Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/5432—Liposomes or microcapsules
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/545—Synthetic resin
• • 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
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
  • G01N33/586—Liposomes, microcapsules or cells
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

• the invention relates to the technical field of detection of biological targets "Specifically, the invention has for an object a new process for the production of capture phases for the detection of s a biological target in a biological sample, and methods of detection and corresponding detection kits.
• the application WO 98/47000 describes a method for detecting a target biological material contained in a sample, according to which a capture phase is available, said target biological material is brought into contact with at least the phase capture, and detects the capture phase-target biological material complex.
• Said method is characterized in that the capture phase is in microparticulate or linear form and is constituted by at least a first particulate or linear polymer, with an apparent hydrophilic character and first compiexan groups, the latter being bound by coordination to a first transition metal, which is itself bound to a first biological ligand, capable of specifically recognizing the target biological material.
• Such a capture phase is proposed to optimize the attachment of the material thereon, while decreasing or eliminating any secondary adsorption reaction dudlt material on said capture phase.
• this document proposes the use of a particulate or linear hydrophilic polymer, and in particular a polymer functionalized product obtained by polymerization of a water-soluble monomer, acrylamide, an acryfamide derivative, methacrylamide or a methacrylamide derivative, at least one reaculatton agent and at least one monomer functional the monomer water-soluble is preferably selected from H-isopropylacrylamide, N-ethyl methacrylamide, propylene glycol, N-propyl methacrylamide, N-isopropylmethacrylamide, ydoprapylacrylamide, diethylacrylamide, methyl; -.
• ⁇ f isopropylacrylamide N-methyl-Nn propylacryiamide
• the monomer preferably being N isopropylacrylamlde (IPAH) and the functional monomer is ,, preferably selected from carboxylic acids, optionally nitrogen, itaconic acid, acrylic derivatives and methacrylic derivatives.
• a particulate polymer of the poly (N-isopropylacrylamide) (PN) type comprising complexing groups derived from itaconic acid or maleic anhydride- ⁇ -methylvinylether is used.
• the application WO 98/59241 proposes to use a capture and / or detection phase which comprises an organic molecule; having at least one reactive function and at least one protein material capable of recognizing or binding specifically and directly or indirectly to the target biological material, said protein material having a specific covalent binding site to the reactive function of the organic molecule, which consists of at least one tag comprising at least six lysine residues or lysine derivative, contiguous, the capture phase is advantageously f immobilized on a solid support by passive or covalent adsorption, the organic molecule may especially be a polymer, particulate or linear, homopolymers such as polylysine, polytyrosine, copolymers such as maleic anhydride copolymers, N-vinylpyrrolidone copolymers, and in particular the maleic anhydride copolymer / methyl-vinyl-stirrup the copolymer M ⁇ vinvI-pyrrolidone / IM-acryloxysuce
• HVE 65 poly (methylvinyl ether / malic anhydride), PEAM 86 poly (maleic anhydride anhydride), SAM 49 poly (styrene / malic anhydride) and NVPAM 3 ⁇ poly (-vinypyrrolidone / malic anhydride),
• WO 03/044533 proposes a method for obtaining a capture phase of a target biological material, comprising a modified protein of interest, capable of specifically binding, directly or indirectly, to said target biological material and immobilized on an immobilization phase comprising reactive groups, in which at least two different peptide sequences, comprising the peptldic sequence of the protein of interest, and one comprising a succession of at least six lysine residues at its N-terminal end; and a succession of at least six residues bistidlne at its C-terminal end,! !
• phase of immoblilsation corresponds to a polymer, which is then itself immobilized on a solid support.
• a poly (methyl vinyl ether-maleic anhydride) amve 57 is used.
• the polymer Since the polymer is not hydrosolubized, it is necessary to dissolve it in anhydrous DMSO (dlmethylsulfoxide) prior to the coupling reaction, carried out in a 95% aqueous medium with the partner protein of the biological target to be detected.
• DMSO dimethylsulfoxide
• the capture phases retain the biological activity of the immobilized or coupled biological ligand, after purification and immobilization or coupling, so that the latter can then interact properly with the biological target to detect and enable reliable detection.
• the capture phase comprises an immobilized protein
• immobilization of the proteins on the capture phase carried out at least partly in organic solvent, as is the case in the application WO 01/92361
• the invention proposes to modify the capture phases used in the detection methods, in particular by developing a new preparation process, in order to increase the detection sensitivity.
• a method is proposed in which the coupling of the biological ligand to the polymer can be carried out in an essentially aqueous solution.
• the invention relates to a process for preparing a capture phase for the detection and / or quantification of a target biological entity, said capture phase comprising a biological ligand for the biological entity, said biological ligand being bound so Covendinge an amphiphilic polymer and being immobilized on a solid support, characterized in that the biological ligand is immobilized on the solid support, by contacting the support: solid with a dispersion of micelles formed of a plurality of chains of the amphiphilic polymer said micelles being surface carriers of a plurality of molecules of the biological IgGand.
• the amphiphilic polymer has a hydrophobic part oriented towards the heart of the micelles and a hydrophilic part on the surface of the micelles, the biological ligand being covalently coupled to the hydrophilic part.
• the immobilization is carried out in a solvent or solvent mixture consisting of at least 90% by weight, preferably at least 95% by weight, and even more preferably at less than 99% by weight of water.
• This solvent or solvent mixture equal to that present in the micelle dispersion used for the immobilization, immobilization
• Such aqueous phase avoids the denaturation of biological iigand as compared to processes of the prior art that use an asset in an aqueous phase / organic phase mixture, with a high proportion of organic phase,
• amphiphilic polymer concentration of 50 to 500 ⁇ g / ml will, for example, be used when the micelles are brought into contact with the ⁇ support. This concentration will correspond to the concentration of the micelle dispersion used.
• the polymer after immobilization, the polymer remains, at least in part, in the form of micelles, such that micelles formed of a plurality of chains of the ampullated polymer, the micelles being surface-bearing. a plurality of molecules of the biological ligand covalently bonded to the amphiphite polymer, soot immobilized on the surface of the support.
• the concentration of amphotile polymer during contacting with the support must be greater than the critical micelle concentration (CMC) of the polymer, so as to maintain the micellar state.
