Exosomes are membrane vesicles of nm in diameter, secreted by various cell types in vivo. Because of their origin and biogenesis, exosomes reflect the content and the normal or pathological physiological state of the cells from which they originate. They are found in many biological fluids such as blood, plasma, serum, urine, saliva, cerebrospinal fluid CSFlymph, bile, bronchoalveolar washings, semen, synovial fluid, amniotic fluid, breast milk, the malignant ascites fluids, Their secretion process is a very liquide blanc selles anormales process for proliferating cells such as cancer liquide blanc selles anormales.
They contain nucleic acid and protein markers of tumor cells, from which they are secreted and are therefore considered as reservoirs of potential new cancer biomarkers. In this, their study shows a growing interest in the liquide blanc selles anormales community. The major limitation to their study lies in obtaining sufficiently purified and enriched preparations from the various aforementioned biological fluids, with current techniques available in the state of the art.
The literature emphasizes indeed the complexity of the evaluation of exosomes due to contaminated preparations, including proteins, microsomal fractions or organelles co-purified by techniques such as ultracentrifugation or nanofiltration, or too little concentrated preparations in exosomes to be analyzed, from immunological separation techniques.
To date, even the combination of these techniques has not been entirely satisfactory. Most of the work in order to characterize the exosomes liquide blanc selles anormales undertaken on the malignant cell cultures.
They are subjected to purification techniques exosomes that the cells secrete in development, and proteomic profiling of exosomes isolated are determined. Thus, under Article Mathivanan S. The culture medium was first subjected to a first series of centrifugations to separate a particle population having a size of nm, which was then engaged in an immunopurification with an antibody humanised A33, recognizing specifically epithelial cells of the colon.
The importance of exosomes having been established, it is essential to have efficient and specific techniques for isolating exosomes, allowing at the same time being reliable, usable in routine and do not require large volumes of biological samples. The invention resides in a method of isolating exosomes which comprises two successive levels of separation which are based on the technique of affinity separation.
This sequential separation gives the method of the invention a high specificity. It enables moreover to be applicable to any sample of a liquid or biological fluid. The process of the invention is a tool that can be used routinely. It overcomes the obstacles that the art was faced to date, opening a way for widespread access to liquide blanc selles anormales characterization of exosomes. The method of the invention comprises at least the following two successive steps:.
Before describing the invention in detail, certain terms used in this text to characterize the invention are defined below. Exosomes belong to a fraction of nano-vesicles secreted by cells in biological fluids. Structurally, they are vesicles having a lipid bilayer comprising surface proteins and sugars. They are defined by their size ranging from 30 to nm, more particularly by a size of at least 40 nm, or even at least 50 nm and at most nm or at most nm and even at most nm.
The biological fluid and organic liquid terms are used interchangeably. A biological fluid is produced by a human or animal being, healthy or diseased, diagnosed or not. It is collected or punctured in humans or animals, directly or indirectly. By liquide blanc selles anormales we understand that can be taken in the human or animal cells or a cellular tissue that is cultured in a suitable medium in which said cells excrete exosomes and all or part will be charged for submission to the liquide blanc selles anormales isolation process.
As a non-limiting example, the cell supernatants include, stool specimens, bone marrow. By purification technique, affinity includes a technique based on specific recognition or interaction between a ligand exosomal carried by the exosome and an antiligand. Recognition may also be of any other kind, for example covalent. This technique allows the isolation of the sample yet exosome bound to said anti-ligand or separate exosome said anti-ligand, for example after elution.
A liquide blanc selles anormales implementation of this technique according to the invention consists of fixing, directly or indirectly, said anti-ligand on a solid support, such as magnetic beads, membranes, matrices of chromatographies, microplates, or devices microfluidics.
In the case of indirect binding, the binding of the anti-ligand to the solid support can be implemented using a magnetic bead as an intermediate between the solid support, for example a membrane, and the antiligand. To do this, the activation of a magnet behind the solid support will determine the magnetic bead to the solid support, and the deactivation of said liquide blanc selles anormales will separate from the solid support liquide blanc selles anormales fixed in anti-ligand, which is itself fixed the magnetic bead.
