined by a commercial sandwich ELISA based on two monoclonal antibodies (Clusterin ELISA, Biovendor) following the indications of the suppliers.
  • the detection limit of the assay was 0.5 ng / ml.
  • Apo J was immunoprecipitated by adding 2 g of a specific monoclonal antibody (Apolipoprotein J, clone CLI-9, Abcam) to pre-washed samples using 50% Protein G Sepharose 4 Fast Flow (GE Healthcare, 1 hour at 4 ° C).
  • the antigen-antibody complexes were precipitated with 50% Protein G Sepharose 4 Fast Flow.
  • the immunoprecipitated complexes were separated from the G protein by heating the sample for 5 minutes under reducing conditions (2.5M Tris-HCI pH 6.8, 100% Glycerol, 20% SDS, bromophenol blue).
  • the samples were resolved by electrophoresis and were electro-transferred to PVDF membranes (Polyvinylidene Difluoride) under semi-dry conditions (Semi-dry transfer system, BioRad). Detection was carried out with monoclonal antibodies against Apo J (clone CLI-9, dilution 1: 2000, Abcam). The anti-mouse antibody was labeled with Cyclo fluorochrome (1: 20000 dilution, GE Healthcare). The fluorescence of the bands was determined with a Typhoon 9400 (GE Healthcare) and the quantification was carried out using the ImageQuant TL (GE Healthcare) program.
  • Human myocardia were obtained from hearts extracted during transplants performed at the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain).
  • the tissue samples were immersed in fixation solution (4% paraformaldehyde) and after immersion in paraffin, they were cut into serial sections 5 mm thick and placed on poly-L-lysine coated supports.
  • a rabbit polyclonal primary antibody against Apolipoprotein J was used at a 1: 50 dilution (Abcam ab69644). Before incubation with the primary antibody (2 hours), the sections were washed, eliminating the activity of endogenous peroxidase with H2O2 and goat serum was used to block nonspecific junctions.
  • Primary antibodies were detected using the avidin-biotin immunoperoxidase technique.
  • the sections were incubated subsequently with a biotinylated secondary antibody (1: 200, Vector ⁇ ).
  • the chromogen used was 3,3'-diaminobenzidine. Hematoxylin was used for staining the nuclei.
  • C-reactive protein (PCR) levels were measured with a commercial kit (Roche) with a detection range between 0.1 and 20 mg / L. When the PCR levels were above the detection limit the samples were diluted. Lipids were measured by conventional enzymatic methods within the first 8 hours from the start of the event. Measurements made in this period of time are considered the baseline level. Total cholesterol levels were analyzed using the CHOD-PAP method (Boehringer-Mannheim, Germany) and triglycerides by TOD-PAP (Boehringer-Mannheim, Germany). HDL cholesterol levels were measured after manual precipitation of lipoproteins with the PEG 6000 method (Boehringer-Mannheim, Germany).
  • LDL cholesterol levels were determined by centrifugation and when they were below 3.36 mmol / L, LDL cholesterol levels were estimated using Fridewald's formula.
  • Troponin T levels were measured with a commercial Roche Diagnostics IV generation kit and analyzed with the Roche Diagnostics Elecsys 2010.

Abstract

The present invention relates to use of the glycosylated forms of apolipoprotein J as markers of tissue damage and of the acute inflammation said damage entails, more particularly the product of acute myocardial infarction, together with a method of diagnosis of said damage and/or inflammation and a kit comprising the elements necessary to effect said diagnosis. In the present invention it is demonstrated that the diverse glycosylated forms vary their expression should there exist tissue damage and/or acute inflammation, permitting the utilisation thereof as markers in the case of the existence of damage and/or inflammation.

Description

Uso de las isoformas de Apo J como biomarcadores de lesión tisular.  Use of Apo J isoforms as biomarkers of tissue injury.
La presente invención se encuentra dentro del campo de la biomedicina. Específicamente, se refiere al uso de las formas glicosiladas de la apolipoproteína J (Apo J) como marcadores de daño tisular e inflamación aguda, más concretamente producidos por infarto agudo de miocardio (IAM), así como a un método de diagnóstico de dicho daño y/o inflamación y a un kit que comprende los elementos necesarios para llevar a cabo dicho diagnóstico. ESTADO DE LA TÉCNICA ANTERIOR The present invention is within the field of biomedicine. Specifically, it refers to the use of glycosylated forms of apolipoprotein J (Apo J) as markers of tissue damage and acute inflammation, more specifically produced by acute myocardial infarction (AMI), as well as a method of diagnosing such damage and / or inflammation and a kit comprising the necessary elements to carry out said diagnosis. STATE OF THE PREVIOUS TECHNIQUE
El IAM es una de las mayores causas de mortalidad y morbilidad en todo el mundo, se puede definir como una enfermedad causada por una situación de estrés que va acompañada de una respuesta inflamatoria aguda del organismo. Multitud de los hábitos de vida actuales representan factores de riesgo para esta enfermedad como por ejemplo la obesidad, el tabaquismo, los elevados niveles de colesterol, etc. Esta patología provoca daños en el tejido cardíaco los cuales se mantienen tras el evento inicial. Debido a la imposibilidad de realizar biopsias en dicho tejido, se hace necesaria la obtención de marcadores que sirvan para diagnosticar el alcance de dicho daño y la respuesta inflamatoria aguda que genera. AMI is one of the major causes of mortality and morbidity worldwide, it can be defined as a disease caused by a stress situation that is accompanied by an acute inflammatory response of the organism. A multitude of current life habits represent risk factors for this disease such as obesity, smoking, high cholesterol levels, etc. This pathology causes damage to the heart tissue which is maintained after the initial event. Due to the impossibility of performing biopsies in said tissue, it is necessary to obtain markers that serve to diagnose the extent of said damage and the acute inflammatory response it generates.
Se han propuesto como marcadores de este daño múltiples proteínas, muchas de las cuales se encuentran implicadas en la formación de placas de ateroma o metabolismo de lípidos como por ejemplo las lipoproteínas de alta densidad (HDL). Además de éstas, también se han propuesto proteínas que se asocian con dichas lipoproteínas como la apolipoproteína A-l (Apo A-l) o la Apo J. Multiple proteins have been proposed as markers of this damage, many of which are involved in the formation of atheroma plaques or lipid metabolism such as high density lipoproteins (HDL). In addition to these, proteins have also been proposed that are associated with said lipoproteins such as apolipoprotein A-l (Apo A-l) or Apo J.
Esta Apo J (clusterina, TRPM2, CLI, NA1/NA2, ku70-binding protein 1 , Aging- associated gene 4 protein, o SP-40,40) se asocia a las HDL mediante la unión a Apo A-l (Stuart et al. 1992). Apo J fue inicialmente descrita como una glicoproteína heterodimérica, secretada por células de Sertoli de ratas, cultivadas in vitro. El producto de la traducción es un precursor de cadena simple el cual sufre una digestión dando lugar a dos subunidades, una de 34 kDa y otra de 47 kDa. Se ha demostrado que Apo J presenta diversas N-glicosilaciones que llevan asociados azúcares complejos los cuales contribuyen a entre un 20% y un 30% del peso total de Apo J. Dicha proteína ha sido asociada con multitud de procesos celulares que tienen como resultado por ejemplo efectos citoprotectores (Schwochau et al. 1998,), antiinflamatorios (McLaughlin et al. 2000), o antiapoptóticos (Zhang et al. 2005). Apo J además, se une a multitud de moléculas con elevada afinidad incluyendo tanto lípidos como péptidos o proteínas. This Apo J (clusterin, TRPM2, CLI, NA1 / NA2, ku70-binding protein 1, Aging-associated gene 4 protein, or SP-40,40) is associated with HDL by binding to Apo Al (Stuart et al. 1992). Apo J was initially described as a heterodimeric glycoprotein, secreted by rat Sertoli cells, grown in vitro. The product of the translation is a simple chain precursor which undergoes a digestion giving rise to two subunits, one of 34 kDa and another of 47 kDa. It has been shown that Apo J has various N-glycosylations that carry complex sugar associates which contribute between 20% and 30% of the total weight of Apo J. Said protein has been associated with a multitude of cellular processes that result in example cytoprotective effects (Schwochau et al. 1998,), anti-inflammatory (McLaughlin et al. 2000), or antiapoptotic (Zhang et al. 2005). Apo J also binds to a multitude of molecules with high affinity including both lipids and peptides or proteins.
Por otro lado, también se ha descrito que Apo J presenta diferentes actividades en función del momento del ciclo celular en el que actúe (Trougakos et al. 2005). On the other hand, it has also been described that Apo J has different activities depending on the time of the cell cycle in which it operates (Trougakos et al. 2005).
Apo J también ha sido relacionada con diversas patologías como pueden ser, por ejemplo, la diabetes o diferentes tipos de cáncer. Esta proteína también es abundante en enfermedades ateroscleróticas. Son también conocidas las propiedades antiinflamatorias de Apo J. De hecho, a la Apo J ya se le habían atribuido efectos anti-inflamatorios a través del NF-κΒ (Santilli G. et al. 2003). Además de su relación con patologías autoinmunes y de carácter inflamatorio, por otro lado Apo J ha sido relacionada con enfermedades cardíacas coronarias. Existe la hipótesis que durante la fase temprana de isquemia del IAM se dan respuestas que tratan de compensar el estado inflamatorio que los síndromes coronarios agudos provocan. De hecho, en un modelo experimental porcino de isquemia/reperfusión, se ha mostrado que tras 30 minutos de isquemia miocárdica ya se pueden detectar alteraciones sistémicas proinflamatorias. (Vilahur G, et al.2009). Aunque aún no se ha confirmado la presencia de Apo J en los capilares de miocardios isquémicos, se ha confirmado la ausencia de dichas señales en tejido miocárdico no isquémico (Vákevá A, et al. 1993). Además tampoco se sabe con certeza si la deposición de Apo J en el miocardio isquémico contribuiría al proceso de cicatrización de dicho miocardio. Apo J has also been related to various pathologies such as, for example, diabetes or different types of cancer. This protein is also abundant in atherosclerotic diseases. The anti-inflammatory properties of Apo J. are also known. In fact, Apo J had already been attributed anti-inflammatory effects through NF-κΒ (Santilli G. et al. 2003). In addition to its relationship with autoimmune and inflammatory diseases, on the other hand Apo J has been related to coronary heart disease. There is a hypothesis that during the early phase of AMI ischemia, responses are given that try to compensate for the inflammatory state that acute coronary syndromes cause. In fact, in a porcine experimental model of ischemia / reperfusion, it has been shown that after 30 minutes of myocardial ischemia, proinflammatory systemic alterations can already be detected. (Vilahur G, et al. 2009). Although the presence of Apo J in the ischemic myocardial capillaries has not yet been confirmed, the absence of such signals in non-ischemic myocardial tissue has been confirmed (Vákevá A, et al. 1993). Furthermore, it is not known with certainty whether the deposition of Apo J in the ischemic myocardium would contribute to the healing process of said myocardium.
Esta última relación entre la Apo J y las enfermedades cardíacas coronarias, no se encuentra muy bien definida ya que existen datos respecto tanto a un aumento como a una disminución de los niveles totales de dicha proteína en caso de existir una patología cardíaca coronaria (Trougakos et al. 2002; Kujiraoka et al. 2006). Adicionalmente, en todas las patologías de carácter inflamatorio también se habla siempre de cambios en los niveles totales de Apo J (McLaughlin L et al. 2000; Newkirk MM. et al.1999). Esta controversia o variedad de datos provoca que los niveles totales en suero y/o plasma de dicha proteína no sean un buen marcador de daño cardíaco ya que su respuesta no es constante cuando se da un evento vascular. Esto hace que sea necesaria la búsqueda de nuevos marcadores de daño tisular así como de la respuesta inflamatoria aguda asociada al infarto, más fiables que los niveles totales de Apo J y que no presenten este inconveniente. This last relationship between Apo J and coronary heart disease is not very well defined since there are data regarding both an increase as to a decrease in the total levels of said protein in the case of coronary heart disease (Trougakos et al. 2002; Kujiraoka et al. 2006). Additionally, in all pathologies of an inflammatory nature, there is always talk of changes in the total levels of Apo J (McLaughlin L et al. 2000; Newkirk MM. Et al. 1999). This controversy or variety of data causes that the total serum and / or plasma levels of said protein are not a good marker of cardiac damage since its response is not constant when a vascular event occurs. This makes it necessary to search for new markers of tissue damage as well as the acute inflammatory response associated with the infarction, more reliable than the total Apo J levels and not presenting this problem.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
La presente invención se refiere al uso de las formas glicosiladas de la Apolipoproteína J, preferiblemente de los conjuntos Apo J-15 y Apo J-29, como marcadores de daño tisular así como de la inflamación aguda asociada al infarto, más concretamente de daño cardíaco producido por infarto agudo de miocardio. También se refiere a un método de diagnostico de dicho daño y/o inflamación y al kit que comprende los elementos necesarios para la realización de dicho método. The present invention relates to the use of the glycosylated forms of Apolipoprotein J, preferably of the sets Apo J-15 and Apo J-29, as markers of tissue damage as well as acute inflammation associated with heart attack, more specifically heart damage caused by acute myocardial infarction. It also refers to a method of diagnosing said damage and / or inflammation and to the kit comprising the elements necessary for performing said method.
En la presente invención se entiende por "infarto", "infarto de miocardio" o "infarto agudo de miocardio" (IAM) a la enfermedad causada por una situación de estrés que va acompañada de una respuesta inflamatoria aguda del organismo. En la presente invención se entiende que el IAM está asociado y/o precedido por un episodio inflamatorio agudo. También conocido en el lenguaje coloquial como ataque al corazón o ataque cardíaco, hace referencia a un riego sanguíneo insuficiente, con daño tisular asociado, en una parte del corazón, producido por una obstrucción en una de las arterias coronarias, frecuentemente por ruptura de una placa de ateroma vulnerable. La isquemia o suministro deficiente de oxígeno que resulta de tal obstrucción produce la angina de pecho, que si se recanaliza precozmente no produce muerte del tejido cardíaco, mientras que si se mantiene esta anoxia se produce la lesión del miocardio y finalmente la necrosis, es decir, el infarto. In the present invention, "infarction", "myocardial infarction" or "acute myocardial infarction" (AMI) is understood as the disease caused by a stress situation that is accompanied by an acute inflammatory response of the organism. In the present invention it is understood that AMI is associated and / or preceded by an acute inflammatory episode. Also known in the colloquial language as heart attack or heart attack, it refers to insufficient blood flow, with associated tissue damage, in a part of the heart, caused by an obstruction in one of the coronary arteries, often by rupture of a plaque of vulnerable atheroma. Ischemia or poor oxygen supply resulting from such obstruction produces angina pectoris, which if recanalized early does not cause death of heart tissue, while if this anoxia is maintained, myocardial injury and finally necrosis occur, that is , the heart attack
En la presente invención se demuestra como en pacientes que han sufrido un síndrome coronario agudo la cantidad de Apo J total se encuentra alterada respecto a individuos que no han sufrido este evento. Dicha Apo J presenta diferentes isoformas con diversos estados de glicosilación. La cantidad de Apo J total puede variar enormemente en un mismo evento entre diversos individuos ya que algunas de sus isoformas aumentan mientras otras disminuyen, y no en todos los individuos las proporciones de las mismas son iguales. Por ello la detección de Apo J total no es un marcador adecuado para el diagnóstico de daño tisular ni de la inflamación aguda asociada y/o previa al infarto. Este problema se puede solventar mediante la detección de una única isoforma glicosilada o de grupos de isoformas que respondan de igual forma en todos los individuos y en la misma enfermedad. In the present invention it is demonstrated how in patients who have suffered an acute coronary syndrome the amount of total Apo J is altered with respect to individuals who have not suffered this event. Said Apo J has different isoforms with various glycosylation states. The amount of total Apo J can vary greatly in the same event among different individuals since some of its isoforms increase while others decrease, and not in all individuals the proportions thereof are equal. Therefore, the detection of total Apo J is not an adequate marker for the diagnosis of tissue damage or of the acute inflammation associated and / or prior to infarction. This problem can be solved by detecting a single glycosylated isoform or groups of isoforms that respond in the same way in all individuals and in the same disease.
Por tanto, un primer aspecto de la invención se refiere al uso de al menos una isoforma glicosilada de Apo J como marcador de daño tisular y/o inflamación aguda asociada a un infarto agudo de miocardio. En una realización preferente, la invención se refiere al uso de al menos una isoforma glicosilada de Apo J como marcador de una inflamación aguda tanto previa como posterior a un IAM. Therefore, a first aspect of the invention relates to the use of at least one glycosylated isoform of Apo J as a marker of tissue damage and / or acute inflammation associated with an acute myocardial infarction. In a preferred embodiment, the invention relates to the use of at least one glycosylated isoform of Apo J as a marker of acute inflammation both before and after an AMI.
En la presente invención se entiende por "daño tisular" aquel daño que se genera en un tejido y que supone una alteración en el mismo de su situación fisiológica, ya sea en estructura o en función. Este daño tisular para el que es útil el uso de las formas glicosiladas de Apo J se da en tejidos que se seleccionan de la lista que comprende, tejido cardíaco, tejido vascular o tejido renal In the present invention, "tissue damage" is understood to mean damage that is generated in a tissue and that implies an alteration in the same of its physiological situation, either in structure or function. This tissue damage for which the use of glycosylated forms of Apo J is useful occurs in tissues that are selected from the list comprising, cardiac tissue, vascular tissue or renal tissue.
Otro aspecto de la invención se refiere al uso de al menos una isoforma glicosilada de Apo J como marcador de daño tisular y/o inflamación aguda asociada y/o previa al infarto agudo de miocardio. Another aspect of the invention relates to the use of at least one glycosylated isoform of Apo J as a marker of tissue damage and / or acute inflammation associated and / or prior to acute myocardial infarction.
En la presente invención se entiende por "inflamación aguda" aquella inflamación que se asocia a cambios en el flujo y calibre vascular, que hacen que aumente el flujo sanguíneo, cambios estructurales en los vasos sanguíneos que aumentan la permeabilidad vascular e inducen la formación de exudado inflamatorio y al paso de los leucocitos del espacio vascular al extravascular alcanzando así el foco de las lesiones. El resultado de todo ello es el acúmulo de un fluido rico en proteínas, fibrina y leucocitos. In the present invention, "acute inflammation" is understood to mean inflammation that is associated with changes in vascular flow and caliber, which increase blood flow, structural changes in blood vessels that increase blood flow. vascular permeability and induce the formation of inflammatory exudate and the passage of leukocytes from the vascular space to the extravascular thus reaching the focus of the lesions. The result of all this is the accumulation of a fluid rich in proteins, fibrin and leukocytes.
