METHOD FOR PRODUCING A CAPTURE PHASE FOR THE DETECTION OF A BIOLOGICAL TARGET, AND ASSOCIATED DETECTION METHODS AND KITS

Abstract

The invention provides a novel method of preparing a capture phase for detecting and/or quantifying a target biological entity, said capture phase including a biological ligand for the biological entity, said biological ligand being covalently bonded to an amphiphilic polymer and being immobilized on a solid support, the method being characterized in that the biological ligand is immobilized on the solid support by bringing the solid support into contact with a dispersion of micelles formed by a plurality of chains of the amphiphilic polymer, said micelles carrying a plurality of molecules of the biological ligand on the surface thereof. The invention also provides corresponding capture phases and associated detection methods and kits.

Claims

1. A method of preparing a capture phase for detecting and/or quantifying a target biological entity, said capture phase including a biological ligand for the biological entity, said biological ligand being covalently bonded to an amphiphilic polymer and being immobilized on a solid support, the method being characterized in that the biological ligand is immobilized on the solid support by bringing the solid support into contact with a dispersion of micelles formed by a plurality of chains of the amphiphilic polymer, said micelles carrying a plurality of molecules of the biological ligand on the surface thereof.

2. A preparation method according to claim 1, characterized in that the amphiphilic polymer has a hydrophobic portion oriented towards the core of the micelles and a hydrophilic portion at the surface of the micelles, the biological ligand being covalently coupled to the hydrophilic portion.

3. A method according to claim 1, characterized in that after immobilization, at least a portion of the polymer remains in the form of micelles, such that micelles formed by a plurality of amphiphilic polymer chains are immobilized at the surface of the support, said micelles carrying a plurality of molecules of the biological ligand on the surface thereof bonded with the amphiphilic polymer in a covalent manner.

4. A preparation method according to claim 1, characterized in that the immobilization is carried out in a solvent or solvent mixture constituted by at least 90% by weight, preferably at least 95% by weight, and more preferably at least 99% by weight of water.

5. A preparation method according to claim 1, characterized in that the micelles in the dispersion and/or the micelles finally immobilized on the support are formed by 100 to 5000 polymer chains and/or carry 10 to 500000 biological ligand molecules.

6. A preparation method according to claim 1, characterized in that it includes a step of covalent coupling between the biological ligand and the amphiphilic polymer, which step is carried out while the polymer is in the form of micelles, so as to form the micelles carrying a plurality of molecules of the biological ligand at the surface thereof.

7. A preparation method according to claim 6, characterized in that coupling is carried out in a solvent or solvent mixture constituted by at least 90% by weight, preferably at least 95% by weight, and more preferably at least 99% by weight of water.

8. A preparation method according to claim 5, characterized in that the coupling is carried out with a polymer concentration corresponding to at least 50 times, preferably to at least 200 times the critical micelle concentration of the polymer and/or with an amphiphilic polymer concentration at least ten times greater than that used when bringing the micelles into contact with the support.

9. A preparation method according to claim 1, characterized in that the amphiphilic polymer is a linear block polymer including at least one hydrophilic block and at least one hydrophobic block, the hydrophilic block being positioned at the surface of the micelles and carrying at least one molecule of the biological ligand by covalent bonding.

10. A preparation method according to claim 1, characterized in that the mean density of biological ligand molecules per polymer chain in the dispersion of micelles is from 0.1 to 100, and in particular from 1 to 100.

11. A preparation method according to claim 1, characterized in that the dispersion of micelles has a polydispersity index from 0 to 0.2 as determined by dynamic light scattering.

12. A preparation method according to claim 1, characterized in that the amphiphilic polymer has a molar mass greater than 5000 g/mol, preferably greater than 10000 g/mol.

13. A preparation method according to claim 1, characterized in that the amphiphilic polymer includes, or indeed is exclusively constituted by, a first linear block consisting in a hydrophobic homopolymer resulting from polymerizing a hydrophobic monomer A; and a second linear block consisting in a hydrophilic copolymer resulting from copolymerizing a monomer B carrying 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 in a covalent manner.