By normal cell, comprises a cell having no detectable labels or a detectable level of markers characteristic of an abnormal state of the cell. Included in liquide blanc selles anormales definition, stem cells, including mesenchymal, neural and hematopoietic, which secrete exosomes features an undifferentiated state. In contrast, a cell is considered abnormal when it has one or more detectable labels, or a detectable level of markers characteristic of a state different from that of a normal cell, including a pathological condition of the cell or state may change to a pathological condition.
In particular, this definition, cancer cells, adenomatous, infectious, inflammatory, immune-stimulated, burned and generally abused by any kind of stimulus. As previously reported, despite their small size, exosomes have the advantage of accumulating many proteins that constitute traceability, and are the epitome of cells that excrete, particularly the nature of the tissue, the state normal or abnormal cell The method of the invention comprises a liquide blanc selles anormales affinity purification step for isolating a population P of exosomes, which is based on the existence of specific markers of exosomes, which may be present or exposed on the surface of those -ci at a stage of development.
These markers that also called generic ligands are antigens, receptors, growth factors, and any other particle or molecule, and any fraction thereof, capable of being liquide blanc selles anormales recognized by an anti-ligand. The first step, thus implementing at least one anti-ligand specifically recognizing at least one of the markers or ligands above. According to a variant of the invention, the first stage comprises only one purification, in which is used the one or more anti-ligands.
According to another variant of the invention, the first step may comprise two consecutive sub-steps or more, each of them engaging one or more specific anti-ligands of one or more generic ligands, respectively. As illustrated in the examples, the first step leads to the obtaining of a population of exosomes P which are separated from the or said specific anti-ligands of one or generic ligands exosomes.
This separation is preferably carried out by elution under conditions liquide blanc selles anormales fall within the general competence of the expert. Previously, this fluid may have been treated. Thus, without departing from the scope of the invention, the biological fluid may be pre-treated by a physical separation step, by size, which allows isolation of a fraction of the biological fluid containing no molecules or particles of a size exceeding nm, preferably nm, which is then subjected to the first step a.
This physical separation step in advance successive steps of affinity purification has the advantage that it allows to enrich the sample prior to the implementation of said steps of affinity purification, yielding exosomes with a purification even higher. After the first step liquide blanc selles anormalesa population is obtained P exosome which is then engaged in the second step b of the inventive method. The second step of purification by affinity of the inventive method for liquide blanc selles anormales from the population P exosome, a sub SP exosome population is based on the existence of specific own markers, or characteristics some exosomes, said markers being capable of being present or exposed on the surface thereof, at a stage of their development.
These markers, referred to particular ligands, as opposed to generic ligands are antigens, receptors, growth factors, and any other particle or molecule, and any fraction thereof, capable of being recognized specifically by an anti-ligand.
The second step b uses at least one anti-ligand specifically recognizing at least one of the markers or particular ligands liquide blanc selles anormales. As illustrated in the examples, and depending on the technology in which the isolated exosomes are then involved according to the invention, the second step results in the production of a SP subpopulation exosome that are related to said specific anti-ligands or ligands of said characteristics of a SP subpopulation exosomes, or are separate from said anti-ligands.
In the latter case, the exosomes obtained are free from liquide blanc selles anormales support and are soluble. The separation of anti-ligands exosomes is preferably carried out by elution under conditions which fall within the general skill of the art.
According to a variant of the invention, the second step b comprises only one purification, in which one uses one or more anti aforesaid ligands. Purification of this second step b can be performed in several sub-steps involving one or more anti-ligands, contributing to the isolation of a subpopulation same SP exosomes, which will therefore be increasingly specific. According to another variant of the invention, liquide blanc selles anormales method of the invention comprises at least a third step c affinity purification, this step liquide blanc selles anormales applied to said SP subpopulation and using at least one anti-ligand specific to an ligand characteristic of a subpopulation SP 'of exosomes, the subpopulation SP' being included in the subpopulation SP, to obtain said sub-population SP.
Like the second step bthe third step c purification may include substeps contributing to the isolation of a subpopulation same Liquide blanc selles anormales 'of exosomes. Of course, one or more additional sequential steps of affinity purification can still complement the process of the invention.
In this case, the exosomes are separate from said anti-ligands in the affinity purifications before the last purification step by affinity, exosomes may then be separated or not said anti-ligands in the last purification step. MS sub-population and the subpopulation SP 'are of particular exosome population. Depending on the particular anti-ligands used, the SP sub-population or subpopulation SP 'may for example collect exosomes from the same organ of the same tissue, or the same type cells.