En los primeros 10-15 minutos de un episodio de inflamación aguda para el que es útil el uso de las formas glicosiladas de Apo J, se produce una hiperhemia por dilatación de arteriolas y vénulas y apertura de los vasos de pequeño calibre. Tras esta fase aumenta la viscosidad de la sangre, lo que reduce la velocidad del flujo sanguíneo. Al disminuir la presión hidrostática en los capilares, la presión osmótica del plasma aumenta, y en consecuencia un líquido rico en proteínas sale de los vasos sanguíneos originando el exudado inflamatorio. In the first 10-15 minutes of an episode of acute inflammation for which the use of glycosylated forms of Apo J is useful, hyperhemia occurs due to dilation of arterioles and venules and opening of small caliber vessels. After this phase the viscosity of the blood increases, which reduces the speed of blood flow. By decreasing the hydrostatic pressure in the capillaries, the osmotic pressure of the plasma increases, and consequently a protein-rich liquid leaves the blood vessels causing the inflammatory exudate.
Dichos cambios se diferencian de la inflamación crónica principalmente en la duración del proceso. Mientras la inflamación aguda puede durar desde unos minutos hasta unos días, la inflamación crónica dura semanas o meses y tiene se caracteriza porque el infiltrado celular está compuesto sobre todo por macrófagos, linfocitos y células plasmáticas, y la reacción inflamatoria es más productiva que exudativa, es decir, que la formación de tejido fibroso prevalece sobre el exudado de líquidos. These changes differ from chronic inflammation mainly in the duration of the process. While the acute inflammation can last from a few minutes to a few days, the chronic inflammation lasts for weeks or months and is characterized in that the cell infiltrate is mainly composed of macrophages, lymphocytes and plasma cells, and the inflammatory reaction is more productive than exudative, that is, the formation of fibrous tissue prevails over fluid exudate.
En la presente invención se entiende por "inflamación crónica" aquella inflamación que se asocia a diversas causas: a) progresión de una inflamación aguda; b) episodios recurrentes de inflamación aguda y c) inflamación crónica desde el comienzo asociada frecuentemente a infecciones intracelulares (tuberculosis, lepra, etc). Microscópicamente la inflamación crónica se caracteriza por la presencia de macrófagos y sus derivados (células epitelioides y gigantes), linfocitos, células plasmáticas, neutrófilos, eosinófilos y fibroblastos. La proteína Apolipoproteína J o Apo J (también denominada clusterina, TRPM2, CLI, NA1/NA2, ku70-binding protein 1 , Aging-associated gene 4 protein, o SP- 40,40), se encuentra codificada en Homo sapiens por el gen CLU con número de identificación en el GenBank 1 191 . Esta proteína incluye tanto las formas sin glicosilar como las formas glicosiladas. Existen 2 formas conocidas de esta proteína generadas por diferentes ARNm a partir del mismo gen. La isoforma 1 es traducida a partir del ARNm con número de acceso al GenBank NM_001831 . La isoforma 2 es traducida a partir del ARNm con número de acceso al GenBank NM_203339. In the present invention, "chronic inflammation" means inflammation that is associated with various causes: a) progression of acute inflammation; b) recurrent episodes of acute inflammation and c) chronic inflammation from the beginning frequently associated with intracellular infections (tuberculosis, leprosy, etc.). Microscopically, chronic inflammation is characterized by the presence of macrophages and their derivatives (epithelioid and giant cells), lymphocytes, plasma cells, neutrophils, eosinophils and fibroblasts. The Apolipoprotein J or Apo J protein (also called clusterin, TRPM2, CLI, NA1 / NA2, ku70-binding protein 1, Aging-associated gene 4 protein, or SP-40,40), is encoded in Homo sapiens by the gene CLU with identification number in GenBank 1 191. This protein includes both glycosylated and glycosylated forms. There are 2 known ways of this protein generated by different mRNAs from the same gene. Isoform 1 is translated from the mRNA with GenBank accession number NM_001831. Isoform 2 is translated from the mRNA with GenBank accession number NM_203339.
En la presente invención se entiende por "Apo J total" la cantidad de Apo J presente incluyendo sus diversas isoformas y diferentes estados de glicosilación. In the present invention, "total Apo J" is understood as the amount of Apo J present including its various isoforms and different glycosylation states.
Se entiende por "isoforma glicosilada de Apo J" o "forma glicosilada de Apo J" en la presente invención aquella proteína cuya secuencia de aminoácidos es igual a Apo J, y que presenta unido al menos un azúcar en cualquiera de sus aminoácidos mediante un enlace N-glicosídico u O-glicosídico. The term "glycosylated isoform of Apo J" or "glycosylated form of Apo J" is understood in the present invention as a protein whose amino acid sequence is equal to Apo J, and which has at least one sugar attached to any of its amino acids via a link N-glycosidic or O-glycosidic.
Existen multitud de estados de glicosilación de Apo J ya sea en cuanto a cantidad de aminoácidos glicosilados, como a la composición en azúcares de dichas glicosilaciones. Estas variaciones le confieren a las diferentes formas glicosiladas de Apo J diversas características en cuanto a tamaño y punto isoeléctrico (pl). El peso molecular varía en función de la glicosilación desde los 37,1 a los 47,3 kDa, y el punto isoeléctrico de 4,6 a 5. Cada una de dichas formas presenta diferentes características y diversos patrones de expresión en función del estado fisiológico de cada individuo. There are many states of Apo J glycosylation either in terms of the amount of glycosylated amino acids, or in the sugar composition of said glycosylations. These variations give the different glycosylated forms of Apo J various characteristics in terms of size and isoelectric point (pl). The molecular weight varies depending on glycosylation from 37.1 to 47.3 kDa, and the isoelectric point from 4.6 to 5. Each of these forms has different characteristics and different patterns of expression depending on the physiological state. of each individual.
En el caso de daño tisular así como de la inflamación aguda asociada y/o previa al IAM, existen grupos de formas que se comportan de igual forma, es decir aumentando o disminuyendo, en la misma patología en todos los individuos. Como se demuestra en la figura 2 de la presente invención, los spots de las formas glicosiladas que presentan una mayor variación y por lo tanto una mayor utilidad para el diagnóstico son las formas correspondientes a los spots 6 y 9 (Apo J-15) las cuales disminuyen, y a los spots 3, 7, 8 y 1 1 (Apo J-29), los cuales aumentan. Por tanto, los spots que conforman el grupo Apo J-15 son el spot número 6 que se corresponde con una forma glicosilada de Apo J con un peso molecular de aproximadamente 43,2 kDa y un punto isoeléctrico (pl) de aproximadamente 4,8 en una electroforesis bidimensional, y el spot número 9 que se corresponde con una forma glicosilada de Apo J con un peso molecular de aproximadamente 41 ,2 kDa y un punto isoeléctrico (pl) de aproximadamente 4,9 en una electroforesis bidimensional. En los ejemplos de la presente invención se muestra que ambos spots sufren una reducción superior al 50% en caso de daño tisular así como de la inflamación aguda asociada y/o previa al IAM. In the case of tissue damage as well as the acute inflammation associated and / or prior to AMI, there are groups of ways that behave in the same way, that is, increasing or decreasing, in the same pathology in all individuals. As shown in Figure 2 of the present invention, the spots of the glycosylated forms that exhibit a greater variation and therefore a greater utility for diagnosis are the forms corresponding to spots 6 and 9 (Apo J-15). which decrease, and spots 3, 7, 8 and 1 1 (Apo J-29), which increase. Therefore, the spots that make up the Apo J-15 group are spot number 6 that corresponds to a glycosylated form of Apo J with a molecular weight of approximately 43.2 kDa and an isoelectric point (pl) of approximately 4.8 in a two-dimensional electrophoresis, and spot number 9 that corresponds to a glycosylated form of Apo J with a molecular weight of approximately 41.2 kDa and an isoelectric point (pl) of approximately 4.9 in a two-dimensional electrophoresis. In the examples of the present invention it is shown that both spots suffer a reduction greater than 50% in case of tissue damage as well as the acute inflammation associated and / or prior to AMI.
Por su parte Apo J-29 engloba cuatro formas glicosiladas de Apo J diferentes. Estas formas se corresponden con los spots 3, 7, 8 y 1 1 de los ejemplos de la presente invención. El spot 3 es una forma glicosilada de Apo J con un peso molecular de aproximadamente 44,8 kDa y un pl de aproximadamente 4,6; el spot 7 es una forma glicosilada de Apo J con un peso molecular de aproximadamente 41 kDa y un pl de aproximadamente 4,8; el spot 8 es una forma glicosilada de Apo J con un peso molecular de aproximadamente 44 kDa y un pl de aproximadamente 4,8; el spot 3 es una forma glicosilada de Apo J con un peso molecular de aproximadamente 42,2 kDa y un pl de aproximadamente 5. Todos los puntos isoeléctricos y pesos moleculares de las formas del conjunto Apo J-15 y del conjunto Apo J-29 se refieren a una movilidad electroforética obtenida mediante la técnica de la electroforesis bidimensional y sometida, por tanto a la variabilidad que esta metodología comporta. El uso de las formas incluidas en los conjuntos Apo J-15 y Apo J-29 bien de forma independiente o bien en combinación, supone una mejora en los marcadores para el estudio del daño tisular así como de la inflamación aguda asociada y/o previa al IAM. Al ser las formas de estos conjuntos las que mejor responden, estas son las formas glicosiladas preferidas para identificar el daño tisular. Por tanto, una realización preferida de este aspecto de la invención, se refiere al uso de al menos una forma glicosilada de Apo J como marcador de daño tisular y/o inflamación donde la forma glicosilada se selecciona del conjunto Apo J-15 y/o del conjunto Apo J-29. En una realización más preferida las formas glicosiladas son todas las del conjunto Apo J-15 y/o todas las del conjunto Apo J-29. On the other hand Apo J-29 encompasses four different glycosylated forms of Apo J. These forms correspond to spots 3, 7, 8 and 1 1 of the examples of the present invention. Spot 3 is a glycosylated form of Apo J with a molecular weight of approximately 44.8 kDa and a pl of approximately 4.6; spot 7 is a glycosylated form of Apo J with a molecular weight of approximately 41 kDa and a pl of approximately 4.8; spot 8 is a glycosylated form of Apo J with a molecular weight of approximately 44 kDa and a pl of approximately 4.8; spot 3 is a glycosylated form of Apo J with a molecular weight of approximately 42.2 kDa and a pl of approximately 5. All isoelectric points and molecular weights of the Apo J-15 and Apo J-29 assembly forms they refer to an electrophoretic mobility obtained by means of the two-dimensional electrophoresis technique and subjected, therefore, to the variability that this methodology entails. The use of the forms included in the Apo J-15 and Apo J-29 sets either independently or in combination, represents an improvement in the markers for the study of tissue damage as well as the associated and / or previous acute inflammation to the IAM. Being the forms of these sets that best respond, these are the preferred glycosylated forms to identify tissue damage. Therefore, a preferred embodiment of this aspect of the invention refers to the use of at least one glycosylated form of Apo J as a marker of tissue damage and / or inflammation where the glycosylated form is selected from the Apo J-15 and / or of the Apo J-29 set. In a more preferred embodiment the glycosylated forms are all of the Apo J-15 set and / or all of the Apo J-29 set.
En la presente invención se demuestra que Apo J total presenta variaciones en tejido cardíaco dañado y/o inflamado frente a un tejido cardíaco sano. Esto viene determinado por las variaciones en las diversas formas glicosiladas de la proteína. Uno de los eventos más importantes que generan daño en el tejido cardíaco es el infarto agudo de miocardio, que es una de las enfermedades de mayor incidencia en el mundo. En la presente invención, se demuestra que este infarto de miocardio provoca variaciones en la cantidad de las formas de Apo J en diversos estados de glicosilación respecto a individuos que no sufren este infarto. Por 5 tanto, en una realización aun más preferida de este aspecto de la invención se refiere al uso de al menos una forma glicosilada de Apo J, preferiblemente seleccionada del conjunto Apo J-15 y/o del conjunto Apo J-29, más preferiblemente todas las del conjunto Apo J-15 y/o del conjunto Apo J-29, como marcador de daño cardíaco y/o inflamación, preferiblemente producido por infarto 10 agudo de miocardio. The present invention demonstrates that total Apo J exhibits variations in damaged and / or inflamed heart tissue versus healthy heart tissue. This is determined by the variations in the various glycosylated forms of the protein. One of the most important events that generate damage to heart tissue is the acute myocardial infarction, which is one of the diseases with the highest incidence in the world. In the present invention, it is demonstrated that this myocardial infarction causes variations in the amount of Apo J forms in various states of glycosylation with respect to individuals who do not suffer from this infarction. Therefore, in an even more preferred embodiment of this aspect of the invention it refers to the use of at least one glycosylated form of Apo J, preferably selected from the Apo J-15 assembly and / or Apo J-29 assembly, more preferably all of the Apo J-15 and / or Apo J-29, as a marker of cardiac damage and / or inflammation, preferably caused by acute myocardial infarction.
Apo J también se puede encontrar alterada en tejido renal. Por tanto, otra realización preferida de este aspecto de la invención se refiere al uso de al menos una forma glicosilada de Apo J, preferiblemente seleccionada del conjunto Apo J- 15 15 y/o del conjunto Apo J-29, más preferiblemente todas las del conjunto Apo J-15 y/o del conjunto Apo J-29, como marcador de daño renal y/o inflamación aguda asociada y/o previa al infarto. Apo J can also be found altered in renal tissue. Therefore, another preferred embodiment of this aspect of the invention relates to the use of at least one glycosylated form of Apo J, preferably selected from the Apo J-15 set and / or the Apo J-29 set, more preferably all of the Apo J-15 set and / or Apo J-29 set, as a marker of renal damage and / or acute inflammation associated and / or prior to infarction.
Otra realización preferida se refiere al uso de las formas glicosiladas de Apo J de 0 peso molecular 43,2 kDa aproximadamente y pl 4,8 aproximadamente, y/o de peso molecular 41 ,2 kDa aproximadamente y pl 4,9 aproximadamente en una electroforesis bidimensional, como marcadores de daño tisular y/o inflamación aguda asociada y/o previa al infarto, preferiblemente de daño cardíaco, más preferiblemente producido por infarto de miocardio.Another preferred embodiment relates to the use of glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8 pl, and / or approximately 41.2 kDa molecular weight and approximately 4.9 4.9 in an electrophoresis two-dimensional, as markers of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably of heart damage, more preferably caused by myocardial infarction.
5  5
Otra realización preferida se refiere al uso de las formas glicosiladas de Apo J de peso molecular 44,8 kDa aproximadamente y pl 4,6 aproximadamente, de peso molecular 41 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 44 kDa aproximadamente y pl 4,8 aproximadamente, y/o de peso 30 molecular 42,2 kDa aproximadamente y pl 5 aproximadamente en una electroforesis bidimensional como marcadores de daño tisular y/o inflamación aguda asociada y/o previa al infarto, preferiblemente de daño cardíaco, más preferiblemente producido por infarto de miocardio. Otra realización preferida se refiere al uso de las formas glicosiladas de Apo J de peso molecular 43,2 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 41 ,2 kDa aproximadamente y pl 4,9 aproximadamente, de peso molecular 44,8 kDa aproximadamente y pl 4,6 aproximadamente, de peso molecular 41 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 44 kDa aproximadamente y pl 4,8 aproximadamente, y/o de peso molecular 42,2 kDa aproximadamente y pl 5 aproximadamente en una electroforesis bidimensional como marcadores de daño tisular y/o inflamación aguda asociada y/o previa al infarto, preferiblemente de daño cardíaco, más preferiblemente producido por infarto de miocardio. Another preferred embodiment relates to the use of glycosylated forms of Apo J of approximately 44.8 kDa molecular weight and approximately 4.6 pl, approximately 41 kDa molecular weight and approximately 4.8 pl, approximately 44 kDa molecular weight and pl Approximately 4.8, and / or molecular weight approximately 42.2 kDa and approximately 5 in a two-dimensional electrophoresis as markers of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably heart damage, more preferably caused by myocardial infarction. Another preferred embodiment relates to the use of glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8 pl, of approximately 41.2 kDa molecular weight and approximately 4.9 pl, of 44.8 molecular weight about kDa and about 4.6 pl, about 41 kDa molecular weight and about 4.8 pl, about 44 kDa molecular weight and about 4.8 pl, and / or about 42.2 kDa molecular weight and about 5 pl in a two-dimensional electrophoresis as markers of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably of heart damage, more preferably caused by myocardial infarction.
Otro aspecto de la presente invención se refiere a un método de obtención de datos útiles para el diagnóstico, pronóstico o monitorización del tratamiento de un daño tisular y/o inflamación aguda asociada y/o previa al infarto, preferiblemente daño cardíaco, más preferiblemente producido por infarto agudo de miocardio que comprende los siguientes pasos: Another aspect of the present invention relates to a method of obtaining useful data for the diagnosis, prognosis or monitoring of the treatment of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably heart damage, more preferably caused by acute myocardial infarction comprising the following steps:
a) obtener una muestra biológica aislada de un individuo, y  a) obtain an isolated biological sample from an individual, and
b) detectar la cantidad de al menos una forma glicosilada de Apo J en la muestra obtenida en el paso (a).  b) detect the amount of at least one glycosylated form of Apo J in the sample obtained in step (a).
En una realización preferida de este aspecto de la invención, el método de obtención de datos útiles para el diagnóstico, pronóstico o monitorización del tratamiento de un daño tisular y/o inflamación aguda asociada y/o previa al infarto, preferiblemente daño cardíaco, más preferiblemente producido por infarto agudo de miocardio que comprende además de los pasos (a) y (b) descritos anteriormente, un paso adicional: In a preferred embodiment of this aspect of the invention, the method of obtaining useful data for the diagnosis, prognosis or monitoring of the treatment of tissue damage and / or acute inflammation associated and / or prior to the infarction, preferably heart damage, more preferably produced by acute myocardial infarction which comprises in addition to steps (a) and (b) described above, an additional step:
c) comparar la cantidad detectada en el paso (b) con una cantidad de referencia obtenida de una muestra control. En una realización más preferida de este aspecto de la invención las formas glicosiladas que se detectan en el paso (b) se seleccionan del conjunto Apo J-15 y/o del conjunto Apo J-29. En una realización aun más preferida de este aspecto de la invención las formas glicosiladas que se detectan en el paso (b) son todas las del conjunto Apo J-15 y/o todas las del conjunto Apo J-29. En otra realización más preferida de este aspecto de la invención las formas glicosiladas que se detectan en el paso (b) son las formas glicosiladas de Apo J de peso molecular 43,2 kDa aproximadamente y pl 4,8 aproximadamente, y/o de peso molecular 41 ,2 kDa aproximadamente y pl 4,9 aproximadamente en una electroforesis bidimensional. c) compare the amount detected in step (b) with a reference amount obtained from a control sample. In a more preferred embodiment of this aspect of the invention the glycosylated forms detected in step (b) are selected from the Apo J-15 assembly and / or the Apo J-29 assembly. In an even more preferred embodiment of this aspect of the invention the glycosylated forms detected in step (b) are all of the Apo J-15 set and / or all of the Apo J-29 set. In another more preferred embodiment of this aspect of the invention the glycosylated forms detected in step (b) are the glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8, and / or of weight molecular 41, approximately 2 kDa and approximately 4.9 in a two-dimensional electrophoresis.