14. A preparation method according to claim 13, characterized in that the monomer A is selected from hydrophobic derivatives of methacrylate, acrylate, acrylamide, methacrylamide, and lactides, or from styrene and its derivatives; the monomer A is preferably n-butyl acrylate, tertiobutyl acrylate, tertiobutyl acrylamide, octadecyl acrylamide, lactide, lactide-co-glycolide, or styrene.

15. A preparation method according to claim 13, characterized in that the monomer B is selected from functional derivatives of acrylate, methacrylate, acrylamide or methacrylamide, and from functional styrene derivatives; the monomer B is preferably N-acryloxy succinimide, N-methyacryloxy succinimide, 2-hydroxyethyl methacrylate, 2-aminoethyl methacrylate, 2-hydroxyethyl acrylate, 2-aminoethyl acrylate, or 1,2:3,4-di-O-isopropylidene-6-O-acryloyl-D-galactopyranose.

16. A preparation method according to claim 13, characterized in that the monomer B carries a reactive function X selected from —NH.sub.2, —COOH, —OH, —SH, and from —C≡CH functions, ester, halogenocarboynyl, sulfhydryl, disulfide, hydrazine, hydrazone, azide, isocyanate, isothiocyanate, alkoxyamine, aldehyde, epoxy, nitrile, maleimide, halogenoalkyl, and maleimide groups, from functions that can be activated by anactivating agent such as carbodiimides, and in particular a carboxylic acid activated in the form of an ester of N-hydroxysuccinimide, pentachlorophenyl, trichlorophenyl, p-nitrophenyl, or carboxyphenyl, or indeed from bifunctional homo- or hetero-compounds.

17. A preparation method according to claim 13, characterized in that the monomer C is selected from hydrophilic derivatives of acrylamide, methacrylamide, N-vinylpyrrolidone, and oxyethylene; the monomer C is preferably N-vinylpyrrolidone or N-acryloyl morpholine.

18. A preparation method according to claim 13, characterized in that the first block has a molar mass between 1000 g/mol and 250000 g/mol.

19. A preparation method according to claim 13, characterized in that the second block has a molar mass greater than 1000 g/mol, and preferably greater than 2000 g/mol.

20. A preparation method according to claim 13, characterized in that the second block is a random copolymer with a composition, expressed as the ratio of the quantity of monomer C divided by the quantity of monomer B, the quantities being expressed in moles, which ratio is preferably in the range 1 to 10, more preferably in the range 1.5 to 4.

21. A method according to claim 1, characterized in that the biological ligand is an antigen, a hapten, or a protein.

22. A phase for capturing a target biological entity, the capture phase being characterized in that it comprises micelles immobilized on a solid support, said micelles being formed by a plurality of chains of an amphiphilic polymer, and said micelles carrying a plurality of molecules of at least one biological ligand for the target biological entity on the surface thereof, said molecules of the biological ligand being bonded to the chains of the amphiphilic polymer in a covalent manner.

23. A capture phase according to claim 22, characterized in that the micelles are immobilized on the solid support by adsorption.

24. A capture phase according to claim 23, characterized in that at least a portion of the micelles are immobilized on the solid support by adsorption by means of an interaction between the biological ligand and the solid support, a portion of the micelles optionally being immobilized on the solid support by adsorption by means of an interaction between the polymer and the solid support, the interactions involved possibly being electrostatic or ionic bonds or hydrophobic interactions, in particular.

25. A capture phase according to claim 22, characterized in that a portion of the biological ligands, corresponding in particular to at least 50% of the biological ligands present on the capture phase, is accessible and available for interacting and bonding with a target biological entity.

26. A capture phase according to claim 22, characterized in that the micelles immobilized on the support are formed by 100 to 5000 polymer chains and/or carry 10 to 500000 biological ligand molecules.

27. A capture phase according to claim 22, characterized in that the amphiphilic polymer is a linear block polymer including at least one hydrophilic block and at least one hydrophobic block, the hydrophilic block being positioned on the surface of the micelles, and carrying at least one molecule of the biological ligand by covalent bonding.

28. A capture phase according to claim 22, characterized in that the amphiphilic polymer has a molar mass greater than 5000 g/mol, preferably greater than 10000 g/mol.