• CMC critical micelle concentration
• the method according to the invention may comprise a coupling step send between the biological ligand and the polymer amp lphile performed while the polymer is in the form of micelles., So as to form the surface-bearing micelles of a plurality of biological Iigand molecules.
• the coupling is advantageously carried out in a solvent or solvent mixture consisting of at least 90% by weight, preferably at least 95% by weight, and even more preferably at least 99% by weight. % by mass of water.
• a solvent or solvent mixture consisting of at least 90% by weight, preferably at least 95% by weight, and even more preferably at least 99% by weight. % by mass of water.
• the coupling is carried out with an ampiphile polymer concentration at least 10 times greater than that used when the micelles are brought into contact with the support, in order to promote the coupling efficiency, to guarantee the preservation of the state. micellar during this coupling (and thus allow an orientation of this coupling to the outer portion of the hydrophilic crown), Such a concentration makes it possible to promote the preservation of the micelle shape during coupling and then during immobilization.
• an amphiphilic polymer concentration of 0.5 to 5 mg / ml during the covalent coupling step between the biological ligand. ?
• the coupling of the biological figand is carried out in such a way as to obtain micelles with the hydrophilic part of the polymer oriented towards the surface of the micelles, covalently coupled to the biological ligand.
• the polymer remains, at least in part, in the form of micelles, so that micelles formed of a plurality of chains of the amphiphilic polymer, are immobilized on the surface of the carrier lesdltes micelles being carriers at the surface of a plurality of molecules of the biological ligand covalently bound to the amphiphilic polymer.
• the micelles is finally immobilized by adsorption on the solid support by means of a biological interaction / solid support interaction, a portion of the micelles possibly being immobilized by adsorption on the solid support by via a polymer / solid support interaction, the interactions involved possibly being electrostatic or ionic bonds, or hydropole interactions,
• part of the biological ligands is free, in particular is not bound to the support.
• a coupling step will be carried out with a polymer concentration corresponding to at least 50 times, preferably at least 200 times the critical mass concentration of the polymer.
• the amphiphilic polymer is preferably a linear block polymer comprising at least one hydrophilic block and at least one hydrophobic block, the hydrophilic block being positioned at the surface of the micelles, and being covalently bonding carrier of at least one molecule of the biological ligand.
• the method will be implemented with one or other of the following features, any combination of the following features, or all of the following features;
• the average density of biological ligand molecules per polymer chain is from 0.1 to 100, and in particular from 1 to 100, the average density of ligand molecules per polymer chain can be deduced from the determination of the residual reactive functions of the ligand involved in the coupling;
• the micelles in the dispersion and / or micelles finally immobilized on the support are formed from 100 to 5000 d chains of polymers and / or carry 10 to 500 000 molecules of biological ligand.
• the dispersion of micelles has a polydispersity index of
• amphiphilic polymer has a molar mass greater than 5000 g / mol, preferably greater than 10,000 g / mol;
• the biological ligand is an antigen, a hapten, or a protein
• the amphiphilic polymer comprises, or even consists exclusively of, a first linear block consisting of a hydrophobic homopolymer resulting from the polymerization of a hydrophobic monomer; a second linear block consisting of a hydrophilic copolymer resulting from the copolymerisatson of a monomer B bearing a reactive function X and a hydrophilic monomer C not carrying a reactive function, said second block being bonded to one end of the first block eovalently; in this case preferably;
• the monomer est is chosen from the hydrophobic derivatives of niethacrylate, acrylate, acrylamide, methacrylamide, acetates, or from styrene and its derivatives and is preferably n-butyl acrylate, ertiobutyl acrylate, tert-butyl acrylamide, ⁇ -dichloride scryfamide, laetide, lactlde-co-glycolide or styrene and / or the monomer 8 is chosen from functional derivatives of aerylate, methacrylate, acrylamide or methacrylamide, styrene functional derivatives, and is preferably N-acryloxy sucdinimide, N-methacryloxy succine, 2-hydroxyethyl methacrylate, 2-aminoethyl methacrylate, 2-hydroxyethyl acrylate, 2-aminoethyl acrylate or 1,2,2,4-di-O-isopropylidene-6-O-
• the monomer S is carrying a reactive functional group X chosen from the functional groups -NH3 ⁇ 4 -CQOH, -OH, -SH, OOCH, ester, C, sulfhydryl, disulfide, hydrazine, hydrazone, azide, isocyanate, isothiocyanate, alkoxyamine, aldehyde, epoxy, nielle, maleimetal / haloalkyl, nalelmide ,.
• a reactive functional group X chosen from the functional groups -NH3 ⁇ 4 -CQOH, -OH, -SH, OOCH, ester, C, sulfhydryl, disulfide, hydrazine, hydrazone, azide, isocyanate, isothiocyanate, alkoxyamine, aldehyde, epoxy, nielle, maleimetal / haloalkyl, nalelmide ,.
• an activating agent such as carbodiimides, and in particular an activated carboxylic acid in the form of an ester of H-hydroxysuccinimide, pentahlorophenyl, trichlorophéoyl, p-nitrophényle, cartayphenyl, or among the compounds homo- or heterobifunctional;
• the monomer C is chosen from hydrophilic derivatives of acrylamide, methacrylamide and ⁇ -methylpyrrolidone; and preferably the oxyethylene, the monomer is N-vinylpyrrolidone or H-acryloylpropylene; the first block has a molar mass of between 1000 and 250 000 g / mol;
• the second block has a molar mass greater than 1000 g / mol and, preferably, greater than 2000 g / mol; the second block is a statistical copolymer, the composition of which, expressed by the ratio of the amounts of monomers in mol, amount of monomer, to amount of monomer B, preferably belongs to the range from 1 to 10, preferably from 1.5 to 4.
• the invention also relates to a capture phase of a target biological entity, characterized in that it comprises micelles immobilized on a solid support, said micelles being formed of a plurality of chains of an amphiphilic polymer, and said micelles being surface-bearing of a plurality of molecules of at least one biological ligand for the target biological entity, said molecules of the biological ligand being covalently bound to the chains of the amphiphilic polymer.