En otra realización más preferida de este aspecto de la invención las formas glicosiladas que se detectan en el paso (b) son las formas glicosiladas de Apo J de peso molecular 44,8 kDa aproximadamente y pl 4,6 aproximadamente, de peso molecular 41 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 44 kDa aproximadamente y pl 4,8 aproximadamente, y de peso molecular 42,2 kDa aproximadamente y pl 5 aproximadamente en una electroforesis bidimensional. In another more preferred embodiment of this aspect of the invention the glycosylated forms detected in step (b) are the glycosylated forms of Apo J of approximately 44.8 kDa molecular weight and approximately 4.6 pl, of 41 kDa molecular weight approximately and pl approximately 4.8, molecular weight approximately 44 kDa and approximately 4.8, and molecular weight approximately 42.2 kDa and approximately 5 in a two-dimensional electrophoresis.
En otra realización más preferida de este aspecto de la invención las formas glicosiladas que se detectan en el paso (b) son las formas glicosiladas de Apo J de peso molecular 43,2 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 41 ,2 kDa aproximadamente y pl 4,9 aproximadamente, de peso molecular 44,8 kDa aproximadamente y pl 4,6 aproximadamente, de peso molecular 41 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 44 kDa aproximadamente y pl 4,8 aproximadamente, y de peso molecular 42,2 kDa aproximadamente y pl 5 aproximadamente en una electroforesis bidimensional. In another more preferred embodiment of this aspect of the invention, the glycosylated forms detected in step (b) are the glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8, of molecular weight 41, About 2 kDa and about 4.9 pl, about 44.8 kDa molecular weight and about 4.6 pl, about 41 kDa molecular weight and about 4.8 pl, about 44 kDa molecular weight and about 4.8 pl , and molecular weight 42.2 kDa approximately and pl 5 approximately in a two-dimensional electrophoresis.
Otro aspecto de la presente invención se refiere a un método de diagnóstico que comprende los pasos (a)-(c) mencionados anteriormente, que además comprende un paso (d) donde una cantidad de al menos una de las formas glicosiladas del conjunto Apo J-15 detectada en el paso (b) menor que la cantidad de referencia del paso (c), es indicativa de daño tisular y/o inflamación aguda asociada y/o previa al IAM, preferiblemente daño cardíaco, más preferiblemente producido por infarto agudo de miocardio. En una realización preferida de este aspecto de la invención las isoformas del paso (b) son todas las formas glicosiladas del conjunto Apo J-15. Otro aspecto de la presente invención se refiere a un método de diagnóstico que comprende los pasos (a)-(c) mencionados anteriormente, que además comprende un paso (d) donde una cantidad de las formas glicosiladas de Apo J de peso molecular 43,2 kDa y pl 4,8 aproximadamente, y/o de peso molecular 41 ,2 kDa aproximadamente y pl 4,9 aproximadamente en una electroforesis bidimensional, detectada en el paso (b) menor que la cantidad de referencia del paso (c), es indicativa de daño tisular y/o inflamación aguda asociada y/o previa al infarto, preferiblemente daño cardíaco, más preferiblemente producido por infarto agudo de miocardio. Another aspect of the present invention relates to a diagnostic method comprising the steps (a) - (c) mentioned above, which further comprises a step (d) where an amount of at least one of the glycosylated forms of the Apo J assembly -15 detected in step (b) less than the reference amount of step (c), is indicative of tissue damage and / or acute inflammation associated and / or prior to AMI, preferably heart damage, more preferably caused by acute heart attack. myocardium In a preferred embodiment of this aspect of the invention the isoforms of step (b) are all glycosylated forms of the Apo J-15 assembly. Another aspect of the present invention relates to a diagnostic method comprising the steps (a) - (c) mentioned above, which further comprises a step (d) where a quantity of the glycosylated forms of Apo J of molecular weight 43, 2 kDa and pl 4.8 approximately, and / or molecular weight 41.2 kDa approximately and pl 4.9 approximately in a two-dimensional electrophoresis, detected in step (b) less than the reference amount of step (c), It is indicative of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably heart damage, more preferably caused by acute myocardial infarction.
Otro aspecto de la presente invención se refiere a un método de diagnóstico que comprende los pasos (a)-(c) mencionados anteriormente, que además comprende un paso (d) donde una cantidad de al menos una de las formas glicosiladas del conjunto Apo J-29 detectada en el paso (b) mayor que la cantidad de referencia del paso (c) es indicativa de daño tisular y/o inflamación aguda asociada y/o previa al IAM, preferiblemente daño cardíaco, más preferiblemente producido por infarto agudo de miocardio. En una realización preferida de este aspecto de la invención las isoformas del paso (b) son todas las formas glicosiladas del conjunto Apo J-29. Another aspect of the present invention relates to a diagnostic method comprising the steps (a) - (c) mentioned above, which further comprises a step (d) where an amount of at least one of the glycosylated forms of the Apo J assembly -29 detected in step (b) greater than the reference amount of step (c) is indicative of tissue damage and / or acute inflammation associated and / or prior to AMI, preferably heart damage, more preferably caused by acute myocardial infarction . In a preferred embodiment of this aspect of the invention the isoforms of step (b) are all glycosylated forms of the Apo J-29 assembly.
Otro aspecto de la presente invención se refiere a un método de diagnóstico que comprende los pasos (a)-(c) mencionados anteriormente, que además comprende un paso (d) donde una cantidad de las formas glicosiladas de Apo J de peso molecular 44,8 kDa aproximadamente y pl 4,6 aproximadamente, de peso molecular 41 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 44 kDa aproximadamente y pl 4,8 aproximadamente, y/o de peso molecular 42,2 kDa aproximadamente y pl 5 aproximadamente en una electroforesis bidimensional, detectadas en el paso (b) mayor que la cantidad de referencia del paso (c) es indicativa de daño tisular y/o inflamación aguda asociada y/o previa al infarto, preferiblemente daño cardíaco, más preferiblemente producido por infarto agudo de miocardio. Otro aspecto de la presente invención se refiere a un método de diagnóstico que comprende los pasos (a)-(c) mencionados anteriormente, que además comprende un paso (d) donde una cantidad de al menos de al menos las formas glicosiladas del conjunto Apo J-15 detectada en el paso (b) menor que la cantidad de referencia del paso (c), y/o una cantidad de al menos de todas las formas glicosiladas del conjunto Apo J-29 detectada en el paso (b) mayor que la cantidad de referencia del paso (c) son indicativas de daño tisular y/o inflamación aguda asociada y/o previa al IAM, preferiblemente daño cardíaco, más preferiblemente producido por infarto agudo de miocardio. En una realización preferida de este aspecto de la invención, las formas glicosiladas detectadas en el paso (b) son todas las formas glicosiladas del conjunto Apo J-15 y/o todas las formas glicosiladas del conjunto Apo J-29 Another aspect of the present invention relates to a diagnostic method comprising the steps (a) - (c) mentioned above, which further comprises a step (d) where a quantity of the glycosylated forms of Apo J of molecular weight 44, About 8 kDa and about 4.6 pl, about 41 kDa molecular weight and about 4.8 pl, about 44 kDa molecular weight and about 4.8 pl, and / or about 42.2 kDa molecular weight and pl 5 approximately in a two-dimensional electrophoresis, detected in step (b) greater than the reference amount of step (c) is indicative of tissue damage and / or acute inflammation associated and / or prior to infarction, preferably heart damage, more preferably caused by acute myocardial infarction. Another aspect of the present invention relates to a diagnostic method comprising steps (a) - (c) mentioned above, which further comprises a step (d) where an amount of at least at least the glycosylated forms of the Apo set J-15 detected in step (b) less than the reference amount of step (c), and / or an amount of at least all of the glycosylated forms of the Apo J-29 set detected in step (b) greater than The reference amount of step (c) are indicative of tissue damage and / or acute inflammation associated and / or prior to AMI, preferably heart damage, more preferably caused by acute myocardial infarction. In a preferred embodiment of this aspect of the invention, the glycosylated forms detected in step (b) are all glycosylated forms of the Apo J-15 assembly and / or all glycosylated forms of the Apo J-29 assembly.
Otro aspecto de la presente invención se refiere a un método de diagnóstico que comprende los pasos (a)-(c) mencionados anteriormente, que además comprende un paso (d) donde una cantidad de las formas glicosiladas de Apo J de peso molecular 43,2 kDa aproximadamente y pl 4,8 aproximadamente y/o de peso molecular 41 ,2 kDa aproximadamente y pl 4,9 aproximadamente en una electroforesis bidimensional, detectadas en el paso (b) menor que la cantidad de referencia del paso (c), y una cantidad de las formas glicosiladas de Apo J de peso molecular 44,8 kDa aproximadamente y pl 4,6 aproximadamente, de peso molecular 41 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 44 kDa aproximadamente y pl 4,8 aproximadamente, y/o de peso molecular 42,2 kDa aproximadamente y pl 5 en una electroforesis bidimensional, detectadas en el paso (b) mayor que la cantidad de referencia del paso (c) son indicativas de daño tisular y/o inflamación aguda asociada y/o previa al infarto, preferiblemente daño cardíaco, más preferiblemente producido por infarto agudo de miocardio. Los pasos (b) y/o (c) de los métodos descritos anteriormente pueden ser total o parcialmente automatizados, por ejemplo, por medio de un equipo robótico sensor para la detección de la cantidad en el paso (b) o la comparación computerizada en el paso (c). Además de los pasos especificados anteriormente puede comprender otros pasos adicionales, por ejemplo relacionados con el pre- tratamiento de la muestra o la evaluación de los resultados obtenidos mediante estos métodos. Another aspect of the present invention relates to a diagnostic method comprising the steps (a) - (c) mentioned above, which further comprises a step (d) where a quantity of the glycosylated forms of Apo J of molecular weight 43, Approximately 2 kDa and approximately 4.8 and / or molecular weight 41.2 approximately 2 kDa and approximately 4.9 in a two-dimensional electrophoresis, detected in step (b) less than the reference amount of step (c), and an amount of the glycosylated forms of Apo J of approximately 44.8 kDa molecular weight and approximately 4.6 pl, approximately 41 kDa molecular weight and approximately 4.8 pl, approximately 44 kDa molecular weight and approximately 4.8 pl , and / or molecular weight 42.2 kDa approximately and pl 5 in a two-dimensional electrophoresis, detected in step (b) greater than the reference amount of step (c) are indicative of tissue damage and / or associated acute inflammation and / or before the infarction, preferably heart damage, more preferably caused by acute myocardial infarction. The steps (b) and / or (c) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the quantity in step (b) or the computerized comparison in step (c). In addition to the steps specified above you can include other additional steps, for example related to the pre Sample treatment or evaluation of the results obtained by these methods.
El término "diagnóstico", tal y como se utiliza en la presente invención, se refiere a la capacidad de discriminar entre muestras procedentes de pacientes que presentan un daño tisular y/o inflamación aguda asociada y/o previa al infarto y muestras procedentes de individuos sanos que no han sufrido este daño y/o inflamación, cuando se aplica un método de clasificación de muestras basado en el análisis de la cantidad de las formas glicosiladas de Apo J y en la comparación de dicha cantidad detectada con respecto a una cantidad de referencia. Esta detección tal y como es entendida por un experto en la materia no pretende ser correcta en un 100% de las muestras analizadas. Sin embargo, requiere que una cantidad estadísticamente significativa de las muestras analizadas sean clasificadas correctamente. La cantidad que es significativamente estadística puede ser establecida por un experto en la materia mediante el uso de diferentes herramientas estadísticas, por ejemplo, pero sin limitarse, mediante la determinación de intervalos de confianza, determinación del valor p, test de Student o funciones discriminantes de Fisher. Preferiblemente, los intervalos de confianza son al menos del 90%, al menos del 95%, al menos del 97%, al menos del 98% o al menos del 99%. Preferiblemente, el valor de p es menor de 0,1 , de 0,05, de 0,01 , de 0,005 o de 0,0001 . Preferiblemente, la presente invención permite detectar correctamente el daño y/o inflamación en al menos el 60%, en al menos el 70%, en al menos el 80%, o en al menos el 90% de los sujetos de un determinado grupo o población analizada. The term "diagnosis", as used in the present invention, refers to the ability to discriminate between samples from patients presenting with tissue damage and / or acute inflammation associated and / or prior to infarction and samples from individuals healthy who have not suffered this damage and / or inflammation, when a sample classification method is applied based on the analysis of the amount of glycosylated forms of Apo J and the comparison of said detected amount with respect to a reference amount . This detection, as understood by one skilled in the art, is not intended to be correct in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly. The amount that is significantly statistical can be established by one skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the p-value, Student's test or discriminant functions of Fisher Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferably, the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention allows to correctly detect the damage and / or inflammation in at least 60%, in at least 70%, in at least 80%, or in at least 90% of the subjects of a certain group or population analyzed.
La expresión "detectar o detección de la cantidad" de una proteína, entendiendo por proteína cada una de las diferentes formas glicosiladas de Apo J en la muestra obtenida, tal y como se utiliza en la presente descripción, hace referencia a la medida de la cantidad o la concentración, preferiblemente de manera semi-cuantitativa o cuantitativa. La medida puede ser llevada a cabo de manera directa o indirecta. La medida directa se refiere a la medida de la cantidad o la concentración de la proteína, basada en una señal que se obtiene directamente de la proteína, y que está correlacionada directamente con el número de moléculas de la proteína, presente en la muestra. Dicha señal -a la que también podemos referirnos como señal de intensidad- puede obtenerse, por ejemplo, midiendo un valor de intensidad de una propiedad química o física de la proteína. La medida indirecta incluye la medida obtenida de un componente secundario (por ejemplo, un componente distinto de la proteína) o un sistema de medida biológica (por ejemplo la medida de respuestas celulares, ligandos, "etiquetas" o productos de reacción enzimática). The expression "detect or detect the amount" of a protein, each protein being understood to mean each of the different glycosylated forms of Apo J in the sample obtained, as used in the present description, refers to the measure of the quantity or the concentration, preferably semi-quantitatively or quantitatively. The measure can be carried out directly or indirectly. Direct measurement refers to the measure of the amount or concentration of the protein, based on a signal that is obtained directly from the protein, and that is directly correlated with the number of protein molecules present in the sample. That signal -to the that we can also refer to as an intensity signal - it can be obtained, for example, by measuring an intensity value of a chemical or physical property of the protein. The indirect measurement includes the measurement obtained from a secondary component (for example, a component other than the protein) or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products).
El término "cantidad", tal y como se utiliza en la descripción, se refiere pero no se limita, a la cantidad absoluta o relativa de la proteína, así como a cualquier otro valor o parámetro relacionado con las mismas o que pueda derivarse de éstas. Dichos valores o parámetros comprenden valores de intensidad de la señal obtenidos a partir de cualquiera de las propiedades físicas o químicas de la proteína mediante medida directa, como por ejemplo, valores de intensidad de espectroscopia de masas o resonancia magnética nuclear. Adicionalmente, dichos valores o parámetros incluyen todos aquellos obtenidos mediante medida indirecta, por ejemplo, cualquiera de los sistemas de medida descritos en otra parte del presente documento. The term "quantity", as used in the description, refers to, but is not limited to, the absolute or relative amount of the protein, as well as any other value or parameter related to or derived from them. . Said values or parameters comprise values of signal intensity obtained from any of the physical or chemical properties of the protein by direct measurement, such as, for example, intensity values of mass spectroscopy or nuclear magnetic resonance. Additionally, said values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.
El término "comparación" o "comparar", tal y como se utiliza en la descripción, se refiere pero no se limita, a la comparación de la cantidad de las formas glicosiladas de Apo J de la muestra biológica a analizar, también llamada muestra biológica problema, con una cantidad de las formas glicosiladas de Apo J de una muestra de referencia deseable descrita en otra parte de la presente descripción. La muestra de referencia puede ser analizada, por ejemplo, simultánea o consecutivamente, junto con la muestra biológica problema. The term "comparison" or "compare", as used in the description, refers to, but is not limited to, the comparison of the amount of Apo J glycosylated forms of the biological sample to be analyzed, also called the biological sample. problem, with a quantity of the glycosylated forms of Apo J of a desirable reference sample described elsewhere in the present description. The reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample.
El término "cantidad de referencia", tal y como se utiliza en la descripción, se refiere a la cantidad de la cantidad absoluta o relativa de las formas glicosiladas de la proteína Apo J que permite discriminar daño tisular y/o inflamación aguda asociada y/o previa al infarto, de una situación no patológica. Las cantidades de referencia adecuadas pueden ser determinadas por el método de la presente invención a partir de una muestra de referencia que puede ser analizada, por ejemplo, simultánea o consecutivamente, junto con la muestra biológica problema. La muestra de referencia puede ser, por ejemplo, un extracto de proteínas obtenido a partir de una muestra biológica aislada de un individuo sano, o una mezcla de extractos de proteínas obtenidos a partir de muestras biológicas aisladas de uno o varios diversos individuos sanos. La cantidad de referencia se obtiene a partir de una muestra de referencia proveniente de un individuo sano, es decir, que no presente daño tisular y/o inflamación aguda asociada y/o previa al IAM. La cantidad de referencia se puede obtener también a partir de una muestra de referencia proveniente del mismo individuo que sufre el daño y/o inflamación, pero previamente a sufrir el daño y/o inflamación. El término "muestra biológica aislada", tal y como se utiliza en la descripción se refiere, pero no se limita, a tejidos y/o fluidos biológicos de un sujeto, obtenidos mediante cualquier método conocido por un experto en la materia que sirva para tal fin. Preferiblemente la muestra biológica aislada comprende: tejido cardíaco, sangre, suero, plasma u orina. The term "reference amount", as used in the description, refers to the amount of the absolute or relative amount of glycosylated forms of the Apo J protein that allows discriminating tissue damage and / or associated acute inflammation and / or before the infarction, of a non-pathological situation. Suitable reference amounts can be determined by the method of the present invention from a reference sample that can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. The reference sample may be, for example, an extract of proteins obtained from a biological sample isolated from a healthy individual, or a mixture of protein extracts obtained from biological samples isolated from one or several different healthy individuals. The reference amount is obtained from a reference sample from a healthy individual, that is, that there is no tissue damage and / or acute inflammation associated and / or prior to AMI. The reference amount can also be obtained from a reference sample from the same individual that suffers the damage and / or inflammation, but prior to suffering the damage and / or inflammation. The term "isolated biological sample", as used in the description, refers to, but is not limited to, tissues and / or biological fluids of a subject, obtained by any method known to a person skilled in the art that serves for such finish. Preferably the isolated biological sample comprises: heart tissue, blood, serum, plasma or urine.