29. A capture phase according to claim 22, characterized in that the amphiphilic polymer includes, or indeed is exclusively constituted by, a first linear block consisting in a hydrophobic polymer resulting from polymerizing a hydrophobic monomer A; and a second linear block consisting in a hydrophilic copolymer resulting from copolymerizing a monomer B carrying a reactive function X with a hydrophilic monomer C not carrying any reactive function, said second block being bonded to one end of the first block in a covalent manner.

30. A capture phase according to claim 26, characterized in that the amphiphilic polymer is as defined in claim 14.

31. A capture phase according to claim 22, characterized in that the biological ligand is an antigen, a hapten, or a protein.

32. A device for detecting and/or quantifying a target biological entity, the device comprising a capture phase according to claim 22, and at least one tracer for detection.

33. A device for detecting and/or quantifying a target biological entity, comprising a capture phase obtained by the method according to claim 1, and at least one tracer for detection.

34. A kit for detecting and/or quantifying a target biological entity, the kit comprising: a solid support; a dispersion in aqueous solution of micelles formed by chains of an amphiphilic polymer, carrying a plurality of molecules of at least one biological ligand for the target biological entity on the surface thereof, said biological ligand molecules being bonded to the chains of the amphiphilic polymer in a covalent manner; and at least one tracer for detection.

35. A method of detecting and/or quantifying a target biological entity in vitro in a biological sample, wherein: a capture phase according to claim 22 is provided; said biological sample is brought into contact with at least the capture phase; and said target biological entity fixed on the capture phase is detected and/or quantified after the biological entity has bonded with a biological ligand molecule covalently bonded to the chains of the amphiphilic polymer of the capture phase.

36. A method of detecting and/or quantifying a target biological entity in vitro in a biological sample, wherein: a capture phase obtained by the method according to claim 1 is provided; said biological sample is brought into contact with at least the capture phase as obtained in this manner; and said target biological entity fixed on the capture phase is detected and/or quantified after the biological entity has bonded with a biological ligand molecule covalently bonded to the chains of the amphiphilic polymer of the capture phase.

37. A method of detecting and/or quantifying a target biological entity in vitro in a biological sample, wherein: a capture phase is prepared by the method according to claim 1, said biological sample is brought into contact with at least the capture phase as prepared in this way; and said target biological entity fixed on the capture phase is detected and/or quantified after the biological entity has bonded with a biological ligand molecule covalently bonded to the chains of the amphiphilic polymer of the capture phase.

38. A detection method according to claim 35, characterized in that it is a direct method in which the sample that might contain the target biological entity is brought into contact with the capture phase and bonding between the biological ligand immobilized on the support and the target biological entity is revealed by the presence of a tracer.

39. A method according to claim 38, characterized in that the tracer is a biological ligand of the target biological entity coupled to a marker.

40. A detection method according to claim 35, characterized in that it is an indirect method in which the sample that might contain the target biological entity is brought into contact with the capture phase in the presence of an analog of the target biological entity, and the bonding between the biological ligand immobilized on the support and the target biological entity is revealed by the presence of a tracer, indirectly by detecting the bonding between the biological ligand immobilized on the support and the analog of the target biological entity.

41. A method according to claim 40, characterized in that the tracer is the analog of the target biological entity coupled to a marker.

42. A method according to claim 39, characterized in that the marker is selected from enzymes, chromophores, radioactive molecules, fluorescent molecules and electrochemiluminescent salts.

43. A method according to claim 41, characterized in that the marker is selected from enzymes, chromophores, radioactive molecules, fluorescent molecules and electrochemiluminescent salts.

Description

[0124] FIG. 1 is a diagrammatic representation of micelles carrying biological ligands, in a block type amphiphilic copolymer.

[0125] FIG. 2 is a diagrammatic representation of micelles carrying biological ligands immobilized on the surface of a support, which highlights the interactions between the micelles and the support: A) via the ligand present on the surface of the micelles; B) via an amphiphilic polymer (via a hydrophilic chain); C) by adsorption in the form of a unimer (via a hydrophobic/hydrophilic/or ligand portion).

[0126] FIG. 3 illustrates the preparation of micelles coupled with p24, used in the examples below.

[0127] FIG. 4 presents the results obtained with an ELISA test with free p24 (o) or p24 coupled with captured micelles (•) as well as with micelles alone (□) (under the same dilution conditions as with p24).