• the capture phase according to the invention will have one or other of the following characteristics, any combination of the following characteristics, or all of the following characteristics:
• the micelles are immobilized by adsorption on the solid support
• the micelles are immobilized by adsorption on the solid support by means of a biological ligand / solid support interaction, a portion of the micelles possibly being immobilized by adsorption on the solid support via a polymer interaction.
• solid support the interactions involved being in particular electrostatic bonds or it ionic, or hydrophobic interactions; however, even if a portion of the biological ligands immobilized to the support surface ,, intervenes to allow the immobilization of mi ⁇ lies on the solid support, part of the biological ligands is free, in particular is not bound to the support; a part of the biological ligands, corresponding in particular to at least 50% of the biological ligands present on the capture phase, remains accessible and will be available to interact and bind with a target biological entity, to allow capture of the latter when the capture phase will be used in a detection method; C 'is why the support on which the micelles are immobilized is referred to as capture phase;
• the micelles immobilized on the support are formed from 100 to 5000 polymer chains and / or carry 10 to 500 000 biological ligand molecules
• the amphipile polymer has a hydrophobic portion oriented to the heart of the micelles and a hydrophilic part on the surface of the micelles, the biological ligand being covalently coupled to the hydrophilic part,
• the amphiphilic polymer is a linear block polymer comprising at least one hydrophilic block and at least one hydrophobic block, the hydrophilic block being positioned on the surface of the micelles, and being covalently bonded, at least one molecule of the biological ligand
• amphiphilic polymer has a molar mass greater than 5,000 g / mol, preferably greater than 10,000 g / mol;
• the amphiphilic polymer includes, or even consists exclusively of, a first linear block consisting of a hydrophobic homopolymer resulting from the polymerization of a hydrophobic monomer A; a second linear block consisting of a hydrophilic copolymer resulting from the copolymerization of a monomer 8 bearing a reactive function X and a hydrophilic monomer C having no reactive function, said second block being bonded to one end of the first block covalently; the monomers A, B, C and the reactive functional groups K f and the blocks are preferably as defined previously in the context of the process;
• the biological ligsnd is an antigen, a hapten, or a protein.
• the invention also relates to a device for detecting and / or quantifying a target biological entity comprising a capture phase according to the invention and at least one tracer for detection.
• Linvenrion also relates to a device for detecting and / or quantifying a target biological entity, comprising a capture phase obtained according to the method of the invention and at least one tracer for detection.
• the invention also relates to a kit for detecting and / or quantifying a target biological entity comprising;
• a dispersion in aqueous solution of micelles formed of chains of an amphiphilic polymer, bearing at the surface of a plurality of molecules of at least one biological figure for the target biological entity, said biological ligand molecules being covalently bonded the chains of the amphiphilic polymer;
• the dispersion and the solid support will preferably have the same characteristics as those in the present description in connection with the method of preparing the capture phase.
• the subject of the invention is also a method for detecting and / or quantifying in vitro a biological target entity in a biological sample, according to which a capture phase according to the invention is available.
• said biological sample with at least the capture phase, and detecting and / or quantifying said target biological entity attached to the capture phase, after binding of the biological entity in a biological ligand molecule covalently bonded channels of the amphiphilic polymer of the capture phase.
• Another subject of the invention is a method for the in vitro detection and / or quantification of a target biological entity in a biological sample, according to which: a capture phase obtained according to the process according to the invention is available; contacting said biological sample with at least the capture plasmid thus obtained, and detecting and / or quantifying said fixed biological entity on the capture phase, after binding of the biological entity to a biological ligand molecule bound to covalently to the chains of the amphiphilic polymer of the capture phase,
• the invention also relates to a process for the in vitro detection and / or quantification of a target biological entity in a biological sample, in which: a capture phase is prepared according to the method of the invention. in contact with said biological sample with at least the capture phase thus prepared, and said target biological entity fixed on the capture phase is detected and / or quantified after binding of the biological entity to a biological ligand molecule bound in a covalently to the chains of the amphiphilic polymer of the capture base.
• Such detection and / or quantification methods may be a direct method, in which the sample, which may contain the target biological entity, is brought into contact with the capture phase and the bond between the biological ligand immobilized on the support and the target biological entity is highlighted by the presence of a tracer.
• the tracer is in particular a biological ligand of the target biological entity coupled to a marker.
• Sandwich immunoassays also called immunometric, are the most commonly used formats.
• Such detection and / or quantification methods may be an indirect method in which the sample likely to contain the target biological entity is brought into contact with the capture phase, in the presence of a biological entity analogue. and binding between the immobilized biological ligand on the capture medium and the target biological entity is: evidenced by the presence of a tracer, indirectly by detection of the binding between the immobilized biological ligand on the support and the analogue of the target biological entity.
• the tracer is in particular the analogue of the target biological entity coupled to a marker.
• Immunoassays in competition are the formats that will be the most conventionally used.
• the label is, for example, selected from enzymes, chromophores, radioactive molecules, fluorescent molecules and electrochemiluminescent salts.
• micelle formed of a plurality of channels of an amphiphilic polymer f is meant an assembly in a spheroidal form chains of an amphiphilic polymer including a hydrophilic part forms ia ring (facing the aqueous solution in which the micelles are located), and a hydrophobic portion forms the heart, as schematically illustrated in FIG.
• An amphiphilic polymer will self-assemble in this form, therefore, it is in aqueous solution at a concentration greater than a characteristic concentration of said polymer, called critical micellar concentration.
• the micelles are characterized by their hydrodynamic diameter. The latter is calculated from the hydrodynamic radius measured by the technique of dynamic diffusion of the light.
• the hydrodynamic radius is the radius of a theoretical sphere that would have the coefficient of diffusion that the particle considered.
• the miceiies generally have a hydrodynamic diameter of 50 to 200 nm, preferably 50 to 150 nm.
• the hydrodynamic diameter of the micelles can be measured in a dispersion of 50 ⁇ g / ml of polymer in an aqueous solution of 1 mM NaQ, for example, using a Zetasizer ano ⁇ S90 (a! Vern, UK) device.
• this polydispersity (defined as the square of the ratio of the standard deviation on the hydrodynamic diameter determined by the same apparatus), representative of the distribution width in a range, is typically less than 0.2 and the most often less than 0.1 ? characteristic of the narrow size distribution of such micelia.