Los términos "sujeto" o "individuo", tal y como se utiliza en la descripción, se refiere a animales, preferiblemente mamíferos, y más preferiblemente, humanos. The terms "subject" or "individual", as used in the description, refer to animals, preferably mammals, and more preferably, humans.
Los términos "sujeto sano" o "individuo sano" en la presente invención se refieren a aquel individuo que no tenga alterados los parámetros bioquímicos estándares para medir daño cardíaco y/o inflamación aguda asociada y/o previa al infarto [troponina, creatina kinasa (CK), creatina kinasa MB (CK-MB) y péptido natriurético tipo B (BNP)] ni signos de patología al realizar pruebas diagnósticas para medir daño cardíaco y/o inflamación (ECG y resonancia magnética cardiovascular). The terms "healthy subject" or "healthy individual" in the present invention refer to that individual who has not altered the standard biochemical parameters for measuring cardiac damage and / or acute inflammation associated and / or prior to infarction [troponin, creatine kinase ( CK), creatine kinase MB (CK-MB) and natriuretic peptide type B (BNP)] or signs of pathology when performing diagnostic tests to measure heart damage and / or inflammation (ECG and cardiovascular magnetic resonance).
En una realización preferida, la detección de la variación en la cantidad de las formas glicosiladas de Apo J se realiza mediante una electroforesis bidimensional (2-DE). In a preferred embodiment, the detection of the variation in the amount of glycosylated forms of Apo J is performed by a two-dimensional electrophoresis (2-DE).
La electroforesis bidimensional es una técnica analítica de separación de fundamento cinético basada en el movimiento o migración de las macromoléculas disueltas en un determinado medio (solución tampón de electroforesis), a través de una matriz o soporte reticulado como resultado de la acción de un campo eléctrico. La separación de las proteínas se da en función a dos características: en primer lugar, se separan las proteínas según su punto isoeléctrico (pl) mediante isoelectroenfoque (IEF) y, en segundo lugar, la separación se realiza según su masa molecular, mediante electroforesis en condiciones desnaturalizantes (SDS-PAGE). Existen numerosas variaciones de esta técnica en función de la técnica de detección de los productos finales tras la separación. Las diferentes muestras se pueden cargar en geles diferentes tras lo que se lleva a cabo la 2-DE manteniendo las mismas condiciones en todos los geles. Posteriormente estos geles se tiñen con una tinción especifica de proteínas, como por ejemplo, aunque sin limitarse azul de Coomassie o tinción de plata, y se comparan los spot correspondientes a la proteína de interés entre los diferentes geles. Otra forma de detección y comparación de la cantidad de proteínas es mediante la técnica DIGE (Differential In Gel Electrophoresis) la cual se basa en el mareaje de las proteínas de las muestras de estudio con uno de los tres fluorocromos (Cy2, Cy3 o Cy5) antes de la separación. A continuación se mezclan las muestras y se separan en un único gel bidimensional, minimizando la variabilidad experimental. Debido al mareaje específico de cada muestra pueden observarse de manera individualizada y realizar un análisis comparativo de la expresión diferencial de proteínas, permitiendo la cuantificación precisa. Two-dimensional electrophoresis is an analytical technique of separation of kinetic foundation based on the movement or migration of macromolecules dissolved in a certain medium (electrophoresis buffer solution), through a matrix or cross-linked support as a result of the action of a field electric. The separation of proteins is based on two characteristics: first, the proteins are separated according to their isoelectric point (pl) by isoelectric focusing (IEF) and, secondly, the separation is carried out according to their molecular mass, by electrophoresis under denaturing conditions (SDS-PAGE). There are numerous variations of this technique depending on the detection technique of the final products after separation. Different samples can be loaded into different gels after which 2-DE is carried out maintaining the same conditions in all gels. Subsequently, these gels are stained with a specific protein stain, such as, but not limited to Coomassie blue or silver staining, and the spots corresponding to the protein of interest between the different gels are compared. Another way of detecting and comparing the amount of proteins is by means of the DIGE (Differential In Gel Electrophoresis) technique, which is based on the protein mapping of the study samples with one of the three fluorochromes (Cy2, Cy3 or Cy5) before separation. The samples are then mixed and separated in a single two-dimensional gel, minimizing experimental variability. Due to the specific marking of each sample they can be observed individually and perform a comparative analysis of the differential expression of proteins, allowing precise quantification.
En otra realización preferida la detección de la variación en la cantidad de las formas glicosiladas de Apo J se realiza mediante la incubación con un anticuerpo específico en un inmunoensayo. El término "inmunoensayo", tal y como se utiliza en la presente descripción se refiere a cualquier técnica analítica que se basa en la reacción de la conjugación de un anticuerpo con la muestra obtenida. Ejemplos de inmunoensayos conocidos en el estado de la técnica son, por ejemplo, pero sin limitarse: inmunoblot, ensayo inmunoabsorbente ligado a enzimas (ELISA), radioinmunoensayo (RIA), inmunohistoquímica o chips de proteína. In another preferred embodiment the detection of the variation in the amount of glycosylated forms of Apo J is performed by incubation with a specific antibody in an immunoassay. The term "immunoassay", as used herein, refers to any analytical technique that is based on the reaction of conjugation of an antibody with the sample obtained. Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry or protein chips.
En una realización más preferida, el inmunoensayo es un inmunoblot o Western blot. Para llevar a cabo un inmunoblot, se obtiene un extracto de proteínas a partir de una muestra biológica aislada de un sujeto y se separan las proteínas en un medio de soporte capaz de retenerlas mediante electroforesis. Una vez separadas las proteínas se transfieren a un soporte diferente donde pueden ser detectadas mediante el uso de anticuerpos específicos que reconocen las formas glicosiladas de la proteína Apo J. La electroforesis es una técnica analítica de separación de fundamento cinético basada en el movimiento o migración de las macromoléculas disueltas en un determinado medio (solución tampón de electroforesis), a través de una matriz o soporte reticulado como resultado de la acción de un campo eléctrico. El comportamiento de la molécula viene dado por su movilidad electroforética y ésta por la carga, tamaño y forma de la misma. Cuanto mayor es la relación carga/tamaño más rápido migra un ión en el seno del campo eléctrico. Existen numerosas variaciones de esta técnica en función del equipo utilizado, soporte y condiciones físico-químicas en las cuales se va a llevar a cabo la separación. Preferiblemente, para llevar a cabo la detección de la cantidad de las formas glicosiladas de la proteína Apo J mediante inmunoblot, las proteínas obtenidas a partir de una muestra biológica aislada de un sujeto se separan mediante la electroforesis bidimensional descrita anteriormente. In a more preferred embodiment, the immunoassay is an immunoblot or Western blot. To carry out an immunoblot, a protein extract is obtained from an isolated biological sample of a subject and the proteins are separated in a support medium capable of retaining them by electrophoresis. Once the proteins are separated, they are transferred to a different support where they can be detected through the use of specific antibodies that recognize the glycosylated forms of the Apo J. protein. Electrophoresis is an analytical technique of kinetic basis separation based on the movement or migration of the macromolecules dissolved in a certain medium (electrophoresis buffer solution), through a matrix or cross-linked support as a result of the action of an electric field. The behavior of the molecule is given by its electrophoretic mobility and this by the charge, size and shape thereof. The higher the charge / size ratio, the faster an ion migrates within the electric field. There are numerous variations of this technique depending on the equipment used, support and physical-chemical conditions in which the separation will be carried out. Preferably, to carry out the detection of the amount of glycosylated forms of the Apo J protein by immunoblot, the proteins obtained from an isolated biological sample of a subject are separated by the two-dimensional electrophoresis described above.
Una vez separadas las proteínas mediante electroforesis, y antes de la detección, las proteínas se transfieren a un soporte o a una membrana, por ejemplo, pero sin limitarse, PDVF, nitrocelulosa o acetato de celulosa. Esta membrana se híbrida con un anticuerpo específico (también llamado anticuerpo primario) que reconoce las formas glicosiladas de la proteína Apo J. A continuación, la membrana se híbrida con un anticuerpo (también llamado anticuerpo secundario) capaz de reconocer de manera específica el anticuerpo primario y que se encuentra conjugado o unido con un compuesto marcador. En una realización alternativa, es el anticuerpo que reconoce las formas glicosiladas de la proteína Apo J el que está conjugado o unido a un compuesto marcador, y no es necesario el uso de un anticuerpo secundario. Una vez detectada la proteína, se puede determinar su tamaño molecular relativo, comparando su migración con la migración de una proteína control que se detecte de forma simultánea, preferiblemente en el mismo soporte, que tiene un tamaño conocido. Por tanto, en una realización aun más preferida, la detección de la variación en la cantidad de las formas glicosiladas de Apo J se realiza mediante una electroforesis bidimensional seguida de un inmunoblot. En otra realización preferida, el inmunoensayo es un ensayo inmunoabsorbente ligado a enzimas o ELISA (Enzyme-Linked ImmunoSorbent Assay). El ELISA se basa en la premisa de que un inmunoreactivo (antígeno de la muestra biológica o anticuerpo) puede ser inmovilizado en un soporte sólido, poniendo luego ese sistema en contacto con una fase fluida que contiene el reactivo complementario que puede unirse a un compuesto marcador. Once the proteins are separated by electrophoresis, and before detection, the proteins are transferred to a support or to a membrane, for example, but not limited to, PDVF, nitrocellulose or cellulose acetate. This membrane is hybridized with a specific antibody (also called primary antibody) that recognizes the glycosylated forms of the Apo J. protein. Next, the membrane is hybridized with an antibody (also called secondary antibody) capable of specifically recognizing the primary antibody. and that it is conjugated or bound with a marker compound. In an alternative embodiment, it is the antibody that recognizes the glycosylated forms of the Apo J protein that is conjugated or bound to a marker compound, and the use of a secondary antibody is not necessary. Once the protein is detected, its relative molecular size can be determined by comparing its migration with the migration of a control protein that is detected simultaneously, preferably on the same support, which has a known size. Therefore, in an even more preferred embodiment, the detection of the variation in the amount of glycosylated forms of Apo J is performed by a two-dimensional electrophoresis followed by an immunoblot. In another preferred embodiment, the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay). The ELISA is based on the premise that an immunoreactive (biological sample antigen or antibody) can be immobilized on a solid support, then putting that system in contact with a fluid phase containing the complementary reagent that can bind to a marker compound .
Existen diferentes tipos de ELISA. En el ELISA directo o ensayo ELISA simple de dos capas, el soporte sólido se recubre con la muestra biológica y se incuba con un anticuerpo que reconoce las formas glicosiladas de la proteína Apo, conjugado o unido a un compuesto marcador. En el ELISA indirecto, el soporte sólido se recubre con la muestra biológica y se incuba con un anticuerpo primario, que reconoce las formas glicosiladas de la proteína Apo J y, a continuación, un anticuerpo secundario, que reconoce al anticuerpo primario, conjugado o unido a un compuesto marcador. En el ELISA sandwich o ensayo de captura de antígeno y detección mediante inmunocomplejos, se recubre el pocilio con un primer anticuerpo que reconoce las formas glicosiladas de la proteína Apo J de manera que las formas glicosiladas de Apo J serán retenidas en el pocilio al ser reconocido por el primer anticuerpo, y después se le aplica un segundo anticuerpo que reconoce a las formas glicosiladas de la proteína Apo J, conjugado o unido a un compuesto marcador. There are different types of ELISA. In the direct ELISA or simple two-layer ELISA assay, the solid support is coated with the biological sample and incubated with an antibody that recognizes glycosylated forms of Apo protein, conjugated or bound to a marker compound. In the indirect ELISA, the solid support is coated with the biological sample and incubated with a primary antibody, which recognizes the glycosylated forms of the Apo J protein and then a secondary antibody, which recognizes the primary antibody, conjugated or bound to a marker compound. In the sandwich ELISA or antigen capture assay and detection by immunocomplexes, the well is coated with a first antibody that recognizes the glycosylated forms of Apo J protein so that glycosylated forms of Apo J will be retained in the well upon recognition. by the first antibody, and then a second antibody is applied that recognizes the glycosylated forms of the Apo J protein, conjugated or bound to a marker compound.
En otra realización preferida, el inmunoensayo es una inmunohistoquímica (IHQ). Las técnicas de inmunohistoquímica permiten la identificación, sobre muestras tisulares o citológicas de determinantes antigénicos característicos. El análisis mediante inmunohistoquímica se realiza sobre cortes de tejido, ya sea congelado o incluido en parafina, procedente de una muestra biológica aislada de un sujeto. Estos cortes se hibridan con un anticuerpo específico o anticuerpo primario que reconoce a las formas glicosiladas de la proteína Apo J. A continuación, la membrana se híbrida con un anticuerpo secundario capaz de reconocer de manera específica el anticuerpo primario y que se encuentra conjugado o unido con un compuesto marcador. En una realización alternativa, es el anticuerpo que reconoce a las formas glicosiladas de las proteínas Apo J el que está conjugado o unido a un compuesto marcador, y no es necesario el uso de un anticuerpo secundario. In another preferred embodiment, the immunoassay is an immunohistochemistry (IHQ). Immunohistochemical techniques allow the identification, on tissue or cytological samples of characteristic antigenic determinants. Immunohistochemical analysis is performed on tissue sections, either frozen or included in paraffin, from an isolated biological sample of a subject. These sections hybridize with a specific antibody or primary antibody that recognizes the glycosylated forms of the Apo J. protein. The membrane is then hybridized with a secondary antibody capable of recognizing specifically the primary antibody and that is conjugated or bound with a marker compound. In an alternative embodiment, it is the antibody that recognizes the glycosylated forms of Apo J proteins that is conjugated or bound to a marker compound, and the use of a secondary antibody is not necessary.
El término "anticuerpo" tal como se emplea en esta memoria, se refiere a moléculas de inmunoglobulinas y porciones inmunológicamente activas de moléculas de inmunoglobulinas, es decir, moléculas que contienen un sitio de fijación de antígeno que se une específicamente (inmunorreacciona) con la formas glicosiladas de la proteína Apo J. Ejemplos de porciones de moléculas de inmunoglobulinas inmunológicamente activas, incluyen fragmentos F(ab) y F(ab')2 que pueden ser generados tratando el anticuerpo con una enzima tal como la pepsina. Los anticuerpos pueden ser policlonales (incluyen típicamente anticuerpos distintos dirigidos contra determinantes o epítopos distintos) o monoclonales (dirigidos contra un único determinante en el antígeno). El anticuerpo monoclonal puede ser alterado bioquímicamente, por manipulación genética, o puede ser sintético, careciendo, posiblemente, el anticuerpo en su totalidad o en partes, de porciones que no son necesarias para el reconocimiento de las formas glicosiladas de Apo J y estando sustituidas por otras que comunican al anticuerpo propiedades ventajosas adicionales. El anticuerpo puede ser también recombinante, quimérico, humanizado, sintético o una combinación de cualquiera de los anteriores. Un "anticuerpo o polipéptido recombinante" (rAC) es un anticuerpo que ha sido producido en una célula hospedadora que ha sido transformada o transfectada con el ácido nucleico codificante del polipéptido, o produce el polipéptido como resultado de la recombinación homologa. Anticuerpos que reconocen a las formas glicosiladas de Apo J conocidos en el estado de la técnica, como los que se describen, pero sin limitarse, en los ejemplos de la presente descripción. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with the forms Apo J. protein glycosylates. Examples of portions of immunologically active immunoglobulin molecules, include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. The antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen). The monoclonal antibody may be biochemically altered, by genetic manipulation, or it may be synthetic, possibly lacking the antibody in whole or in part, from portions that are not necessary for the recognition of the glycosylated forms of Apo J and being replaced by others that give the antibody additional advantageous properties. The antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing. A "recombinant antibody or polypeptide" (rAC) is an antibody that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination. Antibodies that recognize the glycosylated forms of Apo J known in the state of the art, such as those described, but not limited to, in the examples of the present description.
Los anticuerpos empleados para llevar a cabo los métodos de la presente invención pueden ser anticuerpos policlonales o monoclonales capaces de reconocer únicamente cada una de las formas glicosiladas de la proteína Apo J. Estos anticuerpos pueden ser empleados para llevar a cabo los métodos de la presente invención, por ejemplo, pero sin limitarse, mediante inmunoblot, ELISA o inmunohistoquímica. The antibodies used to carry out the methods of the present invention can be polyclonal or monoclonal antibodies capable of recognizing only each of the glycosylated forms of the Apo J protein. These antibodies can be used to carry out the methods of the present invention, for example, but not limited, by immunoblot, ELISA or immunohistochemistry.
El término "compuesto marcador", tal y como se utiliza en la presente descripción, se refiere a un compuesto capaz de dar lugar a una señal cromogénica, fluorogénica, radiactiva y/o quimioluminiscente que permita la detección y cuantificación de la cantidad la proteína Apo J o de sus formas truncadas. El compuesto marcador se selecciona de la lista que comprende radioisótopos, enzimas, fluoróforos o cualquier molécula susceptible de ser conjugada con otra molécula o detectada y/o cuantificada de forma directa. Este compuesto marcador puede unirse al anticuerpo directamente, o a través de otro compuesto. Algunos ejemplos de compuestos marcadores que se unen directamente son, pero sin limitarse, enzimas como la fosfatasa alcalina o la peroxidasa, isótopos radiactivos como 33P o 35S, fluorocromos como fluoresceína o partículas metálicas, para su detección directa mediante colorimetría, auto-radiografía, fluorimetría, o metalografía respectivamente. The term "marker compound", as used herein, refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and quantification of the amount of Apo protein. J or its truncated forms. The marker compound is selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and / or quantified directly. This marker compound can bind to the antibody directly, or through another compound. Some examples of directly binding marker compounds are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as 33P or 35S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, auto-radiography, fluorimetry , or metallography respectively.
Otro aspecto de la presente invención es un kit o dispositivo que comprende los elementos necesarios para llevar a cabo los métodos de la presente invención. El kit de la presente invención comprende los elementos necesarios para: Another aspect of the present invention is a kit or device comprising the elements necessary to carry out the methods of the present invention. The kit of the present invention comprises the elements necessary for:
a) detectar la cantidad de al menos una forma glicosilada de Apo J en la muestra obtenida en (a) y  a) detect the amount of at least one glycosylated form of Apo J in the sample obtained in (a) and
b) comparar la cantidad detectada en el paso (a) con una cantidad de referencia.  b) compare the quantity detected in step (a) with a reference quantity.