[0128] FIGS. 5A and 5B present the results obtained with the same format for the ELISA test as in FIG. 4, but varying the concentration of detection antibody, with immobilization carried out with a concentration of p24 of a):10 μg/mL (FIG. 5A), b): 1 μg/mL (FIG. 5B); graphs of the pure absorbance signal and signal-to-noise ratio as a function of the dilution of antibody (Ab).

[0129] FIGS. 6A and 6B present the influence of the coupling conditions (dilute or concentrated) of p24 on the increase in sensitivity using ELISA after immobilization carried out with a concentration of p24 of: a):10 μg/ml (FIG. 6A), b): 1 μg/ml (FIG. 6B); graphs of the pure absorbance signal and signal-to-noise ratio as a function of the dilution of antibody.

[0130] FIG. 7 demonstrates the stability of micelles-p24 evaluated using ELISA after immobilization carried out with a concentration of p24 of 1 μg/ml; graphs of the pure absorbance signal and signal-to-noise ratio as a function of the dilution of antibody.

[0131] FIG. 8 presents various conditions for immobilization of the antigen S100B on the solid support used below.

[0132] FIGS. 9A and 9B present the ELISA results for the various types of immobilization carried out in accordance with FIG. 8; graphs of the pure absorbance signal and signal-to-noise ratio as a function of the concentration of antibody (Mab).

EXAMPLES

[0133] A study pertaining to the use of micelles formed from polylactide-b-poly(N-vinylpyrrolidone-co-N-acryloxysuccinimide) copolymer (PLA-b-P(NAS-co-NVP)) (with respective molar masses of 19000 and 22000 g.Math.mol.sup.−1 for the PLA and P(NAS-co-NVP)) blocks was carried out. The micelles were prepared using the common solvent method (acetonitrile). Coupling of the proteins, acting as the biological ligand for the target antibody, was carried out via their lysine or N-terminal amine functions (see FIG. 3) with the reactive N-succinimidyl (NS) ester functions of the NAS units present on the crown of the micelles.

[0134] The effect of using micelle-protein conjugates on the increase in the sensitivity of the immuno-enzymatic tests in the capture phase was demonstrated:

[0135] 1. for 3 different sources of antigens:

[0136] a. recombinant p24, capsid protein of HIV;

[0137] b. troponin I cardiac complex I-T-C from Hytest;

[0138] c. native protein S100B bovine brain extract, from HyTest;

[0139] 2. On 2 immunoassay techniques:

[0140] a. manual ELISA technique;

[0141] b. automated VIDAS technique marketed by bioMérieux.

ELISA Study on the Detection of Anti-p24 Antibody

[0142] Coupling of P24 Antigen to Copolymer Micelles

Preparation of Micelles

[0143] The micelles were prepared using the common solvent method (or nanoprecipitation). The copolymer (20 mg) was dissolved in 2 mL of acetonitrile, then this solution was added to 4 mL of milli-Q water at a regular rate. The acetonitrile was evaporated off under reduced pressure. The micellar aqueous solution obtained was typically at a concentration of 5.2 mg.Math.mL.sup.−1 (precise determination by measuring the amount of solid after passage through the oven). The mean micellar size was 56 nm.

Coupling of the Protein (p24)

[0144] The protein was coupled to the micelles (PLA-b-P(NAS-co-NVP)) by adding a volume (typically 500 μL) of 5.2 mg/mL of micelle dispersion to the same volume of p24 in PBS, pH 7.4, at differing concentrations (0 mg.Math.mL.sup.−1 to 2.4 mg.Math.mL.sup.−1). The final coupling medium thus contained 2.6 mg.Math.mL.sup.−1 of micelles and the protein was at concentrations of 0 mg.Math.mL.sup.−1 to 1.2 mg.Math.mL.sup.−1. The samples were placed on a wheel in order to carry out stirring for 20 h at ambient temperature.

[0145] FIG. 3 illustrates a preparation of this type.

