Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
  • G01N2333/765—Serum albumin, e.g. HSA
• • 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 relates to the field of the diagnosis of ischemic states in humans, and in particular to a method for the detection of ischemia-modified albumin ("Ischemia Modified Albumin" or AMI).
• ischemia-modified Albumin Ischemia Modified Albumin
• Ischemia is the decrease in arterial blood supply to an organ. This decrease essentially leads to a decrease in the oxygenation of the tissues of the organ below its needs (hypoxia), and the disruption or even the cessation of its function. Ischemia may be due to a blood clot that obstructs an artery (thrombosis), to an atheromatous plaque, to a hemorrhage that prevents the tissues from being properly fed, to a compression of an artery by an external object (crush of a limb, tourniquet) or by an internal phenomenon (hematoma, tumor, effusion of a liquid). Ischemia can be reversible and cause only limited discomfort. But it can also be irreversible and lead to infarction of the organ, that is to say to the necrosis of part or all of it.
• IMA which is serum albumin with the N-terminal portion modified, is now frequently used as a marker of ischemic heart disease.
• IMA could also be a biomarker of acute stroke 1 .
• Detecting the presence of AMI in the blood of patients who are suspected to be ischemic is therefore of extreme importance and emergency professionals are looking for a specific and reliable test.
• the IMA assay is performed indirectly by a colorimetric test (ACB test) which quantifies the AMI by evaluating the decrease in the cobalt ion fixation capacity by the patient's total albumin. after modification of part of its albumin.
• ACB test colorimetric test
• the current marketed ACB test has the drawbacks of not directly detecting AMI, detecting false positives and it has been considered unreliable in the case where patients have an albumin level below
• HNE 4-hydroxy-2-nonenal
• HHE 4-hydroxy-2-hexenal
• MDA malondialdehyde
• HNE an aldehyde of this type, is toxic and is generated by the ⁇ cleavage of hydroperoxides of ⁇ -6 polyunsaturated fatty acids 4 .
• HHE is also derived from the peroxidation of polyunsaturated fatty acids, specifically acides3 5 fatty acids.
• proteins and peptides are liberated in free form in biological tissues, but diffuse easily from their original site. Because of their ability to bind to the nucleophilic sites of proteins and peptides, principally the histidine, lysine and cysteine residues, to form covalently modified biomolecules, they are thus found either in free form or in bound form. proteins and peptides.
• proteins modified with aldehyde derivatives derived from the lipid peroxidation, and in particular with the HNE include hemoproteins, such as hemoglobin and myoglobin, lipoproteins such as LDL or apolipoprotein B-100, enzymes such as glucose-6-phosphate dehydrogenase or cathepsin B 6 , and albumin which, because of its high concentration in the serum, is a preferred target for these aldehyde derivatives.
• ToyoKuni S. et al. 7 described the importance of HNE-modified albumin for patients with type 2 diabetes. Aldini G. et al.
• HSA human serum albumin
• ToyoKuni S. et al. 9 described monoclonal antibodies recognizing bovine serum albumin modified with HNE and suggested that these antibodies could be useful for the evaluation of ROS (Reactive Oxygen Species) damage, which ROS being involved in a number of biological phenomena such as ischemia-reperfusion. This document does not describe a marker of ischemia itself.
• ROS reactive Oxygen Species
• the modified albumin of ischemia or IMA is actually albumin covalently modified by a reactive aldehyde derivative derived from peroxidation. of lipids and that it was possible to directly detect the IMA, and thus the ischemic states using this property.
• the present invention relates to the use of a binding partner to an aldehyde derivative derived from lipid peroxidation in protein-bound form for the detection of ischemia-modified albumin (IMA) in a biological sample.
• IMA ischemia-modified albumin
• IMA albumin modified with ischemia
• the method of the invention which consists in detecting the AMI by implementing at least one binding partner with an aldehyde derivative derived from the lipid peroxidation of a protein-bound form, has the advantage that it makes it possible to detect directly the IMA, thus increasing the detection sensitivity compared to the indirect detection test of the IMA.