Dicho kit puede contener todos aquellos reactivos necesarios para detectar la cantidad de las formas glicosiladas de la proteína Apo J por medio de cualquiera de los métodos descritos anteriormente en este documento como por ejemplo, pero sin limitarse, anticuerpos específicos de las formas glicosiladas de la proteína Apo J, anticuerpos secundarios o controles positivos y/o negativos. El kit además puede incluir, sin ningún tipo de limitación, tampones, soluciones de extracción de proteínas, agentes para prevenir la contaminación, inhibidores de la degradación de las proteínas, etc. Por otro lado el kit puede incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimización. Preferiblemente, el kit comprende además las instrucciones para llevar a cabo el método de la invención. Said kit may contain all those reagents necessary to detect the amount of glycosylated forms of the Apo J protein by any of the methods described hereinbefore such as, but not limited to, antibodies specific to the glycosylated forms of the protein. Apo J, secondary antibodies or positive and / or negative controls. The kit can also include, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit further comprises instructions for carrying out the method of the invention.
En una realización preferida de este aspecto de la invención las formas glicosiladas a detectar en el paso (a) se seleccionan del conjunto Apo J-15 y/o del conjunto Apo J-29. En una realización más preferida de este aspecto de la invención las formas glicosiladas a detectar en el paso (a) son todas las del conjunto Apo J-15 y/o todas las del conjunto Apo J-29 En otra realización preferida de este aspecto de la invención las formas glicosiladas a detectar en el paso (a) son las formas glicosiladas de Apo J de peso molecular 43,2 kDa aproximadamente y pl 4,8 aproximadamente, y/o de peso molecular 41 ,2 kDa aproximadamente y pl 4,9 aproximadamente en una electroforesis bidimensional . In a preferred embodiment of this aspect of the invention the glycosylated forms to be detected in step (a) are selected from the Apo J-15 assembly and / or the Apo J-29 assembly. In a more preferred embodiment of this aspect of the invention the glycosylated forms to be detected in step (a) are all of the Apo J-15 assembly and / or all of the Apo J-29 assembly. In another preferred embodiment of this aspect of the invention the glycosylated forms to be detected in step (a) are the glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8 pl, and / or approximately 41.2 kDa molecular weight and pl 4, 9 approximately in a two-dimensional electrophoresis.
En otra realización preferida de este aspecto de la invención las formas glicosiladas a detectar en el paso (a) son las formas glicosiladas de Apo J de peso molecular 44,8 kDa aproximadamente y pl 4,6 aproximadamente, de peso molecular 41 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 44 kDa aproximadamente y pl 4,8 aproximadamente, y/o de peso molecular 42,2 kDa aproximadamente y punto isoeléctrico 5 aproximadamente en una electroforesis bidimensional. In another preferred embodiment of this aspect of the invention the glycosylated forms to be detected in step (a) are the glycosylated forms of Apo J of approximately 44.8 kDa molecular weight and approximately 4.6 pl, of approximately 41 kDa molecular weight and pl 4.8 approximately, molecular weight 44 kDa approximately and pl 4.8 approximately, and / or molecular weight 42.2 kDa approximately and isoelectric point 5 approximately in a two-dimensional electrophoresis.
En otra realización preferida de este aspecto de la invención las formas glicosiladas a detectar en el paso (a) son las formas glicosiladas de Apo J de peso molecular 43,2 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 41 ,2 kDa aproximadamente y pl 4,9 aproximadamente, de peso molecular 44,8 kDa aproximadamente y pl 4,6 aproximadamente, de peso molecular 41 kDa aproximadamente y pl 4,8 aproximadamente, de peso molecular 44 kDa aproximadamente y pl 4,8 aproximadamente, y/o de peso molecular 42,2 kDa aproximadamente y pl 5 aproximadamente en una electroforesis bidimensional. A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Las siguientes figuras y ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. In another preferred embodiment of this aspect of the invention the glycosylated forms to be detected in step (a) are the glycosylated forms of Apo J of approximately 43.2 kDa molecular weight and approximately 4.8 pl, of 41.2 kDa molecular weight approximately and pl 4.9 approximately, of molecular weight approximately 44.8 kDa and pl 4.6 approximately, of molecular weight approximately 41 kDa and pl 4.8 approximately, of molecular weight approximately 44 kDa and approximately 4.8, and / or molecular weight 42.2 kDa approximately and pl 5 approximately in a two-dimensional electrophoresis. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.
La presente invención comprende un método de detección/diagnóstico/prevención/tratamiento tanto del daño tisular como de la inflamación aguda asociada al infarto agudo de miocardio mediante el uso de al menos una forma glicosilada de Apo J como marcador. The present invention comprises a method of detection / diagnosis / prevention / treatment of both tissue damage and acute inflammation associated with acute myocardial infarction by using at least one glycosylated form of Apo J as a marker.
Por último la presente invención comprende el uso de al menos una forma glicosilada de Apo J para la fabricación de un medicamento para detección/diagnóstico/prevención/tratamiento tanto del daño tisular como de la inflamación aguda asociada al infarto agudo de miocardio, donde dicha forma glicosilada de Apo J se usa como marcador. DESCRIPCIÓN DE LAS FIGURAS Finally, the present invention comprises the use of at least one glycosylated form of Apo J for the manufacture of a medicament for detection / diagnosis / prevention / treatment of both tissue damage and acute inflammation associated with acute myocardial infarction, wherein said form Apo J glycosylated is used as a marker. DESCRIPTION OF THE FIGURES
Figura 1. Gel de electroforesis bidimensional. Figure 1. Two-dimensional electrophoresis gel.
Imagen representativa de la electroforesis de dos dimensiones. (A) muestra de suero humano tras la extracción de las seis proteínas mayoritarias (albúmina, transferrina, haptoglobina, antitripsina, inmunoglobulina G e inmunoglobulina A) (B) HDL aisladas por ultracentrifugación. Los puntos marcados con un círculo corresponden a proteínas identificadas por MALDI-Tof en ambas muestras.  Representative image of two-dimensional electrophoresis. (A) human serum sample after extraction of the six major proteins (albumin, transferrin, haptoglobin, antitrypsin, immunoglobulin G and immunoglobulin A) (B) HDL isolated by ultracentrifugation. The points marked with a circle correspond to proteins identified by MALDI-Tof in both samples.
Figura 2. Imágenes aumentadas de las zonas de Apo J en controles y pacientes de infarto agudo de miocardio. Figure 2. Increased images of Apo J areas in controls and patients with acute myocardial infarction.
Patrón representativo de Apo J en geles de electroforesis de dos dimensiones de muestras de suero de pacientes IAM (infarto agudo de miocardio) y controles. Apo J se detectó como una serie de 12 - 13 puntos con un rango de punto isoeléctrico entre 4,5 y 5 y una masa molecular entre 37,1 y 47,3 kDa. Los spots identificados como Apo J se numeraron desde el pH más ácido al más básico. El punto número 2 sólo se detectó en los pacientes IAM. Los puntos 1 , 3, 7, 8, 10, 1 1 y 13 mostraron un incremento en los niveles de detección en los pacientes IAM mientras que los puntos 6 y 9 mostraron una intensidad reducida en IAM al compararlos con el grupo control. Los spots 6 y 9 corresponden a Apo J-15, y los spots 3, 7, 8 y 1 1 corresponden a Apo J-29 Representative pattern of Apo J in two-dimensional electrophoresis gels of serum samples from AMI patients (acute myocardial infarction) and controls. Apo J was detected as a series of 12-13 points with an isoelectric point range between 4.5 and 5 and a molecular mass between 37.1 and 47.3 kDa. The spots identified as Apo J they were numbered from the most acidic to the most basic pH. Point number 2 was only detected in AMI patients. Points 1, 3, 7, 8, 10, 1 1 and 13 showed an increase in detection levels in AMI patients while points 6 and 9 showed a reduced intensity in AMI when compared to the control group. The spots 6 and 9 correspond to Apo J-15, and the spots 3, 7, 8 and 1 1 correspond to Apo J-29
Figura 3. Niveles de Apo J Figure 3. Apo J levels
Niveles séricos de Apo J (pg/ml) en pacientes IAM e individuos sanos. Los niveles de Apo J se cuantificaron mediante un ELISA comercial. (A) Diagrama de barras (media ± ES) del grupo control y los pacientes IAM en el momento del ingreso (control: n=51 ; IAM: n=39; * p<0,004; Test de Mann Whitney). (B) Gráfica del seguimiento de los niveles séricos de Apo J en los pacientes IAM desde el momento del ingreso (tiempo 0) hasta las 96 horas. Los valores de cada momento están expresados como media ± ES (§ p<0,0001 vs. control; * p<0,02 vs. time 0; ANOVA y Fisher PSLD como test Post hoc).  Serum Apo J levels (pg / ml) in AMI patients and healthy individuals. Apo J levels were quantified by a commercial ELISA. (A) Bar chart (mean ± ES) of the control group and the AMI patients at the time of admission (control: n = 51; AMI: n = 39; * p <0.004; Mann Whitney test). (B) Graph of the monitoring of serum Apo J levels in AMI patients from the moment of admission (time 0) to 96 hours. The values of each moment are expressed as mean ± ES (§ p <0.0001 vs. control; * p <0.02 vs. time 0; ANOVA and Fisher PSLD as Post hoc test).
Figura 4. Western blot de Apo J. Figure 4. Western blot of Apo J.
Western blot representativo de Apo J en controles y pacientes IAM en suero total después de eliminar las seis proteínas más abundantes(A) y en la fracción de proteínas glicosiladas del suero (B). Se detectaron dos bandas mayoritarias que correspondían al heterodímero de Apo J (65-70kDa) y a las cadenas α y β (40- 45kDa). La banda de mayor intensidad en la fracción total de suero, tanto en pacientes IAM como en controles, era la de menor peso molecular, mientras que la mayor parte de Apo J glicosilada estaba representada por la banda de 65- 70kDa. Las diferencias en la intensidad de las bandas en la fracción glicosilada eran más evidentes en el grupo control.  Western blot representative of Apo J in controls and AMI patients in total serum after eliminating the six most abundant proteins (A) and in the glycosylated whey protein fraction (B). Two major bands corresponding to the Apo J heterodimer (65-70kDa) and the α and β chains (40-45kDa) were detected. The band of greatest intensity in the total serum fraction, both in AMI patients and in controls, was the one with the lowest molecular weight, while the majority of glycosylated Apo J was represented by the 65-70kDa band. Differences in the intensity of the bands in the glycosylated fraction were more evident in the control group.
Figura 5. Imagen de un gel de un western-blot en dos dimensiones. Figure 5. Image of a western-blot gel in two dimensions.
Imagen representativa de Apo J en un gel 2-DE en la fracción glicosilada del suero de individuos sanos (A) y pacientes IAM (B). Se detectaron 6 puntos (1 , 2, 4, 5, 8 y 1 1 ) que correspondían en punto isoeléctrico y masa molecular con los detectados en el suero total . Igual que en la figura 2, los spots se numeraron desde el pH más ácido al más básico. La intensidad de los spots de Apo J era inferior en los pacientes IAM que en los controles. La disminución resulta más evidente en los puntos 4 y 8. Representative image of Apo J in a 2-DE gel in the glycosylated fraction of serum from healthy individuals (A) and AMI patients (B). 6 points (1, 2, 4, 5, 8 and 1 1) were detected that corresponded in isoelectric point and molecular mass with those detected in total serum. As in Figure 2, the spots were numbered from the most acidic pH to the most basic. The intensity of the Apo J spots was lower in AMI patients than in controls. The decrease is most evident in points 4 and 8.
Figura 6. Cuantificación de Apo J. Figure 6. Quantification of Apo J.
Apo J en la fracción de micropartículas y exosomas derivadas del suero (MP-EX) y la fracción del suero libre de MP-EX (fracción soluble) de los pacientes IAM y los individuos control. (A) El análisis mediante western blot reveló la presencia de dos bandas que se corresponden con el heterodímero glicosilado de Apo J (65-70kDa) y las cadenas α y β no glicosiladas (40-45kDa). (B) Diagrama de barras (media ± ES) de los volúmenes relativos de las bandas (calculado como el porcentaje de la intensidad total de las bandas). El porcentaje de la intensidad de las bandas en la fracción soluble variaba significativamente entre los grupos IAM y control (p=0,02). Los resultados se basan en la media ± ES de tres ensayos independientes hechos en una mezcla de extractos de varios pacientes IAM y una mezcla de extractos de varios controles. Apo J in the serum-derived microparticle fraction and exosomes (MP-EX) and the MP-EX free fraction (soluble fraction) of AMI patients and control individuals. (A) Western blot analysis revealed the presence of two bands corresponding to the Apo J glycosylated heterodimer (65-70kDa) and non-glycosylated α and β chains (40-45kDa). (B) Bar chart (mean ± ES) of the relative volumes of the bands (calculated as the percentage of the total intensity of the bands). The percentage of the intensity of the bands in the soluble fraction varied significantly between the AMI and control groups (p = 0.02). The results are based on the mean ± ES of three independent trials done on a mixture of extracts from several AMI patients and a mixture of extracts from several controls.
Figura 7. Inmunohistoquímica. Figure 7. Immunohistochemistry.
Imágenes representativas de la localización de Apo J en tejidos miocárdicos isquémicos y sanos. Apo J se localiza alrededor de los capilares en los miocardios isquémicos. Se detectan señales marrones intensas alrededor de los capilares en secciones miocárdicas de un corazón isquémico, mientras que no se detectan señales en los capilares de un tejido miocárdico no isquémico. Representative images of the location of Apo J in ischemic and healthy myocardial tissues. Apo J is located around the capillaries in the ischemic myocardia. Intense brown signals are detected around the capillaries in myocardial sections of an ischemic heart, while no signals are detected in the capillaries of a non-ischemic myocardial tissue.
BIBLIOGRAFÍA BIBLIOGRAPHY
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Short-term myocardial ischemia induces cardiac modified C-reactive protein expression and proinflammatory gene (cyclo-oxygenase-2, monocyte chemoattractant protein-1 , and tissue factor) upregulation in peripheral blood mononuclear cells. J Thromb Haemost. 2009; 7:485-93. Short-term myocardial ischemia induces cardiac modified C-reactive protein expression and proinflammatory gene (cyclo-oxygenase-2, monocyte chemoattractant protein-1, and tissue factor) upregulation in peripheral blood mononuclear cells. J Thromb Haemost. 2009; 7: 485-93.
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EJEMPLOS EXAMPLES
Los siguientes ejemplos específicos que se proporcionan en este documento de patente sirven para ilustrar la naturaleza de la presente invención. Estos ejemplos se incluyen solamente con fines ilustrativos y no han de ser interpretados como limitaciones a la invención que aquí se reivindica. Por tanto, los ejemplos descritos más adelante ilustran la invención sin limitar el campo de aplicación de la misma. The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.
EJEMPLO 1. PERFIL PROTEÓMICO DE PROTEÍNAS ASOCIADAS A LAS HDL EN MUESTRAS DE SUERO. EXAMPLE 1. PROTEIN PROFILE OF PROTEINS ASSOCIATED WITH HDL IN SAMPLES OF SERUM.
Las muestras de suero (tras la extracción de las seis proteínas más abundantes) de 27 pacientes con IAM y de donantes sanos se analizaron individualmente por duplicado mediante electroforesis de dos dimensiones (2-DE) y las proteínas se identificaron mediante espectrometría de masas tipo MALDI-ToF. En las muestras de suero humano tanto del grupo control como de los pacientes se identificaron proteínas asociadas a las HDL, incluyendo Apo J, PON1 y Apo A-l (Figuras 1A y 1 B; Tabla 1 ). Serum samples (after the extraction of the six most abundant proteins) from 27 patients with AMI and healthy donors were analyzed individually in duplicate by two-dimensional electrophoresis (2-DE) and the proteins were identified by MALDI type mass spectrometry. -ToF. In human serum samples from both the control group and the patients, HDL-associated proteins were identified, including Apo J, PON1 and Apo A-1 (Figures 1A and 1 B; Table 1).
TABLA 1. Proteínas identificadas en muestras de suero y de HDL mediante 2DE y TABLE 1. Proteins identified in serum and HDL samples using 2DE and
MALDI-Tof. MALDI-Tof.
Proteína N° Swissprot MW (kDa) i Suero HDL Swissprot MW No. (kDa) and HDL Serum
Albúmina P02768 69 5.8 - 6.3 Eliminada 11 spots  Albumin P02768 69 5.8 - 6.3 Eliminated 11 spots
Alpha 1 antitripsina P01009 60 - 67 4.9 - 5.2 Eliminada 4 spots  Alpha 1 antitrypsin P01009 60 - 67 4.9 - 5.2 Eliminated 4 spots
Apo A-I P02647 25 5 - 5.8 5 spots 5 spots  Apo A-I P02647 25 5 - 5.8 5 spots 5 spots
75 5 - 5.6 ND 5 spots  75 5 - 5.6 ND 5 spots
Apo A-IV P06727 55 5 - 5.4 1 spot 4 spots  Apo A-IV P06727 55 5 - 5.4 1 spot 4 spots
24 5.6 ND 1 spot  24 5.6 ND 1 spot
Apo E P02649 36 - 37 5.8 3 spots 2 spots  Apo E P02649 36 - 37 5.8 3 spots 2 spots
Apo J P10909 37.1 - 47.3 4.5 - 5 12 - 13 spots 7 spots  Apo J P10909 37.1 - 47.3 4.5 - 5 12 - 13 spots 7 spots
Apo M 095445 23 5.7 1 spot 1 spot  Apo M 095445 23 5.7 1 spot 1 spot
C3 complemento P01024 52 4.8 1 spot 1 spot  C3 complement P01024 52 4.8 1 spot 1 spot
PON 1 P27169 58 4.8 - 5.1 1 spot 3 spots  PON 1 P27169 58 4.8 - 5.1 1 spot 3 spots
Apo A-l = Apolipoproteína A-I; Apo A-IV = Apolipoproteína A-IV; Apo E= Apolipoproteína E; Apo J= Apolipoproteína J; Apo M= Apolipoproteína M; ND = No detectable; PON1 = Paraoxonasa 1. Las proteínas se detectaron mayoritariamente como una serie de spots (Figura 1 B). En ambas fracciones, tanto las HDL como el suero, Apo A-l se detectó como la proteína mayoritaria que consistía en una serie de diferentes spots con una masa aparente de aproximadamente 25 kDa y un punto isoeléctrico (pl) entre 5,0 y 5,8.  Apo A-1 = Apolipoprotein A-I; Apo A-IV = Apolipoprotein A-IV; Apo E = Apolipoprotein E; Apo J = Apolipoprotein J; Apo M = Apolipoprotein M; ND = Not detectable; PON1 = Paraoxonase 1. Proteins were mostly detected as a series of spots (Figure 1 B). In both fractions, both HDL and serum, Apo Al was detected as the majority protein consisting of a series of different spots with an apparent mass of approximately 25 kDa and an isoelectric point (pl) between 5.0 and 5.8 .