• amphiphilic polymer is meant a polymer which has both a hydrophilic part or block and a hydrophobic part or block.
• the amphiphilic character of the polymer is characterized, in aqueous solution and above a concentration called critical critical concentration (CMC), by its aggregation in the form of micelia, this process making it possible to reduce the free energy of the system.
• CMC critical critical concentration
• the amphiphilic polymer chains will preferably be linear and unbranched so as to promote their assembly in the form of micelia.
• An amphiphilic polymer that can be used in the context of the invention is a copolymer consisting of at least two linear blocks, at least one globally hydrophobic block and at least one generally hydrophilic block.
• each monomer In a linear block, each monomer, with the exception of the two end monomers, is bonded to two other monomers, flanking said monomer along the chain.
• One of the ends of the overall hydrophobic block is covalently bonded to one end of the generally hydrophilic block.
• copolymer unless that! otherwise specified, must be understood as a polymer formed by at least two monomers, the term copolymer covers both random copolymers, block or alternating polymers,
• Such a polymer may comprise a first amphiphiie b! Oc hydrophobic, in the form of a hydrophobic homopolymer that is to say comprising a concatenation of a single monomer A hydrophobic.
• Such an amphiphilic polymer may comprise a second bioe bringing its hydrophilic component to the polymer, in the form of a copolymer consisting of two monomers: a first monomer bringing hydrophilicity, in order to promote a maximum deployment of the second block in aqueous phase, and another monomer B providing a reactive function X, in order to perform the covatent coupling of the biological ligand.
• this second block is a random copolymer and will advantageously have a sufficient number of reactive functions close to the end of the hydrophilic block not bound to the hydrophobic block (i.e., in the midle, the end deployed to the solution), to ensure adequate presentation / accessibility of the ligand after coupling, in the solution, for increased recognition efficiency.
• the second block may also consist of a single non-functional monomer, of which only the monomer located at the end of the chain (on the end not bound to the hydrophobic block) will be functionalized. This is the case, for example, of a hydrophilic polyethylene glycol block PEG (monomer unit -CHrCHi-Q- non-functional) whose hydroxyl chain end can be exploited for conjugation to ligands.
• At least 1 reactive function K f and preferably from 1 to 100 reactive functions X are present per amphiphilic polymer chain,
• the second block of the amphiphilic polymer will preferably be a random copolymer (in which the monomer units B and C are statistically distributed along the macromolecular chain) or an alternating copolymer (in which the monomers B and C follow one another. 1? regularly according to a general structure (B €) n in which n is an integer).
• copolymers can be obtained by reaction of polycondensatlon. or by free-radical ionic polymerization, or by group transfer, advantageously by living radical polymerization such as reversible termination polymerization (nitroxide-controlled polymerization, NMP), atom transfer polymerization (ATRP), and preferably reversible addition-fragmentation chain transfer polymerization, called RAPT (see WO 98/01478).
• NMP reversible termination polymerization
• ATRP atom transfer polymerization
• RAPT reversible addition-fragmentation chain transfer polymerization
• hydrophobic monomer is meant a monomer whose homopolymer has in the aqueous phase a compact ball structure, corresponding to a Hark Houwink-Sakurada coefficient (form factor) of less than 0.8.
• hydrophilic monomer is meant a monomer whose homopolymer has in the aqueous phase a deployed structure, corresponding to a Mark-Houwink Sakurada coefficient greater than 0.8.
• biological ligand is meant a biological entity capable of recognizing or binding to the target biological entity.
• the biological ligand is therefore a binding partner for the target biological entity as exemplary of such biological ligands.
• a protélque glycoprotéiqu material or such as an antigen, a hapten, an antibody, a protein, a oanofitine, a peptide "an enzyme, u sugar and fragments thereof, a lectin..; but also a nucleic material such as a nucleic acid (DNA or AN), a nucleic acid fragment, a probe or a primer.
• the invention is particularly suitable for biological ligands chosen from antigens, haptens and proteins.
• target biological entity refers to a biological material of interest
• biological material of interest examples include antibodies, receptors ,, , haptens antigens, proteins, peptldes” the enzymes, sugars, nucleic acids.
• Antibodies acting as a biological ligand for the target biological entity are polyclonal or monoclonal antibodies.
• the polyclonal antibodies can be obtained by immunizing an animai with, as an immunogen, the biological entity target "a fragment thereof or a close equivalent in terms structurai, followed by recovery of the desired antibodies in purified form" by collection of the serum from the animal, and separation of said antibodies from the other components of the serum, in particular by affinity chromatography on a column, on which is fixed an antigen specifically recognized by the antibodies, especially immunogen.
• 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.
• antibody fragments As examples of antibody fragments, mention may be made of Fab Fab f! f F (ab ! ) 2 as well as the chains scFv (of the English “Single chain variable fragment”), dsFv (of the English “Doubie-stranded variable fragment”), These functional fragments can in particular be obtained by genetic engineering .
• Nanofitins are small proteins that, like antibodies, are able to bind to a biological target that can detect, capture, or simply target within an organism.
• hapten refers to non-immunogenic compounds, i.e., incapable by themselves of promoting an immune reaction by production of antibodies, but capable of being recognized by antibodies obtained by immunization of animals. under known conditions, in particular, by immunization with a hapten-protein conjugate, these compounds generally have a molecular weight of less than 3000 Da, and most often less than 2000 Da and may be, for example, glycosylated peptides. meta olites, vitamins, hormones, prostaglandins, toxins or various drugs, nucleosides and nudeotides.
• lectin refers to proteins that bind specifically and reversibly to certain sugars. They play a major role in (Immunity, recognizing the specific carbohydrates of some pathogenic infectious agents, eg a lectin is Concanavalin A (HA), which is responsible among others for the s hémagglut! Nafion,
• HA Concanavalin A
• the biological ligands used may or may not be specific to the target biological entity. They are said to be specific when they are able to bind exclusively or almost exclusively to the target biological entity. They are said to be nonspecific when the binding selectivity to the target biological entity is low and they are then able to bind to other biological entities such as other proteins or antibodies. In general, it will be preferred to use a biological ligand specific for the target biological entity.