• an aldehyde derivative resulting from the peroxidation of lipids there may be mentioned 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (HHE) and malondialdehyde, which constitutes a particular embodiment of the invention.
• the biological sample in which the method of the invention is implemented is any sample likely to contain IMA.
• IMA any sample likely to contain IMA.
• the aldehyde derivatives can be found in the biological sample either in free form or in bound form and it is desired to detect in said sample only said aldehyde derivatives bound to albumin form.
• the binding partner used in the process of the invention is either specific for said derivative in albumin-bound form, or it is not specific for said aldehyde derivative in albumin bound form, but it recognizes of course, said aldehyde derivative in form bound to another protein or peptide.
• the detection method must also implement a means for isolating albumin from the biological sample.
• a means for isolating albumin from the biological sample mention may be made of a specific binding partner for albumin, such as an anti-human serum albumin antibody, which constitutes a mode of particular embodiment of the invention.
• the detection method of the invention implements both a specific binding partner of an aldehyde derivative in bound form and a specific binding partner of albumin, said binding partner being able to to be specific for an aldehyde derivative in albumin bound form.
• Non-molecule binding partners are called when their binding specificity to this molecule is low and they are then able to bind to other ligands, such as, in the case of aldehyde derivatives in bound form, an aldehyde derivative in form bound to a protein other than albumin.
• the detection method of the IMA of the invention can be implemented by any biochemical test widely known to those skilled in the art involving molecular interactions, namely reactions between said aldehyde derivative derived from lipid peroxidation linked to albumin and one or more binding partner (s), specific or not, of the same said aldehyde derivative derived from the lipid peroxidation.
• the method for detecting the AMI of the invention therefore comprises the following steps: a biological sample that may contain IMA is available, this biological sample is brought into contact with at least one partner of the invention. binding of the aldehyde derivative from lipid peroxidation in protein-bound form and detecting the binding of IMA / binding partner of the aldehyde derivative from lipid peroxidation in protein-bound form.
• a biological sample that may contain IMA is available
• binding of the aldehyde derivative from lipid peroxidation in protein-bound form and detecting the binding of IMA / binding partner of the aldehyde derivative from lipid peroxidation in protein-bound form.
• the method of the invention uses a specific partner of the albumin to make it possible to isolate the albumin of said sample, as indicated previously, either in detection or in capture.
• the biochemical test is an immunoassay known to those skilled in the art involving immunological reactions between the aldehyde derivative derived from the lipid peroxidation, which is the antigen, and one or more specific binding partner (s) ( s) what are the antibodies directed against this antigen.
• immunoassay known to those skilled in the art involving immunological reactions between the aldehyde derivative derived from the lipid peroxidation, which is the antigen, and one or more specific binding partner (s) ( s) what are the antibodies directed against this antigen.
• immunological tests as defined above, mention may be made of "sandwich” methods such as ELISA, IRMA and RIA, so-called competitive methods and direct immunodetection methods such as immunohistochemistry, immunocytochemistry , Western-blot and Dot-blot.
• the specific or non-specific binding partners of the aldehyde derivative (s) resulting from the lipid peroxidation sought in the process of the invention, and, where appropriate, the specific binding partners of albumin, are any partner likely to bind to this protein. or these molecules.
• the binding partner antibodies are, for example, either polyclonal antibodies or monoclonal antibodies.
• the polyclonal antibodies can be obtained by immunization of an animal with the molecule concerned, followed by the recovery of the desired antibodies in purified form, by taking the serum of said animal, and separating said antibodies from the other constituents of the serum, in particular by chromatography. affinity on a column on which is fixed an antigen specifically recognized by the antibodies, in particular said aldehyde derivative derived from lipid peroxidation.
• the monoclonal antibodies can be obtained by the hybridoma technique widely known to those skilled in the art.
• the monoclonal antibodies can also be recombinant antibodies obtained by genetic engineering, by techniques well known to those skilled in the art.
• anti-aldehyde derivative antibodies derived from lipid peroxidation in protein bound form other than albumin and anti-albumin antibodies are widely known to those skilled in the art and are sold for example by JaICA and Hytest, respectively.