EJEMPLO 2. PATRÓN PROTEÓMICO DIFERENCIAL DE APO J ENTRE PACIENTES IAM E INDIVIDUOS CONTROL. EXAMPLE 2. APO J DIFFERENTIAL PROTEOMIC PATTERN BETWEEN IAM PATIENTS AND INDIVIDUALS CONTROL.
Apo J se identificó en el suero mediante 2-DE y espectrometría de masas MALDI- ToF como una serie de 12-13 spots diferentes con una masa molecular aparente de entre 37,1 y 47,3 kDa y en un rango de pl entre 4,5 y 5,0 (Figura 2). El volumen relativo de la mayoría de los spots variaba de forma significativa entre los pacientes IAM (muestras obtenidas en el momento del ingreso) y los controles (Tabla 2). Apo J was identified in serum by 2-DE and MALDI-ToF mass spectrometry as a series of 12-13 different spots with an apparent molecular mass between 37.1 and 47.3 kDa and in a range of pl between 4 , 5 and 5.0 (Figure 2). The relative volume of most spots varied significantly between AMI patients (samples obtained at the time of admission) and controls (Table 2).
TABLA 2. Perfil proteómico de Apolipoproteína J en el suero de pacientes IAM e individuos controles obtenido mediante análisis por 2-DE de muestras de suero después de eliminar las seis proteínas mayoritarias. N° Spot pl MW (kDa) Control IAM valor p * TABLE 2. Proteomic profile of Apolipoprotein J in the serum of AMI patients and control individuals obtained by 2-SD analysis of serum samples after eliminating the six major proteins. Spot number pl MW (kDa) IAM control p value *
1 4.6 47.3 446.3 [339.4-969.8] 820.7 [455.2-1098.9] 0.007  1 4.6 47.3 446.3 [339.4-969.8] 820.7 [455.2-1098.9] 0.007
2 4.5 44.4 — 485.9 [428-545.7] NS  2 4.5 44.4 - 485.9 [428-545.7] NS
3 4.6 44.8 684.7 [525.2-1489.8] 1217.5 [591.7-1797.8] 0.01  3 4.6 44.8 684.7 [525.2-1489.8] 1217.5 [591.7-1797.8] 0.01
4 4.7 45.1 1990 [1923.3-2203.4] 1982.9 [1566.2-3011.5] NS  4 4.7 45.1 1990 [1923.3-2203.4] 1982.9 [1566.2-3011.5] NS
5 4.7 43.5 115.5 [1 15.5-115.5] 459.6 [459.6-459.6] NS  5 4.7 43.5 115.5 [1 15.5-115.5] 459.6 [459.6-459.6] NS
6 4.8 43.2 31 13.1 [2138.3-4087.8] 1583.3 [768.7-2450.1] 0.009  6 4.8 43.2 31 13.1 [2138.3-4087.8] 1583.3 [768.7-2450.1] 0.009
7 4.8 41 447.3 [423.2-471.5] 971.5 [717.4-1586.9] 0.001  7 4.8 41 447.3 [423.2-471.5] 971.5 [717.4-1586.9] 0.001
8 4.8 44 1929.4 [1576.7-4067.4] 3350.5 [2195.2-4014.0] <0.001  8 4.8 44 1929.4 [1576.7-4067.4] 3350.5 [2195.2-4014.0] <0.001
9 4.9 41.2 2602.7 [1377.8-2915.3] 1282.2 [934.5-2025.7] <0.001  9 4.9 41.2 2602.7 [1377.8-2915.3] 1282.2 [934.5-2025.7] <0.001
10 4.9 39 262.3 [258.1-266.6] 739.2 [434.3-1018] 0.001  10 4.9 39 262.3 [258.1-266.6] 739.2 [434.3-1018] 0.001
11 5 42.2 1287.3 [1 108.4-2364.9] 2650.7 [2287.7-3130.5] <0.001  11 5 42.2 1287.3 [1 108.4-2364.9] 2650.7 [2287.7-3130.5] <0.001
12 5 41 1494.3 [1328.9-1659.8] 1446.6 [1 172.4-1595.2] NS  12 5 41 1494.3 [1328.9-1659.8] 1446.6 [1 172.4-1595.2] NS
13 5 37.1 117.4 [94-140.7] 361 [279-499.7] <0.001 13 5 37.1 11 7 .4 [94-140.7] 361 [279-499.7] <0.001
* Pacientes con infarto agudo de miocardio (IAM) vs. Controles: Mann Whitney.  * Patients with acute myocardial infarction (AMI) vs. Controls: Mann Whitney.
NS = No significativo. Se detectó un cambio hacia las formas con mayor movilidad electroforética en el grupo IAM (ver tabla 2). Al comparar los pacientes IAM con los controles se vio que el volumen relativo de los spots números 6 y 9 (PM 43,2-41 ,2 kDa) disminuía más del doble (p < 0,001 ), mientras que los spots 7, 10 y 13 (PM 41 ,0 - 37,1 kDa) presentaban un incremento de más del doble (p < 0,001 ). NS = Not significant. A change was detected towards the most electrophoretic mobility forms in the AMI group (see table 2). When comparing the AMI patients with the controls, it was found that the relative volume of spots 6 and 9 (PM 43.2-41, 2 kDa) decreased more than double (p <0.001), while spots 7, 10 and 13 (PM 41, 0 - 37.1 kDa) presented an increase of more than double (p <0.001).
EJEMPLO 3. NIVELES SÉRICOS DE APO J EN PACIENTES IAM E INDIVIDUOS SANOS. EXAMPLE 3. SERIAL LEVELS OF APO J IN PATIENTS IAM AND HEALTHY INDIVIDUALS.
Los niveles séricos de Apo J se midieron mediante ELISA en 39 pacientes IAM en el momento del ingreso (dentro de las 6 primeras horas desde el inicio del evento). Al compararlos con el grupo control, los niveles de Apo J eran significativamente más bajos en los pacientes (Media ± ES; IAM: 37,3 ± 2,9 g/ml vs. Controles: 45,9 ± 1 ,2 pg/ml; p=0,004). Las diferencias en Apo J también se observaron al ajusfar por los efectos de otras variables como los niveles de colesterol total, LDL y HDL (p=0,009). En 35 pacientes se hizo un seguimiento de los niveles séricos de Apo J a diferentes tiempos hasta las 96 horas desde el momento del ingreso. En el 60% de estos pacientes los niveles más bajos de Apo J se detectaron en el momento del ingreso (34,6 ± 3,04 pg/ml). A las 8 y 24 horas después del ingreso los niveles de Apo J aumentaron significativamente (8h: 42,9 ± 2,5 pg/ml, p=0,01 ; 24h: 41 ,9 ± 1 ,7 pg/ml, p=0,02) hasta que alcanzaron los niveles de los controles a las 72-96 horas tras el ingreso (72h: 43,3 ± 2,9 pg/ml, p=0,02; 96h: 49,4 ± 4,3 pg/ml, p=0,001 ). Serum Apo J levels were measured by ELISA in 39 AMI patients at the time of admission (within the first 6 hours from the start of the event). When compared with the control group, Apo J levels were significantly lower in patients (Mean ± ES; AMI: 37.3 ± 2.9 g / ml vs. Controls: 45.9 ± 1.2 pg / ml ; p = 0.004). The differences in Apo J were also observed when adjusting for the effects of other variables such as total cholesterol levels, LDL and HDL (p = 0.009). In 35 patients, serum Apo J levels were monitored at different times up to 96 hours after time of admission In 60% of these patients the lowest levels of Apo J were detected at the time of admission (34.6 ± 3.04 pg / ml). At 8 and 24 hours after admission, Apo J levels increased significantly (8h: 42.9 ± 2.5 pg / ml, p = 0.01; 24h: 41, 9 ± 1, 7 pg / ml, p = 0.02) until they reached the levels of the controls at 72-96 hours after admission (72h: 43.3 ± 2.9 pg / ml, p = 0.02; 96h: 49.4 ± 4, 3 pg / ml, p = 0.001).
En el grupo de estudio (individuos sanos y pacientes IAM) los niveles séricos de Apo J no variaban significativamente en relación a la edad ni al sexo y no se correlacionaban con los niveles de colesterol total, LDL y HDL (Tabla 3). In the study group (healthy individuals and AMI patients), serum levels of Apo J did not vary significantly in relation to age or sex and did not correlate with total cholesterol levels, LDL and HDL (Table 3).
TABLA 3. Correlaciones de la Apolipoproteína J con la edad y parámetros lipidíeos del suero en las poblaciones IAM y control. TABLE 3. Correlations of Apolipoprotein J with age and serum lipid parameters in the AMI and control populations.
Población total Población control Población IAM  Total population Control population IAM population
valor r valor p valor r valor p valor r valor p  r value p value r value p value r value p value
Edad - 0.003 NS 0.046 NS 0.318 NS  Age - 0.003 NS 0.046 NS 0.318 NS
Total-c 0.164 NS 0.274 0.05 0.161 NS  Total-c 0.164 NS 0.274 0.05 0.161 NS
LDL-c 0.178 NS 0.160 NS 0.157 NS  LDL-c 0.178 NS 0.160 NS 0.157 NS
HDL-c 0.082 NS 0.226 NS 0.028 NS  HDL-c 0.082 NS 0.226 NS 0.028 NS
Necrosis * - - - - -0.134 NS  Necrosis * - - - - -0.134 NS
TnTmax† - - - - -0.029 NS  TnTmax † - - - - -0.029 NS
PCR t = 0 } - - - - 0.444 0.01  PCR t = 0} - - - - 0.444 0.01
PCR max. § - - - - -0.086 NS  PCR max. § - - - - -0.086 NS
Necrosis (%): Porcentaje de masa necrótica del miocardio (calculado como el porcentaje de la masa total del corazón medida por resonancia magnética cardiovascular). † TnTmax: Valor máximo de los niveles de troponina T desde el momento del ingreso, t PCR t = 0: Niveles de proteína C reactiva en el momento del ingreso. § PCR max.: Valor máximo de los niveles de proteína C reactiva desde el momento del ingreso. Necrosis (%): Percentage of necrotic myocardial mass (calculated as the percentage of the total mass of the heart measured by cardiovascular magnetic resonance). † TnTmax: Maximum value of troponin levels T from the moment of admission, t PCR t = 0: Levels of C-reactive protein at the time of admission. § Max PCR: Maximum value of C-reactive protein levels from the moment of admission.
A pesar de ello, se vio una correlación baja pero positiva entre los niveles de colesterol total y los niveles de Apo J en la población control (r=0,274, p=0,05). Despite this, a low but positive correlation was seen between total cholesterol levels and Apo J levels in the control population (r = 0.274, p = 0.05).
En el grupo IAM, no se vio correlación entre los niveles séricos de Apo J en el momento del ingreso y el pico de troponina T (definido como el valor máximo de troponina T desde el momento del ingreso), ni tampoco con el porcentaje de masa necrótica del miocardio (calculado como un porcentaje de la masa cardíaca total medida por resonancia magnética cardiovascular). In the IAM group, there was no correlation between serum Apo J levels at the time of admission and the troponin T peak (defined as the maximum troponin T value from the time of admission), nor with the mass percentage Necrotic myocardium (calculated as a percentage of the total cardiac mass measured by cardiovascular magnetic resonance).
Los niveles de Apo J mostraron una correlación positiva con los niveles de 5 Proteína c reactiva (PCR) en el momento del ingreso (r=0,444, p=0,01 ) pero no se obtuvo correlación si los niveles de Apo J se analizaban en relación al pico de PCR (definido como el valor máximo de PCR desde el momento del ingreso; r=- 0,086, p=NS). El pico de PCR se correlacionaba positivamente con los niveles de troponina T y con el porcentaje de masa necrótica del miocardio (r=0,383, p=0,04; 10 r=0,454, p=0,01 , respectivamente). Los niveles de PCR en el momento del ingreso eran significativamente más altos en los pacientes con diabetes (14,4 ± 4,4 mg/L, p=0,006) e hipertensión (10,8 ± 2,6 mg/L; p=0,01 ) que los niveles medios de toda la población IAM (7,5 ± 1 ,6 mg/L). Apo J levels showed a positive correlation with levels of 5 C-reactive protein (CRP) at the time of admission (r = 0.444, p = 0.01) but no correlation was obtained if Apo J levels were analyzed in ratio to the PCR peak (defined as the maximum PCR value from the moment of admission; r = - 0.086, p = NS). The PCR peak correlated positively with troponin T levels and with the percentage of myocardial necrotic mass (r = 0.383, p = 0.04; 10 r = 0.454, p = 0.01, respectively). CRP levels at admission were significantly higher in patients with diabetes (14.4 ± 4.4 mg / L, p = 0.006) and hypertension (10.8 ± 2.6 mg / L; p = 0.01) than the average levels of the entire AMI population (7.5 ± 1.6 mg / L).
15 El dato de correlación positiva entre los niveles de PCR y los niveles de Apo J en el momento del ingreso de los pacientes apoya la hipótesis que durante la fase temprana de isquemia del IAM se dan respuestas que tratan de compensar el estado inflamatorio que los síndromes coronarios agudos provocan. De hecho, existe ya un modelo experimental de isquemia/reperfusión, que ha mostrado que15 The positive correlation data between CRP levels and Apo J levels at the time of admission of patients supports the hypothesis that during the early phase of AMI ischemia, responses are given that try to compensate for the inflammatory state of the syndromes. Acute coronaries provoke. In fact, there is already an experimental ischemia / reperfusion model, which has shown that
20 tras 30 minutos de isquemia miocárdica ya se pueden detectar alteraciones sistémicas proinflamatorias (Vilahur G. et al. 2009). Dicho estudio está desarrollado en un modelo porcino y no en un estudio en humanos como es el caso de la presente invención. 20 after 30 minutes of myocardial ischemia, proinflammatory systemic disorders can already be detected (Vilahur G. et al. 2009). Said study is developed in a pig model and not in a human study as is the case of the present invention.
25 EJEMPLO 4. INMUNODETECCIÓN DE APO J TOTAL Y GLICOSILADA 25 EXAMPLE 4. TOTAL AND GLYCOSILATED APO J IMMUNODETECTION
Tal y como se muestra en la figura 4A, el análisis mediante western blot de Apo J sérica reveló una banda de aproximadamente 40-45 kDa, que representa el 65% del total de Apo J detectada, y una segunda banda con una masa aparente de 65- 30 70kDa, que coincide con la masa molecular del heterodímero glicosilado de Apo J. En el análisis mediante western blot de la fracción glicosilada de Apo J sérica también se observaron dos bandas con una movilidad electroforética similar a la observada en Apo J sérica total aunque con predominio de la banda de mayor peso molecular que representa el 83% de Apo J detectada (Figuras 4A y 4B). EJEMPLO 5. PATRÓN PROTEÓMICO DE LA FRACCIÓN GLICOSILADA DE APO J SÉRICA El patrón proteómico de la fracción glicosilada de Apo J sérica se estudió tras la purificación de las proteínas glicosiladas a través de su unión a lectinas. El subproteoma de las glicoproteínas del suero del grupo de los pacientes IAM y del grupo control (pooles de 10 individuos) se analizó mediante 2-DE (Figura 5). Los pacientes con diabetes fueron excluidos de este análisis. Mediante MALDI-ToF se identificaron como Apo J, tanto en el grupo control como en los pacientes, 6 spots con una masa molecular entre 40 y 45 kDa y un rango de pl entre 4,5 y 5,0. Estos spots son los numerados como 1 , 2, 4, 5, 8 y 1 1 en el análisis mediante 2-DE de la fracción total del suero (comparar Figura 5 con Figura 2A). Los pacientes IAM mostraron una disminución (p = 0,05 Mann Whitney) en la intensidad de Apo J glicosilada al compararlos con la población control (IAM vs. control, media ± ES: 9072 ± 166 vs. 12351 ± 1717, los valores indican la intensidad total de las formas de Apo J normalizadas por la intensidad del volumen de todos los spots en el gel). Al comparar los spots de forma individual, encontramos una disminución significativa de los spots 4 y 8 (los spots mayoritarios) en los pacientes IAM comparados con los controles (media de la intensidad ± ES: 3379 ± 142 vs. 2794 ± 188 y 4246 ± 793 vs. 2694 ± 101 , respectivamente; Mann-Whitney, p < 0,05 para los dos spots). As shown in Figure 4A, analysis by western blot of serum Apo J revealed a band of approximately 40-45 kDa, representing 65% of the total Apo J detected, and a second band with an apparent mass of 65-3070kDa, which coincides with the molecular mass of the glycosylated heterodimer of Apo J. In the western blot analysis of the glycosylated fraction of serum Apo J, two bands were also observed with an electrophoretic mobility similar to that observed in total serum Apo J although with a predominance of the band of greater molecular weight that represents 83% of Apo J detected (Figures 4A and 4B). EXAMPLE 5. PROTEOMIC PATTERN OF THE GLOSSYLED FRACTION OF APO J SERICA The proteomic pattern of the glycosylated fraction of Apo J serum was studied after the purification of glycosylated proteins through their binding to lectins. The serum glycoprotein subproteome of the group of AMI patients and the control group (pools of 10 individuals) was analyzed by 2-DE (Figure 5). Patients with diabetes were excluded from this analysis. Through MALDI-ToF, 6 spots with a molecular mass between 40 and 45 kDa and a range of pl between 4.5 and 5.0 were identified as Apo J, both in the control group and in the patients. These spots are numbered as 1, 2, 4, 5, 8 and 1 1 in the 2-SD analysis of the total serum fraction (compare Figure 5 with Figure 2A). The AMI patients showed a decrease (p = 0.05 Mann Whitney) in the intensity of glycosylated Apo J when compared with the control population (AMI vs. control, mean ± ES: 9072 ± 166 vs. 12351 ± 1717, the values indicate the total intensity of Apo J forms normalized by the intensity of the volume of all spots in the gel). When comparing the spots individually, we found a significant decrease in spots 4 and 8 (the majority spots) in AMI patients compared to controls (mean intensity ± ES: 3379 ± 142 vs. 2794 ± 188 and 4246 ± 793 vs. 2694 ± 101, respectively; Mann-Whitney, p <0.05 for both spots).