• the biological ligand behaving as a binding partner for the target biological entity is chosen according to the target biological entity that is to be detected:
• the biological ligand is an antigen that recognizes said antibody, preferably specifically;
• the target biological entity is an antigen
• the biological ligand is an antibody that recognizes the antigen, preferably specifically
• the biological ligand is a protein which binds to said receptor, preferably specifically;
• the biological ligand is an antibody or a protein that recognizes said hapten, preferably specifically.
• the invention is particularly suited to cases where the biological ligand is an antigen, a hapten, or a protein.
• reactive function present, on the one hand, on the polymer and on the other hand, on the biologic ligand.
• Such reactive functions X are chosen, by way of example, from ester, halogenocardony, sulfhydrite, disulfide, amine (MHz), carboxylic acid (COOH), hydrazine, hydrazone, azide, isocyanate, isothiocyanate, alkoxyamine, aldehyde groups.
• an activated carboxylic acid in the form of an ester of N-hydraxystrcimide, pentachlorophenyl, trichlorophenyl, p-nitrophenite or arboxyphenyl.
• the reactive function (s) present on the biological ligand and allowing to form a slow cove bond between the micelle polymer and the biological ligand may exist naturally on the biologic ligand.
• amines borne by the side chain of lysine can be used for coupling.
• the reactive functions must be "Introduced" previously for example in the form of a tag, according to techniques well known to those skilled in the art.
• a tag may be defined as an amino acid sequence.
• the biological ligand is a proteinaceous material
• a tag comprising six lysine or lysine derivative residues, or more and, optionally, other amino acids
• tags may be anywhere in the protein
• the tag will be located at its end-or C-terminus ale.
• a polynucleotide is synthesized by a solid support chemical method having a reactive function at any point in the chain, such as, for example, the 5 'end . or the 3 'end or on a base or on an internucleotide phosphate or on position 2 !
• a primary amine function can be coupled to an activated carboxylic acid, especially N-hydroxy succinimide or an aldehyde; an alkoxyamine function with a ketone or an aldehyde; a hydrazine function with an aldehyde; or else a thiol function on a haloalkyl or a maleimide.
• the micelles will carry, on average, 10 to 500,000 molecules of biological ligand depending on their molecular weight and nature.
• the micelles of polylactide ⁇ b "PGly copolymer ( ⁇ vinylpyrrolidone ⁇ co ⁇ - acryloxysuednlmide) ⁇ PLA ⁇ hF (VP ⁇ co-MAS)) used in the examples which follow (including a chain comprises in 80 reactive functions of the N-hydroxy succyrimide ester type), it was possible to couple (in PBS pH 7.4) approximately 7500 p24 proteins per micelle (molar mass p24: 24000 g / mol), or approximately one protein per chain.
• This number of ligands can be determined from the residual amino function assay of the ligand after the coupling reaction (2,4,6-trinitrobenzene sulfonic acid or fluorescamine assay method) and can be supplemented with a gel SDS-PGE in the case of protein coupling, of course, since most of the time the polymer chains will carry several reactive functions, several molecules of biological ligand may therefore be attached to a polymer chain, thus multiplying the number of biological ligand molecules fixed by micelle,
• the coupling is carried out on the copolyoid micelies, the ligand binding on the end of the hydrophilic block, strongly deployed in solution, is favored, thus facilitating its future recognition by the target.
• the coupling of the ligand will be the same. more efficient than the number of reactive functions on the hydrophilic block (particularly on its outermost part deployed in solution) will be high.
• the micellar state of the polymer during the coupling of the ligand makes it possible to orient the ligand to the hydrophilic end of the polymer, and thus to promote its accessibility in solution.
• controlled polymerization techniques will be used for the preparation of the polymers, such as the polymerization controlled by nitroxides, MNP, atom transfer polymerization (ATRP), radical polymerization by reversible chain transfer by addition / fragmentation (RAPT) or ionic polymerization.
• the copolymer is obtained in two stages.
• the first hydrophobic block ⁇ ! ⁇ (0.1-lactide) is obtained by ring opening polymerization of a monomer D, t-lactide, functionalized at the end of the chain by a nitroxide fragment 561, capable of initiating the NMP copolymerization of the pair of monomers I-vinyl-pyrrolidone (VP, hydrophilic monomer) and N-acryloxysuccinimide (NAS, functional monomer, carrying hydroxyl succinic acid ester functions
• VP polyvinyl-pyrrolidone
• NAS N-acryloxysuccinimide
• miceiles from amphiphilic polymer can be done according to any known technique.
• the common solvent method the most widely used, is described below (G. Riess., Prog, Polym., Sci. 1107-1170. (2003) ⁇ .
• the polymer will be dissolved in a solvent, most often organic, which solubilizes both the hydrophobic part and the hydrophilic part of the polymer, so as to obtain the sterilization of the polymer.
• One such solvent may be chosen from those miscible with water (acetone, acetonitrile, 1,4-dioxane, tetrahydrofuran (THF) or dimethylsulfoxide (DMSO)) and will be selected according to the nature of the amphophilic copolymer . Water is then added, leading to the formation of the miceiles, and then the initial solvent is removed by evaporation under reduced pressure (in the case of sufficiently volatile solvent (s), acetone type, acetonitrile, THF ) or dialysis against water (in the case of solvent (s) more "heavy (s)", type DMSO).
• the copolymer concentration range in the organic solvent is ⁇ to 15 mg / ml and the organic solvent / water volume ratios are 1 to 0.2.
• Such a technique is described in the case of a polymer of the polylacid-b type " poly ⁇ -vinylpyrrolidone ⁇ co- ⁇ acryloxysuccinimide) ⁇ , LA- -P ⁇ A5 ⁇ co-VP) ⁇ f in the publication N. Handké et al. Macromol. Biosci, 13, 1213-1220 (2013) ,. to which we can refer for more details.
• miceiles Pourron Other well known methods for preparing miceiles Pourron be used, in particular, the dialysis method of placing the copolymer in solution in a common solvent in a dialysis device, and performing dialysis against water.
• the method of direct dissolution of the copolymer in water may also be used in the case of copolymers having a high hydrophilic balance / hydrophere.
• the solid support may be in any suitable form such as a plate, a cone, a ball, the ball being optionally radioactive, fluorescent, magnetic and / or conductive, a bar, a glass tube, a well , a sheet, a chip, a micro-titration plate or the like.