• the antibodies specifically binding to aldehyde derivatives derived from the peroxidation of lipids in albumin-bound form are new and constitute another subject of the invention.
• Antibodies specifically binding to aldehyde derivatives derived from lipid peroxidation in albumin-bound form means any antibody capable of binding to said derivatives with a high specificity or even a specificity of 100%, and unable to bind to aldehyde derivatives derived from the peroxidation of lipids in form bound to a protein other than albumin.
• the binding partner to said aldehyde derivative derived from the peroxidation of lipids in bound form is an antibody, preferably an anti-aldehyde derivative antibody derived from the lipid peroxidation of lipid-bound form. albumin.
• at least one aldehyde derivative bonding partner of the bound form lipid peroxidation it is meant that the process of the invention can employ two or more such partners.
• the method of the invention can implement a binding partner bound to the HNE, in particular linked to albumin, and a binding partner HHE bound form , in particular linked to albumin, or a binding partner bound to the HNE, in particular linked to albumin, and an MDA binding partner in bound form, in particular bound to albumin.
• the binding partners of the aldehyde derivative derived from the lipid peroxidation in protein-bound form used in the method of the invention may be used as a capture reagent or as a detection reagent.
• a specific binding partner of albumin it may be used as a capture reagent or as a detection reagent depending on whether the binding partner of the aldehyde derivative derived from the lipid peroxidation of a bound form of protein is used respectively as a detection reagent or as a capture reagent.
• the visualization of immunological reactions i.e., the IMA binding / binding partner, may be performed by any means of detection, such as direct or indirect means.
• the immunological reactions are observed for example by surface plasmon resonance or by cyclic voltammetry on an electrode bearing a conductive polymer.
• the indirect detection is done by means of a marking, either of the binding partner called the revealing reagent, or of the IMA itself. In the latter case, we speak of a competition method.
• labeling is meant the attachment of a marker reagent capable of directly or indirectly generating a detectable signal.
• a nonlimiting list of these marker reagents consists of:
• enzymes that produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose-6-phosphate dehydrogenase,
• chromophores such as fluorescent compounds, luminescent compounds, dyes, radioactive molecules such as 32P, 35S or 1251, and
• fluorescent molecules such as Alexa or phycocyanines.
• Indirect detection systems can also be used, such as, for example, ligands capable of reacting with an anti-ligand.
• the ligand / anti-ligand pairs are well known to those skilled in the art, which is the case, for example, of the following pairs: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin, polynucleotide / complementary polynucleotide. In this case, it is the ligand that carries the binding partner.
• the anti-ligand can be detectable directly by the labeling reagents described in the preceding paragraph or be itself detectable by a ligand / antiligand.
• the in vitro diagnostic method of ischemic states also implements the detection of a cardiac marker.
• cardiac markers include, without limitation, commonly used cardiac markers such as troponin, such as troponin I or troponin T, and CK-MB (isoform MB of creatine kinase), troponin I being the preferred heart marker.
• cardiac markers such as troponin, such as troponin I or troponin T, and CK-MB (isoform MB of creatine kinase), troponin I being the preferred heart marker.
• the IMA being one of them, they can be separately demonstrated, for example by means of different biochemical tests, or else Simultaneously, in multiplex assay, according to the techniques described above.
• the IMA was purified using its property of no longer binding the divalent ions in order to isolate it from normal HSA.
• a pool of 10 plasmas of patients who had unstable angina (ischemia) or a healthy plasma were each deposited on a column of Nickel-Agarose resin (Pharmacia).
• the unmodified HSA binds to the resin while the IMA is not adsorbed and passes into the filtrate.
• the IMA contained in the filtrates was then concentrated and immunopurified using the anti-HSA 15C7 monoclonal antibody (HyTest) coupled to cyanogen bromide-activated Sepharose resin.