EJEMPLO 6. ANÁLISIS DE APO J EN MICROPARTÍCULAS-EXOSOMAS EXAMPLE 6. APO J ANALYSIS IN MICROPARTICLES-EXOSOMES
La fracción de micropartículas-exosomas del suero (MP-EX) y la fracción del suero libre de MPs-EX (fracción soluble) de individuos sanos (n=10) y pacientes IAM (n=10) se analizaron por ELISA y western blot. Los niveles de Apo J se cuantificaron mediante ELISA en la fracción soluble y en una fracción diez veces concentrada de MP-EX. En la fracción soluble se detectó una concentración de Apo J de 56,5 ± 1 ,4 g/ml mientras que los niveles de Apo J en la fracción MP-EX estaban por debajo del límite de detección del método (0,005 pg/ml). El análisis mediante western blot de la fracción soluble y la fracción MP-EX (10 veces concentrada) en el grupo control y los pacientes IAM (Figura 6A) mostró las dos mismas bandas que aparecían en el análisis de Apo J total y glicosilada (comparar Figura 6A con Figuras 4A y B). En la fracción soluble se detectó una diferencia significativa en la distribución de las bandas entre los pacientes IAM y los controles (p=0,02), en los pacientes IAM Apo J estaba igualmente representada por ambas bandas, mientras que en los controles la banda de mayor peso molecular representaba el 63% de Apo J total detectada. En la fracción MP-EX la banda más representativa era la de menor peso molecular (92%) y no se detectaron diferencias significativas entre los pacientes IAM y los controles (Figura 6B). The serum microparticle-exosome fraction (MP-EX) and the free serum fraction of MPs-EX (soluble fraction) from healthy individuals (n = 10) and AMI patients (n = 10) were analyzed by ELISA and western blot . Apo J levels were quantified by ELISA in the soluble fraction and in a tenfold fraction of MP-EX. In the soluble fraction, an Apo J concentration of 56.5 ± 1.4 g / ml was detected while Apo J levels in the MP-EX fraction were below the detection limit of the method (0.005 pg / ml) . Western blot analysis of the soluble fraction and the MP-EX fraction (10 times concentrated) in the control group and the AMI patients (Figure 6A) showed both same bands that appeared in the analysis of total and glycosylated Apo J (compare Figure 6A with Figures 4A and B). In the soluble fraction a significant difference was detected in the distribution of the bands between the AMI patients and the controls (p = 0.02), in the IAM patients Apo J was also represented by both bands, while in the controls the band of greater molecular weight represented 63% of total Apo J detected. In the MP-EX fraction, the most representative band was the one with the lowest molecular weight (92%) and no significant differences were detected between AMI patients and controls (Figure 6B).
EJEMPLO 7. Inmunohistoquímica EXAMPLE 7. Immunohistochemistry
Apo J se detectó en el miocardio de los pacientes IAM pero no en el tejido sano (Figura 7). La señal de Apo J se detectó mayoritariamente en los microvasos, pero también se vieron trazas en el resto del tejido miocárdico. Apo J was detected in the myocardium of AMI patients but not in healthy tissue (Figure 7). The Apo J signal was mostly detected in the microvessels, but traces were also seen in the rest of the myocardial tissue.
Materiales y Métodos Materials and methods
Población de estudio Study population
Los pacientes con un diagnóstico de infarto agudo de miocardio de nueva presentación fueron ingresaron en urgencias del Hospital de la Santa Creu i Sant Pau. Los criterios de inclusión en el estudio fueron la presencia de dolor precordial (dolor en el lado izquierdo del pecho a la altura del corazón) de más de treinta minutos e ingreso en el centro clínico dentro de las seis primeras horas desde el inicio del evento. Se excluyeron los pacientes con un infarto de miocardio previo. Finalmente, 39 pacientes se incluyeron en el estudio (29 hombres y 10 mujeres) con una edad media de 63 años ± 14. Los pacientes presentaron una elevación del segmento ST del electrocardiograma (> 0,2 mV) en al menos dos derivaciones contiguas niveles de creatina kinasa (CK) y creatina kinasa MB normales en el momento del ingreso, seguidos posteriormente de una duplicación de los mismos. En el momento del ingreso no se detectaron niveles de troponina-T (indicando ausencia de un infarto previo). Treinta y tres pacientes presentaban al menos un factor de riesgo cardiovascular (hipertensión, dislipemia, diabetes o tabaquismo). Los pacientes analizados presentaban diferentes características clínicas así como diversos factores de riesgo y tratamiento (tabla 4). Patients with a diagnosis of newly presented acute myocardial infarction were admitted to the emergency department of the Hospital de la Santa Creu and Sant Pau. The inclusion criteria in the study were the presence of precordial pain (pain in the left side of the chest at the height of the heart) of more than thirty minutes and admission to the clinical center within the first six hours from the start of the event. Patients with a previous myocardial infarction were excluded. Finally, 39 patients were included in the study (29 men and 10 women) with a mean age of 63 years ± 14. Patients presented an elevation of the ST segment of the electrocardiogram (> 0.2 mV) in at least two contiguous shunts levels of normal creatine kinase (CK) and creatine kinase MB at the time of admission, followed subsequently by a duplication thereof. At the time of admission no troponin-T levels were detected (indicating absence of a previous infarction). Thirty-three patients had at least one cardiovascular risk factor (hypertension, dyslipidemia, diabetes or smoking). The patients analyzed had different clinical characteristics as well as various risk factors and treatment (Table 4).
TABLA 4. Detalles clínicos y parámetros lipidíeos de los individuos del estudio. TABLE 4. Clinical details and lipid parameters of the study individuals.
Pacientes IAM Donadores sanos IAM patients Healthy donors
(n = 39) (n = 51)  (n = 39) (n = 51)
Edad (media ± desvest) 63 ± 14 45 ± 10.8  Age (mean ± undress) 63 ± 14 45 ± 10.8
Mujeres / Hombres 10 / 29 17 / 34  Women / Men 10/29 17/34
Colesterol total (mg/dL) 201.7 ± 47.3 200.3 ± 42.1  Total cholesterol (mg / dL) 201.7 ± 47.3 200.3 ± 42.1
Colesterol HDL (mg/dL) 44.2 ± 1 1.8 43 ± 13.5  HDL cholesterol (mg / dL) 44.2 ± 1 1.8 43 ± 13.5
Colesterol LDL (mg/dL) 127.8 ± 40.1 139.5 ± 38.7  LDL cholesterol (mg / dL) 127.8 ± 40.1 139.5 ± 38.7
Factores de riesgo, n (%)  Risk factors, n (%)
Fumadores 51 - Smoking 51 -
Hipertensión 42 -Hypertension 42 -
Dislipemia 49 -Dyslipidemia 49 -
Diabetes Mellitus 27 -Diabetes Mellitus 27 -
Medicación, n (%) Medication, n (%)
ASA 9.8 - ASA 9.8 -
IECA 17.1 -IECA 17.1 -
Estatinas 17.1 -Statins 17.1 -
ARA2 4.9 - β-bloqueantes 7.3 -ARA2 4.9 - β-blockers 7.3 -
ACA 9.8 -ACA 9.8 -
NIT 2.4 -NIT 2.4 -
ADO 17.1 -ADO 17.1 -
Niveles PC (mg/L) * 7.6 ± 9.9 -PC levels (mg / L) * 7.6 ± 9.9 -
TnTmax ^g/L)† 7.3 ± 5.9 -TnTmax ^ g / L) † 7.3 ± 5.9 -
Necrosis % J 13.5 ± 9.8 - Necrosis% J 13.5 ± 9.8 -
* Niveles PCR: Niveles de proteína C reactiva en el momento del ingreso.† TnTmax: Valor máximo de los niveles de troponina T desde el momento del ingreso, t Necrosis (%): Porcentaje de masa necrótica del miocardio (calculado como el porcentaje de la masa total del corazón medida por resonancia magnética cardiovascular). * PCR levels: Levels of C-reactive protein at the time of admission. † TnTmax: Maximum value of troponin T levels from the time of admission, t Necrosis (%): Percentage of myocardial necrotic mass (calculated as the percentage of the total mass of the heart measured by cardiovascular magnetic resonance).
ASA = Ácido acetilsalicílico; IECA = Inhibidores enzima conversión angiotensina; ARA2 = ASA = Acetylsalicylic acid; ACEI = angiotensin conversion enzyme inhibitors; ARA2 =
Antagonistas receptores angiotensina II; ACA = Antagonistas Calcio; NIT = Nitroglicerina; ADO = antidiabéticos orales.  Angiotensin II receptor antagonists; ACA = Calcium Antagonists; NIT = Nitroglycerin; ADO = oral antidiabetics.
El grupo control constaba de cincuenta y un individuos (34 hombres y 17 mujeres) con una edad media de 45 años ± 1 1 , que se presentaron a una revisión médica rutinaria. El comité ético del Hospital de la Santa Creu i Sant Pau aprobó el proyecto y los estudios se llevaron a cabo de acuerdo con los principios de la Declaración de Helsinki. Todos los participantes dieron su consentimiento informado por escrito para formar parte del estudio. The control group consisted of fifty-one individuals (34 men and 17 women) with an average age of 45 years ± 1 1, who underwent a routine medical review. The ethical committee of the Hospital de la Santa Creu i Sant Pau approved the project and the studies were carried out in accordance with the principles of the Declaration of Helsinki. All participants gave written informed consent to be part of the study.
Estudio del infarto mediante resonancia magnética Myocardial infarction study
Los estudios de resonancia magnética cardíaca de contraste se realizaron en la fase crónica tras IAM en todos aquellos pacientes que presentaron IAM con elevación del segmento ST mediante un escáner Philips Intera 1 .5 Teslas (Philips Medical Systems, Best, Países Bajos). Tras obtener los planos de reconocimiento, se adquirieron en modo cine y estado estacionario de alta precisión los ejes largos mediante la realización de múltiples cortes longitudinales (eje corto) a razón de un espesor de corte de 10mm. Estos cortes se realizaron desde el anillo atrio- ventricular hasta el ápex del ventrículo izquierdo. Se adquirieron un total de dieciséis fases de ciclo cardíaco para cada corte. Posteriormente se administró Gadobutrol (Gadovista, Schering, Berlín, Alemania) de manera intravenosa a razón de 0,1 mmol/kg. Diez minutos después de la administración del contraste se adquirieron las secuencias de inversión-recuperación con eco de gradiente en los mismos planos que las secuencias de modo cine a fin de evaluar el realce tardío. El tiempo de inversión se ajustó individualmente para suprimir el miocardio sano (200 a 300 ms). Se realizó una valoración de la motilidad segmentaria del ventrículo izquierdo según las Sociedades Norteamericanas de Imagen, las cuales dividen el ventrículo izquierdo en dos zonas (anteroseptal e inferolateral), 4 paredes (anterior, septal, inferior y lateral) y 17 segmentos. Finalmente se analizaron a doble ciego las secuencias de realce tardío de contraste por tres intérpretes expertos y se evaluó cada segmento.  Contrast cardiac magnetic resonance studies were performed in the chronic phase after AMI in all those patients who presented AMI with ST segment elevation using a Philips Intera 1 .5 Teslas scanner (Philips Medical Systems, Best, The Netherlands). After obtaining the recognition planes, the long axes were acquired in cinema mode and high precision stationary state by means of multiple longitudinal cuts (short axis) at a cutting thickness of 10mm. These cuts were made from the atrioventricular ring to the apex of the left ventricle. A total of sixteen cardiac cycle phases were acquired for each cut. Subsequently, Gadobutrol (Gadovista, Schering, Berlin, Germany) was administered intravenously at a rate of 0.1 mmol / kg. Ten minutes after the administration of the contrast, the inversion-recovery sequences with gradient echo were acquired in the same planes as the cinema mode sequences in order to evaluate the late enhancement. The inversion time was adjusted individually to suppress the healthy myocardium (200 to 300 ms). An assessment of the segmental motility of the left ventricle was performed according to the American Image Societies, which divide the left ventricle into two zones (anteroseptal and inferolateral), 4 walls (anterior, septal, inferior and lateral) and 17 segments. Finally, the double contrast enhancement sequences were analyzed double blind by three expert interpreters and each segment was evaluated.
Recogida de sangre y preparación de muestra Blood collection and sample preparation
Obtención de muestras de sangre: Las muestras de sangre venosa de los pacientes IAM fueron recogidas antes del inicio de la medicación. Se obtuvo el suero, se alicuotó y se guardó a -80°C.  Obtaining blood samples: Venous blood samples from AMI patients were collected before the start of the medication. Serum was obtained, aliquoted and stored at -80 ° C.
Preparación de HDL: Las HDL humanas se obtuvieron a partir de suero normocolesterolémico mediante ultracentrifugación diferencial. Se determinaron las concentraciones de lipoproteínas y de colesterol de las HDL. La fracción proteica se obtuvo mediante la precipitación de 2mg de HDL con acetona pura a - 20°C durante dos horas. Preparación del suero: Para los estudios proteómicos, las muestras de suero se sonicaron (seis ciclos de 15 segundos cada uno) en hielo y se filtraron (0,22μηη) mediante centrifugación para evitar la presencia de impurezas. Las seis proteínas más abundantes del suero se eliminaron utilizando una columna de afinidad específica con capacidad de unirse a la albúmina, inmunoglobulina G, inmunoglobulina A, transferrina, a1 -antitripsina y haptoglobina (Múltiple Affinity Removal Spin Cartridge, Agilent Technologies). La fracción de suero sin las seis proteínas mayoritarias (denominada fracción de suero total) se concentró y se centrifugó con filtros de 5kDa para la eliminación de sales y el cambio a un tampón urea (8M Urea, 2% CHAPS (3-[(3-Cholamidopropil)dimetilamonio]-1 - propanosulfonato)). La fracción de proteínas glicosiladas del suero se purificó mediante el uso de un kit comercial (Qproteome Total Glycoprotein Kit, QIAGEN). La concentración de proteína presente en los extractos se cuantificó con el 2D- Quant Kit (GE Healthcare). Todas las muestras se guardaron a -80°C hasta su uso. Preparation of HDL: Human HDLs were obtained from normocholesterolemic serum by differential ultracentrifugation. They were determined the concentrations of lipoproteins and cholesterol of the HDL. The protein fraction was obtained by precipitation of 2mg of HDL with pure acetone at -20 ° C for two hours. Serum preparation: For proteomic studies, the serum samples were sonicated (six cycles of 15 seconds each) on ice and filtered (0.22μηη) by centrifugation to avoid the presence of impurities. The six most abundant whey proteins were removed using a specific affinity column capable of binding albumin, immunoglobulin G, immunoglobulin A, transferrin, a1-antitrypsin and haptoglobin (Multiple Affinity Removal Spin Cartridge, Agilent Technologies). The serum fraction without the six major proteins (called total serum fraction) was concentrated and centrifuged with 5kDa filters for the removal of salts and the change to a urea buffer (8M Urea, 2% CHAPS (3 - [(3 -Cholamidopropyl) dimethylammonium] -1-propanesulfonate)). The glycosylated whey protein fraction was purified by the use of a commercial kit (Qproteome Total Glycoprotein Kit, QIAGEN). The protein concentration present in the extracts was quantified with the 2D-Quant Kit (GE Healthcare). All samples were stored at -80 ° C until use.
Aislamiento de Micropartículas y Exosomas (MP-EX): El suero se centrifugó a 100.000 g durante 45 minutos a 20°C (OPTIMAX Ultracentrifuge, Beckman Coulter) para la obtención de la fracción MP-EX. Después de dos lavados, los pellets de MP-EX se resuspendieron en un tampón de urea/tiourea (7mol/L urea, 2mol/L tiourea, 2% w/v CHAPS). Las MP-EX del suero se concentraron y se centrifugaron con filtros de 5kDa para la eliminación de sales y el cambio a un tampón urea (8M Urea, 2% CHAPS). Las fracciones de MP-EX derivadas del suero y el suero libre de MP-EX se alicuotaron y se guardaron a -80°C. Análisis proteómico Isolation of Microparticles and Exosomes (MP-EX): The serum was centrifuged at 100,000 g for 45 minutes at 20 ° C (OPTIMAX Ultracentrifuge, Beckman Coulter) to obtain the MP-EX fraction. After two washes, the MP-EX pellets were resuspended in a urea / thiourea buffer (7mol / L urea, 2mol / L thiourea, 2% w / v CHAPS). Serum MP-EXs were concentrated and centrifuged with 5kDa filters for salt removal and change to a urea buffer (8M Urea, 2% CHAPS). MP-EX fractions derived from serum and MP-EX free serum were aliquotted and stored at -80 ° C. Proteomic analysis
Electroforesis bidimensional (2-DE): 120 g (en los geles analíticos), o 300 g (en los preparativos) de proteína del extracto soluble en Urea/CHAPS fueron cargados en tiras de isoelectroenfoque de 18 cm y un rango de pH linear de 4 a 7 (GE Healthcare). Las proteínas en los geles se visualizaron mediante tinción fluorescente (geles analíticos) o con azul Coomassie (geles preparativos). Para cada experimento independiente, la 2-DE de los extractos proteicos de controles y pacientes se procesaron en paralelo para garantizar que fuesen comparables. Cada tanda de 2-DE se repitió al menos dos veces. El análisis de las diferencias entre los patrones proteicos de las muestras controles y las de infarto se llevó a cabo con el programa PD-Quest (BioRad) utilizando un solo master que incluía todos los geles de cada experimento independiente. A cada spot proteico, tras la extracción de la señal de fondo y la normalización entre geles, se le asignó un valor relativo que correspondía al valor de cada spot individual comparado al volumen de todos los spots presentes en el gel. Two-dimensional electrophoresis (2-DE): 120 g (in analytical gels), or 300 g (in preparations) of Urea / CHAPS soluble extract protein were loaded into 18 cm isoelectric focusing strips and a linear pH range of 4 to 7 (GE Healthcare). The proteins in the gels were visualized by staining fluorescent (analytical gels) or with Coomassie blue (preparative gels). For each independent experiment, the 2-ED of the protein extracts from controls and patients were processed in parallel to ensure that they were comparable. Each batch of 2-DE was repeated at least twice. The analysis of the differences between the protein patterns of the control and infarction samples was carried out with the PD-Quest program (BioRad) using a single master that included all the gels of each independent experiment. Each protein spot, after the extraction of the background signal and the normalization between gels, was assigned a relative value that corresponded to the value of each individual spot compared to the volume of all the spots present in the gel.