• the support is in the form of beads, these have, most often, a diameter ranging from one hundred micrometers to the nanometer.
• the support material is preferably selected from latices, polystyrenes, copolymers styrene-butadiene; stene-butadiene copolymers in admixture with one or more polystyrenes, polypropylene, polycarbonates, polystyrene-acrylonitrile copolymers, methyl styrene-methyl methacrylate copolymers among synthetic and natural fibers; among the polysaccharides and drifts of cellulose; among glass, silicon and their derivatives.
• the micelle-biological ligand conjugate will be immobilized on the solid support, by any appropriate means, most often by adsorpdon. Such immobilization can be carried out by "passive" absorption on the solid phase, in particular by means of hydrophobic, electrostatic, Van der Waals or hydrogen bond interactions, the relative contribution of which will depend on the nature of the amphiphilic polymer, the ligand coupled and solid support dlmmoNilsation.
• the same conditions as those applied, and conventionally used by those skilled in the art, for the immobilization of a biological ligand on a solid support can be implemented. In particular, a dispersion of the micelia carrying biological ligands can be deposited in a buffered aqueous solution.
• the buffered aqueous solution will be formed of a solvent or mixture of solvents consisting of at least 90% by weight, preferably at least 95% by weight, and still more preferably at least 99% by weight water.
• Buffers conventionally used in the field of diagnosis may be implemented, so as to obtain and stabilize the pH in the desired range depending on the nature of the biological ligand. It is possible, for example., Using a PBS buffer (phosphate buffered saline ⁇ or tris (tris hydroxyméthylaminomé hane). In the copolymer concentrations used in excess of the CMC polymer., It is reasonable to estimate that i f adsorpt!
• micellar state is very generally preserved on the support, as shown by most studies of visualization of micelles by electron microscopes and / or atomic force microscopes, which require a deposition on support (Cho et ai, 3 Am Chem Soc, 128, 9935-9942 (2006)). ), Gensel et al, Soft Matter, 7, 11144 (2011)).
• the micelles are still formed and are immobilized on the support, mainly by support-ligand interactions, and to a lesser extent potentially by support-polymer interactions as shown in FIG.
• the capture phases described or obtained in the context of the invention may be used for the purpose of detecting and / or assaying and / or purifying a target biological entity.
• the user can directly dispose of the solid support on which the combined organic micelie-ligands is immobilized or kit comprising a solid support and a dispersion of the organic micelle-llgands conjugates described in the context of the invention in an aqueous solution,
• the aqueous solution will advantageously consist of a solvent or solvent mixture consisting of at least 90% by weight, preferably at least 95% by weight, and even more preferably at least 99% by weight of water.
• it will be a buffered aqueous solution, as described previously,
• the capture phases described or obtained in the context of the invention may be implemented in any technique for detecting and / or quantifying a target biological entity in a biological sample.
• Quantification means that the concentration of the present biological entity is determined.
• biological sample is meant any animal biological sample, preferably human, likely to contain a biological entity of interest. These samples are widely known to those skilled in the art, they may correspond to a biological fluid sample, for example whole blood, serum, plasma, urine, cerebrospinal fluid, organic secretion, Tissue collection or isolated cells. This sample can be used as it is, or it can undergo prior to the implementation of the method of detection and / or quantification, an enrichment type preparation or ass u; according to methods known to those skilled in the art.
• samples used in the detection and / or quantification methods may, in fact, be modified beforehand or not before their use.
• sample that have not previously been modified mention may be made of biological fluids such as whole blood, and as examples of a sample that has been modified beforehand, mention may be made of serum, plasma and cells that It is recovered from a biopsy, or after surgery, and cultured in vitro.
• the detection or quantification method can then be carried out in the culture supernatant or in the cell lysate.
• the detection and / or quantification techniques generally use, in addition to the capture phase, a tracer or detection phase, which makes it possible to detect the immobilization of the target biological entity on the capture phase.
• a tracer or detection phase which makes it possible to detect the immobilization of the target biological entity on the capture phase.
• detection or tracer phases comprise a marker.
• the detection and / or quantification method may implement direct or indirect detection.
• the sample likely to contain the target biological entity is brought into contact with the capture and binding phase between the biological ligand immobilized on the support and the target biological entity is then highlighted. thanks to the presence of a tracer.
• the tracer is usually a biological Hgand the target biological entity (most often different from the biological ligand immobilized on the support at the level of the capture base) coupled to a marker,
• Indirect detection methods also called competitive methods, are also assays widely known to those skilled in the art and used, in particular, when the target biological entity is a hapten. It consists in assaying the target biological entity in the sample by creating a competition between the target biological entity of the sample and an analogue of this target biological entity. In this case, the sample that may contain the target biological entity is contacted with the capture phase in the presence of an analogue of the target biological entity. The binding between the immobilized biological ligand on the support and the target biological entity is then demonstrated by the presence of a tracer, indirectly by detecting the binding between the immobilized biological ligand on the support and the like of target biological entity.
• the target biological entity analog is used in the competition reaction after coupling to a label to form a conjugate or tracer.
• the measured signal emitted by the tracer is then inversely proportional to the amount of target biological entity of the sample.
• marker any molecule capable of directly or indirectly generating a detectable signal.
• a non-limiting list of these direct detection markers consists of;
• enzymes that produce a detectable signal for example, by colorimetry ,. fluorescence, luminescence f such as horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase., glueose ⁇ 6 ⁇ phosphate dehydrogenase ,.
• Cbroniophores such as fluorescent, luminescent, dyes,
• radioactive molecules such as 32 P f 5 S or n %
• Fluorescent molecules such as Alexa or His cycocyanins, and * The hydroelectrolytic salts such as organometallic derivatives based acridinlum or ruthenium.
• Indirect detection systems may also be used, such as, for example, ligands capable of reacting with an antigen.