[0146] Characterization of Couplings

SDS PAGE

[0147] The SDS-PAGE analysis showed that, for an introduced quantity of 0.12 mg of p24 per mg of copolymer, the coupling was total. When this quantity was increased (from 0.24 mg/g to 0.48 mg/g), more and more free protein was detected, indicating a “saturation” of the surface of the micelles, and thus non-quantitative coupling. The condition selected thereafter was that corresponding to a quantitative coupling, i.e. 0.12 mg of p24 per mg of copolymer, i.e. 0.3 mg/mL of p24 and 2.6 mg/mL of copolymer.

Assay of the Residual Amine Functions Remaining on the Protein after Coupling (% Modification of Amines)

[0148] 90 μL of coupling medium was placed in a 96-well plate (black, NUNC) and 30 μL of 0.4 mg/mL fluorescamine (in DMSO) was added. After 20 minutes (away from the light), the fluorescence was read with a TECAN fluorimeter at an emission wavelength of 477 nm (excitation wavelength: 416 nm). The percentage of modified amines was determined by the ratio

100−(I.sub.fluo of coupling medium/I.sub.fluo of free protein*100).

[0149] Following coupling, the percentage of modified amines obtained was approximately 100%.

Hydrodynamic Diameter of p24 Micelles (DLS)

[0150] The hydrodynamic diameter of the micelles, diluted by 1/50 in a 1 mM solution of NaCl, was measured by dynamic light scattering (DLS) using a ZetasizerNano S90 instrument (Malvern, UK).

[0151] The hydrodynamic diameter of the micelles was 100 nm for the PBS coupling medium control (micelles without p24); the hydrolysis of the reactive ester functions of NS to carboxylates involves deploying hydrophilic chains), and 111 nm for the micelles that had coupled to the p24.

[0152] Table 1. The low polydispersity index (PI<0.05) indicates very homogeneous sizes, whether before or after coupling. The critical micelle concentration was determined by DLS and by fluorescence by means of the hydrophobic fluorophore Nile Red, as reported previously (Handké et al. Macromol. Biosci., 13, 1213-1220 (2013)). It was of the order of 10 μg/mL, with no significant difference between the micelles and the micelles-p24.

TABLE-US-00001 TABLE 1 Size of reference micelles (under coupling conditions of 2.6 mg/mL copolymer in PBS buffer but without p24) and micelles-p24. Hydrodynamic p24 (mg/mg CMC Micelle diameter (nm) PI copolymer) (μg/mL) Micelle-ref  99.8 ± 5.5 0.03 ± 0.01 — 12 ± 4 Micelle-p24 111.3 ± 5.1 0.04 ± 0.01 0.115 ± 0.005 10 ± 3

[0153] ELISA Protocol for the Detection of Anti-P24 Antibody

[0154] The coupled or free p24 was immobilized on the solid phase (Nunc MaxiSorp F microtitration plate) at different concentrations of PBS (cascade dilutions) for 12 hours at ambient temperature; passivation was carried out in 10% PBS—horse serum (HS); detection with a biotinylated anti-p24 antibody (rabbit) diluted in 10% PBS-Tween-HS, followed by adding streptavidin-peroxidase (horseradish peroxidase, HRP) in 10% PBS-Tween-HS 10%, and revealing with 3,3′,5,5′-tetramethylbenzidine (TMB) (absorbance at 450 nm); free p24 and single micelle controls were systematically prepared under the same conditions as those for coupling, but in the respective absence of micelles and of p24.

[0155] ELISA Results

[0156] The results obtained are represented in FIGS. 4 and 5 and demonstrate a substantial increase in the antibody detection signal compared with free p24. Furthermore, the copolymer tested in the absence of p24 at different dilutions did not involve significant background noise. This increase in sensitivity was confirmed by working with an immobilization carried out with a concentration of p24 (coupled or free) of 10 μg/mL or 1 μg/mL and varying the concentration of detection antibody.

[0157] Influence of Coupling Conditions on the Gain in Sensitivity for ELISA

[0158] The study below shows that the micellar state (i.e. nanoscale object of approximately 100 nm) was indispensable during coupling of the protein in order to increase the sensitivity of the diagnostic test (i.e. to allow the protein to become oriented towards the liquid phase by means of preferential coupling towards the end of the hydrophilic block).