The fabric or the original cells are normal or abnormal. In practice liquide blanc selles anormales before a picture of the implementation and the interests of a method of the invention, in the examples that follow, the population P, after the first step a may be subjected to the second step b the method in the presence of one or more specific anti-ligands to ligands characteristic of a pathological organ, e.
In another implementation, the population P may be subjected to the second step bliquide blanc selles anormales the presence of one or more specific anti-ligands to ligands characteristic of an organ, e. Liquide blanc selles anormales refine the isolation process, the SP sub-population may be subjected to a third step c with specific anti-ligands to ligands markers of abnormal cells to enable the isolation of a subpopulation SP 'd' exosomes characteristics of a prostate tumor.
It is also conceivable that this subpopulation SP 'is obtained by treating a population P after the first step ain a second step b in the presence of specific anti-ligands to ligands markers of abnormal cells to isolate SP subpopulation of exosomes characteristics of abnormal cells; then, the SP sub-population is subjected to a third step c with specific anti-ligands to ligands characteristics of the prostate.
According to the invention, the or at least one of the generic ligands are different from the or at least one of the characteristic ligands. In particular, analogs of antibodies include small proteins such as antibodies are capable of binding to a biological target and to detect, to capture or just the target within an organization or biological sample.
The fields of application of these antibodies analogues are almost as large as those antibodies. As indicated above, exosomes isolated according to the invention can be separated from or anti-ligands involved in step b affinity purification or affinity in the last purification step if other steps of affinity purification are performed.
The exosomes isolated according to the method of the invention, separated from any medium and soluble are particularly useful in the following applications, by way of illustration and non-limiting data:. The disease can be chronic or acute, infectious or non-infectious origin. In one use of the method, the disease is adenoma, cancer, inflammation, sepsis, neurological disease such as Alzheimer's, Parkinson's, multiple sclerosis and prion diseases, or pathology of pregnancy liquide blanc selles anormales as pre-eclampsia.
The treatment can be a drug therapy, radiation therapy or transplantation. The invention is illustrated in the following examples where it is applied to different body fluids of healthy or sick patients, ie with cancer of the prostate, anti-ligands used in the second stage being characteristic of the prostate.
These examples refer to the following figures:. This is of 2 sera from patients P1 and P2 having liquide blanc selles anormales Gleason score of liquide blanc selles anormales and 8, respectively.
The purification method was applied to a patient urine pool with cancer of the prostate with a Gleason score of 7, after post digital rectal examination massage post-DRE CaP and patients benign prostatic hyperplasia BPH. A biotinylated antibody step is performed prior to the commercial kit, One-step Antibody Biotinylation, commercialized by Miltenyi Biotec, according to the supplier's recommendations.
The tube is placed on a magnetic medium to remove the supernatant. For blocking free sites, liquide blanc selles anormales biotin solution at 10 mM is added and incubated for 30 minutes under rotary shaking. A filtration step 0. The urine sample is also subjected to a pretreatment of differential centrifugation and filtration through 0. The urine is then concentrated 5X on Vivaspin 20 10 kD was off, Vivasciences.
The anti-ligand used is an anti-tetraspanin antibody, specifically an anti-CD The first immunopurification by anti-CD63 tetraspanin is carried out in two incubations of the pre-treated blood sample. The remaining volume of 1, 25 ml of pretreated liquide blanc selles anormales is incubated with the bioconjugate in batch overnight under rotary shaking at room temperature. Under the same conditions, this step immunopurification was performed with the anti-tetraspanin, anti-CD81, on pools of sera from sick patients CaP.
This second specific immunopurification step is performed at from elution fractions P P CD63 and CD81, respectively, obtained in 4. This gives the following subpopulations SP: In order to detect the presence of exosomes in fractions purified by the invention process described above, the test ExoTEST liquide blanc selles anormales by HansaBiomed company was used.
The sandwich format allows the specific capture exosomes by reducing the detection of contaminating proteins. In addition, the test allows the quantification of exosomes from biological samples liquide blanc selles anormales purified preparations and enriched in exosomes, thanks to the presence of a calibration standard included in the kit.
The assay samples was performed according to the manufacturer's recommendations. Using a laser nm, the nanoparticles are excited and their Brownian motion is followed by an optical microscope and filmed by a camera.
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