• the ligand then corresponds to the marker to constitute, with the analogue of the target biological entity, the tracer,
• Hgand / anti-ligand couples are well known to those skilled in the art, which is the case, for example, in the following pairs: biotin / streptavidin, hatene / antibody, antigen / antibody, peptide / antibody, sugar / lectin, polynucleotide / complementoler of polyneuteotide,
• the antMigand can then be detectable directly by the direct detection markers described above or be itself detectable by another pair ligand / antl-ligand, and so on,
• reagents allowing the visualization of the marking or the emission of a detectable signal by any type of suitable measuring apparatus, such as, for example, a spectrophotometer, a spectrofluoron meter or a high definition camera.
• the signal proportional (in the case of a direct process) or inversely proportional (in the case of an indirect process) to the amount of The target biological entity of the sample may be compared with a calibration curve obtained beforehand by techniques widely known to those skilled in the art. So; for example, the calibration curve is obtained by performing an assay using the same biological ligand, as well as known increasing amounts of target biological entity. A short is thus obtained by placing on the abscissa, the target biological entity concentration and on the ordinate, the corresponding signal obtained after assaying,
• the detection / quantification method according to the invention can be directly applied to the format of commercially available tests for the detection / quantification of target biological entity
• a biological ligand for which the invention can be applied include p24 and g i20 HIV, the core, NS3 f S4 and S5 of hepatitis C, ORF2 and ORF3 proteins of hepatitis E f l3 ⁇ 4ligosaccharide gaiactomannan fungi of the genus AspergMus,
• the targeted biological entities are the immunoglobulins (humoral response) directed against these pathogens.
• biomarkers of human pathologies such as S10GS protein, troponin I and T, anti-muhlerian hormone (AMH), procaicitonin, PSA (prostate - specific antigen) and other tumor markers.
• AMH anti-muhlerian hormone
• procaicitonin PSA (prostate - specific antigen)
• other tumor markers such as S10GS protein, troponin I and T, anti-muhlerian hormone (AMH), procaicitonin, PSA (prostate - specific antigen) and other tumor markers.
• the targeted biological entities are autoantibodies.
• FIG. 1 is a diagrammatic representation of micelia carrying biological ligands, in the case of a block-type amphiphilic copolymer
• Fi ⁇ yre 2 is a schematic representation of micellas carrying biological ligands immobilized on the surface of a support, which highlights the interactions between the micelia and the support; A) by (Intermediate of the membrane present on the surface of the micelles, B) via the amphiphilic polymer (via a hydrophilic chain); VS) by adsorpdon in a monomeric form (via hydrophobic / hydrophilic part
• FIG. 3 illustrates the preparation of p24-coupled micelia, used in the examples below,
• Figure 4 shows the results obtained with an ELISA test with free p24 (o) or p24 coupled with micelia ( ⁇ ) in capture as well as with micelies alone ( ⁇ ) (under the same dilution conditions as with p24) .
• Flgyre S and SB present the results obtained with the same ELISA test format as in the case of FIG. 4, but by varying the detection antibody concentration, with immobilization carried out with a p24 concentration of a): 10 pg / ml (FIG. 5A), b): 1 ⁇ g / ml (FIG. SB); pure absorbance signal graphs and Signal / Noise ratio as a function of antibody dilution (Ab).
• FIGS. 1A and 6I show the influence of the coupling conditions (diluted or concentrated) of the p24 on the sensitivity gain in ELISA after immobilization performed with a p24 concentration of: a): 10 ⁇ g / ml (Figura SA) b): 1 ⁇ g / ml ( Figure SB); pure absorbance graphs and signal-to-noise ratio as a function of antibody dilution.
• Figure 7 demonstrates the stability of the micelia tested in ELISA after immobilization performed with a p24 concentration of 1 ⁇ g / ml; graphs in pure absorbance signal and signal-to-noise ratio as a function of antibody dilution,
• Figure 8 shows different conditions of immobilization of the S10 ⁇ B antigen on the solid support used hereinafter.
• Figures ⁇ A and ⁇ show the ELISA results for the different types of dimmbllisation made according to Figur ⁇ 8; pure absorbent signal graphs and Signal / Noise ratio as a function of antibody concentration (ab). Examples
• micei-protein conjugates The effect of the use of micei-protein conjugates on the increased sensitivity of immunoenzymatic assays, in the capture phase, has been demonstrated:
• the micelles are prepared by the common solvent method (or nanoprecipitation).
• the copolymer (20 mg) is dissolved in 2 ml of acetonitrile and then this solution is added with a regular flow rate to 4 ml of milli-Q water.
• the acetonitrile is evaporated under reduced pressure.
• the micellar aqueous solution obtained is typically 5.2 mg. (precise determination by measurement of the solid level after passage in an oven).
• the average micelle case is 56 rsm.
• Coupling of the protein on the micelles is achieved by adding a volume (typically 500 ⁇ L) of micelle dispersion at 5.2 mg / mL to the same volume of p2 solution.
• PBS pH 7.4 at varying concentrations (0 to 2.4 mg.mL -1 )
• the final coupling medium thus contains the micelles at 2.6 mg.mL and the protein at concentrations ranging from 0 to 1, 2 mg.mL * 1.
• the samples are placed on a stirring wheel for 20 hours at room temperature.
• the coupling is total When increasing this amount (0.24 to D; 4B mg / g), more and more free protein is detected, indicating a "saturation" the surface of the micelles, and thus a non-quantitative coupling.
• the condition retained for the continuation is that corresponding to a quantitative coupling, the. 0.12 mg of p24 per mg of copolymer, ie p24 at 0.3 mg / mL and 2.6 mg / mL copolymer
• the hydrodynamic diameter of the micelles diluted 1/50 in a solution of iMM aCI is measured by dynamic light scattering (DLS, for Dynamic Light Scattering) using a device.
• the hydrodynamic diameter of the samples is 100 nm for control in PBS coupling medium (mlels without p24); the hydrolysis of the reactive functions of esters of NS to carboxyates leads to the unfolding of the hydrophilic chains, and of 11 nm for the micelles having coupled the p24,
• Mieeife Dsameter fp 24 (mg rog CMC hydrodynsmii u co polymer) (Mg mL)
• the coupled or free p24 was immobilized on the solid phase (Nunc MaxISorp F mlcrotltration plate) at different concentrations. in PBS (Cascade dilutions) f for 12 hours at room temperature; passivation is carried out in PBS - horse serum (SC) 10%; detection with a biotioylated anti-p24 antibody (rabbit) diluted in PBS-Tween-SC 10%, followed by addition of streptavidin-peroxidase CHRP) in PBS-T een-HS 10%, and revelation with 3,3 '5,5'-tetramethylbenzidine (TMB) (absorbance at 450 nm); free p24 controls and micelles alone are systematically prepared under the same conditions as those of the coupling, but in the absence of micelles and p24, respectively.