Análisis mediante espectrometría de masas: Los spots proteicos de interés se recortaron de los geles, se lavaron, deshidrataron, secaron y se digirieron enzimáticamente con tripsina porcina (Promega). Los péptidos resultantes de la digestión con tripsina se mezclaron en una proporción 1 :1 con 5mg/ml de a-ciano- 4-hidroxi-cinámico y fueron cargados en una tira de acero inoxidable para espectrometría de masas. La identificación de las proteínas se llevó a cabo mediante su huella peptídica utilizando un Ettan MALDI-ToF Pro (matrix-assisted láser desorption/ionisation time-of-flight mass spectrometer, GE-Healthcare) trabajando en el modo reflector. Los espectros generados por el MALDI se calibraron internamente utilizando los picos de los productos de autolisis de la tripsina, Ang III (angiotensina III) y ACTH (hormona adrenocorticotropica). Las masas peptídicas se buscaron a través de la base de datos de mamífero no redundante del National Center for Biotechnology Information utilizando ProFound™ y se confirmaron utilizando una búsqueda en Mascot de Matrixscience seleccionando la base de datos SwissProt. Para este estudio, la identificación de proteínas se basó en la medición de al menos 10 péptidos con un mínimo de un 50% de coincidencia y una cobertura de más de un 35%. La expectación mínima para una identificación válida fue de 0,0001 y p < 0,05. Analysis by mass spectrometry: Protein spots of interest were trimmed from the gels, washed, dehydrated, dried and enzymatically digested with porcine trypsin (Promega). The peptides resulting from trypsin digestion were mixed in a 1: 1 ratio with 5mg / ml of a-cyano-4-hydroxy-cinnamic and loaded onto a stainless steel strip for mass spectrometry. Protein identification was carried out by means of their peptide fingerprint using an Ettan MALDI-ToF Pro (matrix-assisted laser desorption / ionisation time-of-flight mass spectrometer, GE-Healthcare) working in the reflector mode. The spectra generated by MALDI were internally calibrated using the peaks of the trypsin autolysis products, Ang III (angiotensin III) and ACTH (adrenocorticotropic hormone). Peptide masses were searched through the non-redundant mammalian database of the National Center for Biotechnology Information using ProFound ™ and confirmed using a Matrixscience Mascot search by selecting the SwissProt database. For this study, protein identification was based on the measurement of at least 10 peptides with a minimum of 50% coincidence and coverage of more than 35%. The minimum expectation for a valid identification was 0.0001 and p <0.05.
Cuantificación de los niveles séricos de Apo J Quantification of serum Apo J levels
La concentración sérica de Apo J se determinó mediante un ELISA comercial tipo sandwich basado en dos anticuerpos monoclonales (Clusterin ELISA, Biovendor) siguiendo las indicaciones de los proveedores. El límite de detección del ensayo era de 0,5 ng/ml. The serum Apo J concentration was determined by a commercial sandwich ELISA based on two monoclonal antibodies (Clusterin ELISA, Biovendor) following the indications of the suppliers. The detection limit of the assay was 0.5 ng / ml.
Inmunoprecipitación de proteína Protein immunoprecipitation
Apo J se inmunoprecipitó añadiendo 2 g de un anticuerpo monoclonal específico (Apolipoprotein J, clone CLI-9, Abcam) a muestras previamente pre-lavadas usando 50% Protein G Sepharose 4 Fast Flow (GE Healthcare, 1 hora a 4 °C). Los complejos antígeno-anticuerpo se precipitaron con 50% Protein G Sepharose 4 Fast Flow. Los complejos inmunoprecipitados se separaron de la proteína G calentando la muestra durante 5 minutos en condiciones reductoras (Tris-HCI 2,5 M pH 6,8, 100% Glicerol, 20% SDS, azul de bromofenol). Apo J was immunoprecipitated by adding 2 g of a specific monoclonal antibody (Apolipoprotein J, clone CLI-9, Abcam) to pre-washed samples using 50% Protein G Sepharose 4 Fast Flow (GE Healthcare, 1 hour at 4 ° C). The antigen-antibody complexes were precipitated with 50% Protein G Sepharose 4 Fast Flow. The immunoprecipitated complexes were separated from the G protein by heating the sample for 5 minutes under reducing conditions (2.5M Tris-HCI pH 6.8, 100% Glycerol, 20% SDS, bromophenol blue).
Análisis por Western Blot Western Blot Analysis
Las muestras se resolvieron mediante electroforesis y fueron electro-transferidas a membranas de PVDF (Polyvinylidene Difluoride) en condiciones semi-secas (Semi-dry transfer system, BioRad). La detección se llevó a cabo con anticuerpos monoclonales contra Apo J (clone CLI-9, dilución 1 :2000, Abcam). El anticuerpo anti-ratón estaba marcado con el fluorocromo Cy3 (1 :20000 dilution, GE Healthcare). La fluorescencia de las bandas se determinó con un Typhoon 9400 (GE Healthcare) y la cuantificación se llevó a cabo utilizando el programa ImageQuant TL (GE Healthcare).  The samples were resolved by electrophoresis and were electro-transferred to PVDF membranes (Polyvinylidene Difluoride) under semi-dry conditions (Semi-dry transfer system, BioRad). Detection was carried out with monoclonal antibodies against Apo J (clone CLI-9, dilution 1: 2000, Abcam). The anti-mouse antibody was labeled with Cyclo fluorochrome (1: 20000 dilution, GE Healthcare). The fluorescence of the bands was determined with a Typhoon 9400 (GE Healthcare) and the quantification was carried out using the ImageQuant TL (GE Healthcare) program.
Inmunohistoquímica Immunohistochemistry
Los miocardios humanos se obtuvieron a partir de corazones extraídos durante transplantes realizados en el Hospital de la Santa Creu i Sant Pau (Barcelona, España). Las muestras de tejido se sumergieron en solución de fijación (4% paraformaldehído) y después de embeberse en parafina se cortaron en secciones seriadas de 5 mm de grosor y se colocaron en soportes recubiertos con poli-L- lisina. Se utilizó un anticuerpo primario policlonal de conejo contra la Apolipoproteína J a una dilución 1 :50 (Abcam ab69644). Antes de la incubación con el anticuerpo primario (2 horas), las secciones se lavaron, eliminando la actividad de la peroxidasa endógena con H2O2 y se utilizó suero de cabra para bloquear uniones inespecíficas. Los anticuerpos primarios se detectaron usando la técnica de la avidina-biotina inmunoperoxidasa. Las secciones se incubaron posteriormente con un anticuerpo secundario biotinilado (1 :200, VectorÓ). El cromógeno usado fue la 3,3'-diaminobenzidina. La hematoxilina se utilizó para la tinción de los núcleos. Análisis bioquímico Human myocardia were obtained from hearts extracted during transplants performed at the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain). The tissue samples were immersed in fixation solution (4% paraformaldehyde) and after immersion in paraffin, they were cut into serial sections 5 mm thick and placed on poly-L-lysine coated supports. A rabbit polyclonal primary antibody against Apolipoprotein J was used at a 1: 50 dilution (Abcam ab69644). Before incubation with the primary antibody (2 hours), the sections were washed, eliminating the activity of endogenous peroxidase with H2O2 and goat serum was used to block nonspecific junctions. Primary antibodies were detected using the avidin-biotin immunoperoxidase technique. The sections were incubated subsequently with a biotinylated secondary antibody (1: 200, VectorÓ). The chromogen used was 3,3'-diaminobenzidine. Hematoxylin was used for staining the nuclei. Biochemical analysis
Los niveles de proteína C reactiva (PCR) se midieron con un kit comercial (Roche) con un intervalo de detección entre 0,1 y 20 mg/L. Cuando los niveles de PCR estaban por encima del límite de detección las muestras se diluían. Los lípidos se midieron por métodos enzimáticos convencionales dentro de las primeras 8 horas desde el inicio del evento. Las mediciones hechas en este periodo de tiempo se consideran el nivel basal. Los niveles totales de colesterol se analizaron mediante el método CHOD-PAP (Boehringer-Mannheim, Germany) y los triglicéridos por TOD-PAP (Boehringer-Mannheim, Germany). Los niveles de colesterol HDL se midieron después de la precipitación manual de las lipoproteínas con el método PEG 6000 (Boehringer-Mannheim, Germany). Cuando los niveles de triglicéridos superaban los 3,36 mmol/L, los niveles de colesterol LDL se determinaron mediante centrifugación y cuando estaban por debajo de 3,36 mmol/L, los niveles de colesterol LDL se estimaban usando la fórmula de Fridewald. Los niveles de troponina T se midieron con un kit comercial de Roche Diagnostics IV generación y se analizaron con el Roche Diagnostics Elecsys 2010.  C-reactive protein (PCR) levels were measured with a commercial kit (Roche) with a detection range between 0.1 and 20 mg / L. When the PCR levels were above the detection limit the samples were diluted. Lipids were measured by conventional enzymatic methods within the first 8 hours from the start of the event. Measurements made in this period of time are considered the baseline level. Total cholesterol levels were analyzed using the CHOD-PAP method (Boehringer-Mannheim, Germany) and triglycerides by TOD-PAP (Boehringer-Mannheim, Germany). HDL cholesterol levels were measured after manual precipitation of lipoproteins with the PEG 6000 method (Boehringer-Mannheim, Germany). When triglyceride levels exceeded 3.36 mmol / L, LDL cholesterol levels were determined by centrifugation and when they were below 3.36 mmol / L, LDL cholesterol levels were estimated using Fridewald's formula. Troponin T levels were measured with a commercial Roche Diagnostics IV generation kit and analyzed with the Roche Diagnostics Elecsys 2010.
Análisis estadístico Statistic analysis
Los datos están expresados como media y error estándar (ES) a menos que se indique lo contrario. N indica el número de individuos analizados. Los análisis estadísticos realizados para la búsqueda de las diferencias entre los grupos control e infarto se realizaron mediante ANOVA de un solo factor o incluyendo covariantes y el test t de Student o el no paramétrico Mann-Withney, según esté indicado. Las correlaciones entre variables se determinaron mediante modelos de regresión simple o múltiple. Se consideró significativo p < 0,05. Data are expressed as mean and standard error (ES) unless otherwise indicated. N indicates the number of individuals analyzed. Statistical analyzes performed to search for differences between the control and infarction groups were performed using single-factor ANOVA or including covariates and the Student t test or the non-parametric Mann-Withney test, as indicated. The correlations between variables were determined by simple or multiple regression models. It was considered significant p <0.05.
Resultados Results
Junto con los cambios ya conocidos asociados a la respuesta inflamatoria que se dan tras un IAM, en la presente invención se han detectado cambios específicos en isoformas de la Apo J asociados tanto al daño tisular provocado por dicho evento como a la respuesta inflamatoria aguda que conlleva. Previamente a Apo J se le han atribuido propiedades antiinflamatorias y ha sido relacionada a patologías autoinmunes y de carácter inflamatorio. Sin embargo, en todas estas patologías siempre se habla de cambios en los niveles totales de Apo J y no de sus isoformas específicas, como es el caso de la presente invención (McLaughlin L. et al. 2000; Newkirk MM. et al. 1999). Together with the known changes associated with the inflammatory response that occur after AMI, specific changes have been detected in the present invention. in Apo J isoforms associated with both the tissue damage caused by said event and the acute inflammatory response that it entails. Previously, Apo J has been attributed anti-inflammatory properties and has been related to autoimmune and inflammatory diseases. However, in all these pathologies there is always talk of changes in the total levels of Apo J and not of its specific isoforms, as is the case of the present invention (McLaughlin L. et al. 2000; Newkirk MM. Et al. 1999 ).
A su vez en la presente invención se ha confirmado la presencia de Apo J en los capilares de miocardios isquémicos, mientras que no se detectan dichas señales en tejido miocárdico no isquémico (Vákevá A. et al. 1993). De hecho a la Apo J ya se le habían atribuido efectos anti-inflamatorios a través del NF-κΒ (Santilli G. et al. 2003). Pero aún no se sabe con certeza si la deposición de Apo J en el miocardio isquémico contribuye al proceso de cicatrización de dicho miocardio. En la presente invención se muestra por primera vez que diferentes isoformas de Apo J sufren modificaciones en la temprana del infarto agudo y que por tanto pueden servir de marcador del daño tisular provocado por dicho evento y la respuesta inflamatoria aguda asociada y/o previa al infarto. In turn, the presence of Apo J in the capillaries of ischemic myocardia has been confirmed, while said signals are not detected in non-ischemic myocardial tissue (Vákevá A. et al. 1993). In fact, Apo J had already been attributed anti-inflammatory effects through NF-κΒ (Santilli G. et al. 2003). But it is not yet known with certainty if the deposition of Apo J in the ischemic myocardium contributes to the healing process of said myocardium. In the present invention it is shown for the first time that different isoforms of Apo J undergo modifications in the early acute infarction and that they can therefore serve as a marker of tissue damage caused by said event and the acute inflammatory response associated and / or prior to the infarction .

Claims

REIVINDICACIONES
1 . Uso de al menos una forma glicosilada de Apo J como marcador de daño tisular y/o inflamación aguda asociada al infarto agudo de miocardio. one . Use of at least one glycosylated form of Apo J as a marker of tissue damage and / or acute inflammation associated with acute myocardial infarction.
2. Uso según la reivindicación 1 donde las formas glicosiladas de Apo J se seleccionan del conjunto Apo J-15 y/o del conjunto Apo J-29. 2. Use according to claim 1 wherein the glycosylated forms of Apo J are selected from the Apo J-15 assembly and / or the Apo J-29 assembly.
3. Uso según cualquiera de las reivindicaciones 1 ó 2 donde las formas glicosiladas son todas las del conjunto Apo J-15 y/o todas las del conjunto Apo3. Use according to any of claims 1 or 2 wherein the glycosylated forms are all of the Apo J-15 set and / or all of the Apo set
J-29. J-29
4. Uso según cualquiera de las reivindicaciones 1 a 3 donde el daño tisular es un daño cardíaco. 4. Use according to any of claims 1 to 3 wherein the tissue damage is cardiac damage.
5. Uso según cualquiera de las reivindicaciones 1 a 4 donde el daño es producido por infarto agudo de miocardio. 5. Use according to any of claims 1 to 4 wherein the damage is caused by acute myocardial infarction.
6. Uso según cualquiera de las reivindicaciones 1 a 3 donde el daño tisular es un daño renal. 6. Use according to any of claims 1 to 3 wherein the tissue damage is renal damage.
7. Método de obtención de datos útiles para el diagnóstico, pronóstico o monitorización del tratamiento de daño tisular y/o inflamación aguda asociada al infarto agudo de miocardio que comprende los siguientes pasos: 7. Method of obtaining useful data for the diagnosis, prognosis or monitoring of the treatment of tissue damage and / or acute inflammation associated with acute myocardial infarction comprising the following steps:
a) obtener una muestra biológica aislada de un individuo, y  a) obtain an isolated biological sample from an individual, and
b) detectar la cantidad de al menos una forma glicosilada de Apo J en la muestra obtenida en el paso (a).  b) detect the amount of at least one glycosylated form of Apo J in the sample obtained in step (a).
8. Método según la reivindicación 7 que además comprende un paso: 8. Method according to claim 7 further comprising a step:
c) comparar la cantidad detectada en el paso (b) con una cantidad de referencia obtenida de una muestra control. c) compare the amount detected in step (b) with a reference amount obtained from a control sample.
9. Método según cualquiera de las reivindicaciones 7 u 8 donde la forma glicosilada de Apo J que se detecta en el paso (a) se selecciona del conjunto Apo J-15 y/o del conjunto Apo J-29. 9. Method according to any of claims 7 or 8 wherein the glycosylated form of Apo J detected in step (a) is selected from the Apo J-15 assembly and / or the Apo J-29 assembly.
10. Método según cualquiera de las reivindicaciones 7 a 9 donde en el paso (a) se detectan todas las formas glicosiladas del conjunto Apo J-15 y/o todas las del conjunto Apo J-29. 10. Method according to any of claims 7 to 9 wherein in step (a) all glycosylated forms of the Apo J-15 assembly and / or all of the Apo J-29 assembly are detected.
1 1 . Método de diagnóstico según cualquiera de las reivindicaciones 9 o 10 que además comprende un paso adicional (d) donde una cantidad de al menos una de las formas glicosiladas del conjunto Apo J-15 detectadas en el paso (b) menor que la cantidad de referencia del paso (c) y/o donde una cantidad de al menos una de las formas glicosiladas del conjunto Apo J-29 detectadas en el paso (b) mayor que la cantidad de referencia del paso (c) son indicativas de daño tisular y/o inflamación aguda asociada al infarto agudo de miocardio. eleven . Diagnostic method according to any of claims 9 or 10, further comprising an additional step (d) wherein an amount of at least one of the glycosylated forms of the Apo J-15 set detected in step (b) less than the reference amount of step (c) and / or where an amount of at least one of the glycosylated forms of the Apo J-29 set detected in step (b) greater than the reference amount of step (c) are indicative of tissue damage and / or acute inflammation associated with acute myocardial infarction.
12. Método según cualquiera de las reivindicaciones 7 a 1 1 donde el daño tisular es un daño cardíaco. 12. Method according to any of claims 7 to 1 1 wherein the tissue damage is cardiac damage.
13. Método según cualquiera de las reivindicaciones 7 a 12 donde el daño es producido por infarto agudo de miocardio. 13. Method according to any of claims 7 to 12 wherein the damage is caused by acute myocardial infarction.
14. Método según cualquiera de las reivindicaciones 7 a 1 1 donde el daño tisular es un daño renal . 14. Method according to any of claims 7 to 1 1 wherein the tissue damage is renal damage.
15. Método según cualquiera de las reivindicaciones 7 a 14 donde la detección de la cantidad de la forma glicosilada de Apo J del paso (b) se lleva a cabo mediante electroforesis bidimensional . 15. Method according to any of claims 7 to 14 wherein the detection of the amount of the glycosylated form of Apo J in step (b) is carried out by two-dimensional electrophoresis.
16. Método según cualquiera de las reivindicaciones 7 a 15 donde la detección de la cantidad de la forma glicosilada de Apo J del paso (b) se lleva a cabo mediante un inmunoensayo. 16. Method according to any of claims 7 to 15 wherein the detection of the amount of the glycosylated form of Apo J in step (b) is carried out by an immunoassay.
17. Método según cualquiera de las reivindicaciones 7 a 16 donde la muestra biológica aislada del paso (a) se selecciona de la lista que comprende tejido cardíaco, suero, sangre, plasma o tejido renal. 17. Method according to any of claims 7 to 16 wherein the biological sample isolated from step (a) is selected from the list comprising cardiac tissue, serum, blood, plasma or renal tissue.
18. Kit para llevar a cabo el método según cualquiera de las reivindicaciones 7 a 17 que comprende los elementos necesarios para: 18. Kit for carrying out the method according to any of claims 7 to 17 comprising the elements necessary for:
a) detectar la cantidad de al menos una forma glicosilada de Apo J en una muestra biológica aislada obtenida de un individuo, y  a) detecting the amount of at least one glycosylated form of Apo J in an isolated biological sample obtained from an individual, and
b) comparar la cantidad detectada con una cantidad de referencia obtenida de una muestra control.  b) compare the amount detected with a reference amount obtained from a control sample.
19. Kit según la reivindicación 18 donde las formas glicosiladas a detectar se seleccionan del conjunto Apo J-15 y/o del conjunto Apo J-29. 19. Kit according to claim 18 wherein the glycosylated forms to be detected are selected from the Apo J-15 assembly and / or the Apo J-29 assembly.
20. Kit según la reivindicación 19 donde las formas glicosiladas a detectar son todas las del conjunto Apo J-15 y/o todas las del conjunto Apo J-29. 20. Kit according to claim 19 wherein the glycosylated forms to be detected are all those of the Apo J-15 set and / or all of the Apo J-29 set.
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