A. Coupling in Dilute/Concentrated Medium

[0159] “Concentrated” coupling: coupling of the protein under the above conditions (2.6 mg.Math.mL.sup.−1 of micelles and 0.3 mg.Math.mL.sup.−1 of p24 in PBS). As demonstrated by SDS-PAGE, coupling was total under these conditions and the micelles-p24 were 111 nm (PI=0.04) in size. The coupling medium was then diluted in PBS, in order to obtain concentrations of p24 of 10 μg.Math.mL.sup.−1 (88 μg.Math.mL.sup.−1 of micelle) or 1 μg.Math.mL.sup.−1 (8.8 μg.Math.mL.sup.−1 of micelle) for immobilization on the ELISA plate. [0160] “Dilute” coupling: coupling of the p24 was carried out directly under the conditions used for immobilization on the ELISA plate, i.e.: [0161] 10 μg.Math.mL.sup.−1 of p24 and 88 μg.Math.mL.sup.−1 of micelle in PBS; for this purpose, 100 μL of 0.6 mg.Math.mL.sup.−1 p24 and 100 μL of 5.2 mg.Math.mL.sup.−1 micelle were added to 5.8 mL of PBS and the solution was stirred on a rotating wheel for 20 h. [0162] 1 μg.Math.mL.sup.−1 of p24 and 8.8 μg.Math.mL.sup.−1 of micelle in PBS; for this purpose, 10 μL of 0.6 mg.Math.mL.sup.−1 p24 and 10 μL of 5.2 mg.Math.mL.sup.−1 micelle were added to 5.98 mL of PBS and the solution was stirred on a rotating wheel for 20 h.

[0163] As can be seen in Table 2, the hydrodynamic diameters obtained for the various coupling media (direct measurement on 1 μg.Math.mL and 10 μg.Math.mL of p24 concentrations of coupling media) showed that the micellar state was not retained in “dilute” coupling, as indicated by the sizes of the net increase and the very high polydispersity indices, which are indicative of aggregation phenomena, in contrast to media obtained from coupling p24 under concentrated conditions. This may be explained by the fact that coupling of the p24 occurs under conditions relatively close to the CMC of the copolymer (88 μg/mL and 8.8 μg/mL of copolymer respectively for the couplings at 10 μg/mL and 1 μg/mL of p24, the CMC being 10 μg/mL). Hence, the p24, by coupling, further accentuates the capacity of the copolymer, which is already high (because of its concentration close to the CMC) to leave the micelle and form unimers. Since the micelles have been destabilized, this may be followed by a reorganization of the system with aggregation processes that are controlled to a greater or lesser extent. It should be noted that for micelles placed under the same coupling conditions but in the absence of p24, the sizes observed by DLS were 91 nm (PI=0.05) for the concentration of 88 μg/mL, and 77 nm (PI=0.3) for the concentration of 8.8 μg/mL (indicating a weakened micellar state at concentrations close to the CMC, because the standard size of the micelles of copolymer alone is 100 nm with PI=0.03, Table 1). Thus, a priori, the presence of p24 for coupling under these conditions is indeed what accentuates the micelle destabilization process.

[0164] These dilute medium coupling conditions, resulting in a “non-micellar” medium, lead to a drop in sensitivity in ELISA compared with free p24 (FIG. 6), while a significant gain was confirmed for the standard conditions (concentrated state coupling), which maintained the micellar state.

TABLE-US-00002 TABLE 2 Sizes obtained for various coupling media (measured directly on coupling media with 1 μg .Math. ml.sup.−1 and 10 μg .Math. ml.sup.−1 of p24) Coupling [p24] Hydrodynamic type (μg .Math. mL.sup.−1) diameter (nm) PI Concentrated 10 105 0.13 Concentrated 1 100 0.2 Diluted 10 200 0.5 Diluted 1 210 0.5

Conclusion:

[0165] These studies show that in order to have a significant gain in sensitivity in ELISA, it is vital to carry out coupling of the protein to the copolymer organized into the form of micelles. Thus, it is necessary to react the p24 with the micelles in an aqueous medium with a copolymer concentration substantially higher than the CMC in order to preserve the micellar state.