• FIGS. 4 and 5 show an important gain of the antibody detection signal, relative to the free p24. Moreover, the copolymer tested in the absence of p24 at the different dilutions does not cause significant background. This sensitivity gain is confirmed by working with an immobilization carried out with a concentration of p24 (coupled or free) of 10 ⁇ g / ml or 1 ⁇ g / ml and by varying the detection antibody concentration.
• the molecular state (the nanoparticle of about 100 nm) is indispensable when coupling the protein to increase the sensitivity of the diagnostic test (Le. the liquid phase due to the preferential coupling towards the end of the hydrophilic block).
• Coupling "diluted” the coupling of p24 is carried out directly under the conditions used for immobilization on the ELISA plate, Le. :
• P24 micelles (0.3 mg / ml) of p24 were stored at 4 ° C for 1 month. Compared to free p24 control, more suitable for optimal sensitivity (p24 supplier given at 2.4 mg / ml, stored at ⁇ 2Q € °) f-p24 micelles maintain their superiority in terms of sensitivity ( Figure 7) .
• the Tn1 protein was coupled to Pt ⁇ b ⁇ P miceifcs (A $ ⁇ co ⁇ NVP) in PBS at a concentration of 0.137 mg / ml in Tnl and 0.868 mg / ml micelles (0/158 mg Tnl per ml). mg of copolymer). Coupling was analyzed by SDS-PAGE gel which shows that the coupling of the micelles seem almost total, since the free TnI n f is not detected. The TnI thus coupled to the micelles was tested on VIDAS® (bioeric) and compared with TnI alone, with micelles alone on the solid phase.
• VIDAS® bioeric
• the VIDAS automated test is composed of 2 elements:
• the cartridge is a plastic strip containing 10 wells sealed with an aluminum film in which the different solutions are distributed,
• the cone serves as a pipetting system and solid phase. Each reagent of the cartridge is sucked up and then discharged by the cone.
• the cones are immobilized either with free TnI (ITC) at 0.03 ⁇ g / ml or micelles alone at 0.190 ⁇ g / ml or mlcelles-Tnl at 0.03 ⁇ g / ml in Tnl and 0.190 ⁇ g / ml. in copolymer, in a volume of 300 ⁇ L.
• the cones are emptied, then brought into contact with the passlvation buffer (0.2 M Tris buffer, pH 6.2) containing a protein or peptide saturation agent.
• the cones are then seeded and stored at + 4 ° C until use.
• the 3 capture phases prepared were compared by reacting them with a tracer which is a mixture of two anti-Tl mono-lonal antibodies (clones 16 ⁇ and 789 marketed by HYTEST, Sweden).
• a tracer which is a mixture of two anti-Tl mono-lonal antibodies (clones 16 ⁇ and 789 marketed by HYTEST, Sweden).
• this antibody is directly coupled to the alkaline phosphatase enzyme, making it possible to reduce the number of stages of the immunological reaction and to reduce the duration thereof (DEX2 protocol of VIDAS®, total duration approximately 40 min).
• the concentration of use of this tracer is 0.14 ⁇ g / ml in a volume of 400 ⁇ l.
• the generation of the signal is done by adding the substrate 4-Hymethyl mbellif eryf phosphate; the enzyme of the conjugate catalyzes the hydrolysis reaction of this substrate to 4-methylheferone whose emission is measured at 450 nm.
• the signal-to-noise ratio is improved when the TnI is coupled to the polymer
• the objective of this study is to demonstrate the need to use antigen-bearing copolymer micelles to increase the sensitivity of diagnostic tests for antigen immobilization.
• the antigen selected for this study is the $ 1008 protein (native antigen, bovine brain extract, HyTest).
• the polymer is PLA-b-P (NAS-co-NVP) as in the previous examples. It is used either in a micellarre form (in dispersion in 100% aqueous buffer) or dissolved in DMSO ("copolymer" form) for coupling with the 51008 antigen in a 5% DMSO-95% aqueous buffer medium. These two protocols, carried out in parallel, are carried out to evaluate the influence of the coupling conditions, that is to say coupling in 100% aqueous media with micelles versus coupling in a semi-organic medium DMSO / water with an initially dissolved polymer.
• the polymer-antigen couplings are made either in solution in an Eppendorf tube or on an ELISA microplate after immobilization of the mole or copolymer.
• the assay is carried out as previously reported for the p24 protein. Following the coupling, the% of modified amines is approximately 100%.
• 96 wells (unc Haxisor F96) are distributed 100 .mu.L / well of only micelies (l st step condition 2 ⁇ or copolymer alone (1 st condition of step 3) diluted in water to 74 pg / L
• the microplate are Incubated 12h at temperature
• the reaction mixture is then stirred for 1 hour in order to obtain the adsorption and then drained in order to carry out the couplings under dilute conditions, in the microplate directly, 100 ⁇ l / well of a solution of SIQ0B at 5 ⁇ g / ml are added to the wells.
• the microplate is incubated for an additional 12 hours.
• microplate is incubated for 1 h at 37 ° C f followed by 3 washes in TBS,
• An anti-SlOOB antibody (8D5 clone) as Fab 'and coupled to alkaline phosphatase is distributed (100 pL / well, concentration ranging from 0.2 to 1.2 mg / ml), incubated for 1 h at 37 ° C., followed by 3 washes in T8S, Finally, 100 ⁇ l / well of the substrate -nitropenyl phosphate are added, the reading of the colorimetric signal is carried out at 405 nm on microplate reader,
• the coupling of the antigen to the copolymer in the form of micelles can improve the detection sensitivity with respect to the free S100B system, unlike the same coupling on the copolymer in non ⁇ miceliaire (semi-organic conditions DMSO / water polymer initially dissolved in DMSO), or with respect to the posterior coupling of the antigen on a solid phase modified with the same or the same non -cellular copolymer.

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