• the IMA was isolated after electrophoresis SDS-PAGE on 12% acrylamide gel and transfer to a PVDF membrane. It was then sequenced by the Edman technique, in order to determine if the modification that characterizes the IMA is a cleavage amino acids component its N-terminal, as indicated in the patent application WO00 / 20840. Results: Position 1 2 3 4 5 6 7 8 9 10
• HSA N-term- D A H K S E VA H R IMA: N-term- D A H K X E VA X X
• the SELDI-TOF technique (Ciphergen) was used to identify a mass addition related to the binding of a group to the modified albumin plasma albumin relative to the albumin of healthy samples. Interaction with the anti-hydroxynonenal antibody was then characterized with these same 2 angor and healthy samples.
• the non-adsorbed Nickel-Agarose resin fractions from a pool of 4 angina plasmas, as well as a pool of 14 healthy sera, are immunopurified on anti-HSA resin (mAb 15C7 monoclonal antibody).
• eluates enriched in IMA, resulting from this double purification, are analyzed by SELDI-TOF on hydrophilic strips (NP20) and on epoxide grouped arrays (PS20) on which are grafted covalently, via their amine function, anti-antibodies.
• NP20 hydrophilic strips
• PS20 epoxide grouped arrays
• -HNE HNEJ-2, JaICA
• the results in the table show a significant mass shift between albumin from pooled healthy sample eluates and albumen from the pool of angina samples.
• the albumin pool albumin has a higher albumin mass than the normal samples (healthy pool eluate and commercial HSA), which means that the modification that characterizes the MAI would be the addition of an undefined pool causing an increase massive.
• the anti-HNE-protein monoclonal antibody HNEJ-2 (JaICA) was coupled on a PS20 surface via its epoxide groups.
• the 2 non-adsorbed fractions adsorbed on a nickel-agarose resin column, of the same pool of 6 angor plasmas, are immunopurified with an anti-HSA affinity resin (AcM 15C7).
• AcM 15C7 anti-HSA affinity resin
• the 2 purified fractions (IMA and HSA) are dialysed in PBS, concentrated, quantified by spectrometry D ⁇ 27 9nm and stabilized by a reduction to NaBD 4 .
• Anti-HNE-protein monoclonal antibody solution HNEJ-2 from JaICA
• anti-HHE-protein JaICA HHE53
• anti-MD A-protein Jaica 1F83
• diluted to 10 ⁇ g / mL buffer Tris 0.2 M maleic acid 0.2 M at pH 6.2 was incubated for 2 h at 37 0 C on a black plate of 96 wells (capture antibody). After 2 hours of saturation at 37 ° C. with a solution of PBS containing 0.2% of gelatin, plasmas diluted 1/10 in PBS-Tween 0.05% were incubated overnight at 40 ° C.
• IMA, or albumin modified by the HNE, HHE or MDA, specifically selected by the monoclonal capture antibody was detected by an antibody F (ab ') 2 anti-human serum albumin (MAb 10C3, bioMérieux, France ) coupled to biotin, diluted to 1 ⁇ g / ml in PBS containing 0.05% Tween and 0.1% gelatin and incubated for 2h at 37 ° C.
• Streptavidin coupled to alkaline phosphatase diluted at 1 ° C. / 10000 in TBS (Saline Tris Buffer) containing 0.05% Tween and 0.1% gelatin was incubated for 1 h at 37 ° C.
• a measurement of the fluometric signal was then carried out after the introduction of a substrate fluorogenic alkaline phosphatase.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• General Health & Medical Sciences (AREA)
• Hematology (AREA)
• Medicinal Chemistry (AREA)
• Urology & Nephrology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Organic Chemistry (AREA)
• Biochemistry (AREA)
• Biomedical Technology (AREA)
• Cell Biology (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Biophysics (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Peptides Or Proteins (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=39790990

Family Applications (1)

Country Status (3)

Families Citing this family (5)

Family Cites Families (3)

• 2009
  • 2009-03-10 WO PCT/FR2009/050386 patent/WO2009115756A2/fr active Application Filing
  • 2009-03-10 US US12/865,562 patent/US20110039353A1/en not_active Abandoned
  • 2009-03-10 EP EP09721625A patent/EP2252897A2/de not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events