[0166] Stability Study

[0167] The micelles-p24 (0.3 mg/mL of p24) were stored at 4° C. for 1 month. By comparison with the free p24 control, more appropriately at an optimized sensitivity (p24 from supplier stated to be 2.4 mg/mL, stored at −20° C.), the micelles-p24 retained their superiority in terms of sensitivity (FIG. 7).

VIDAS Study on the Detection of Anti-Troponin I Antibody

[0168] The same PLA-b-P(NAS-co-NVP) copolymer as before was used to couple TnI (ITC) with a view to detecting anti-TnI antibody on an automated VIDAS® immunoassay instrument marketed by bioMérieux.

[0169] The TnI protein was coupled onto micelles of PLA-b-P(NAS-co-NVP) in PBS at a concentration of 0.137 mg/mL of TnI and 0.868 mg/mL of micelles (0.158 mg of TnI per mg of copolymer). The coupling was analyzed by SDS-PAGE gel, which showed that coupling to the micelles appeared to be almost total because free TnI was not detected. The TnI coupled thereby to the micelles was tested using VIDAS®(bioMérieux) and compared with micelles alone on the solid phase.

[0170] The automated VIDAS test was composed of 2 elements:

[0171] 1—the cartridge was a plastic bar containing 10 wells sealed with an aluminum film into which the various solutions were distributed;

[0172] 2—the cone, termed the SPR (Solid Phase Receptacle), acted as the pipetting system and the solid phase. Each reagent of the cartridge was aspirated then discharged via the cone. Either free TnI (ITC) in an amount of 0.03 μg/mL, or the micelles alone in an amount of 0.190 μg/mL, or the micelles-TnI in an amount of 0.03 μg/mL of TnI and 0.190 μg/mL of copolymer, in a volume of 300 μL, were immobilized on the cones.

[0173] At the end of the immobilization step, which was carried out at ambient temperature over 12 hours, the cones were emptied then brought into contact with the passivation buffer (Tris 0.2 M buffer, pH 6.2) containing a protein or peptide type saturation agent. The cones were then dried and stored at +4° C. until use.

[0174] Next, the three prepared capture phases were compared by reacting them with a tracer that was a mixture of two anti-TnI monoclonal antibodies (clones 16A11 and 7B9 marketed by HYTEST, Sweden). For practical reasons, this antibody was directly coupled to the enzyme alkaline phosphatase, in order to reduce the number of steps of the immunological reaction and reduce the duration (DEX2 protocol from VIDAS®, total duration approximately 40 min). The concentration at which this tracer was used was 0.14 μg/mL in a volume of 400 μL. The signal was generated by adding the substrate 4-methylumbelliferyl phosphate; the enzyme of the conjugate catalyzes the hydrolysis reaction of this substrate to form 4-methylumbelliferone; the fluorescence it emitted was measured at 450 nm.

[0175] Table 3 summarizes the results obtained using VIDAS® on TnI coupled to the polymer in the form of micelles and not coupled.

[0176] The signal-to-noise ratio was improved when the TnI was coupled to the polymer.

TABLE-US-00003 TABLE 3 VIDAS results for TnI coupled to polymer compared with non-coupled TnI. Result SPR control 0 SPR TnI free 0.03 μg/ml 72 Signal/noise ratio 72 SPR micelles alone 11 0.190 μg/ml of copolymer SPR micelle-TnI, 1156 0.03 μg/ml of TnI, 0.190 μg/ml of copolymer Signal/noise ratio 105

Conclusion

[0177] Coupling of the antigen to the copolymer, organized in the form of micelles, can be used to improve the detection sensitivity.

ELISA Study on the Detection of Anti-S100B Antibody

[0178] The aim of this study was to demonstrate the necessity of using micelles of copolymer carrying antigen in order to increase the sensitivity of diagnostic tests for immobilization of the antigen.

[0179] The antigen retained for this study was the protein S100B (native antigen, bovine brain extract, HyTest).

[0180] The polymer was PLA-b-P(NAS-co-NVP), the same as in the preceding examples. It was used either in a micellar form (in dispersion in 100% aqueous buffer), or dissolved in DMSO (“copolymer” form) for coupling with the antigen S100B in a 5% DMSO-95% aqueous buffer medium. These two protocols, implemented in parallel, were carried out in order to evaluate the influence of the coupling conditions, i.e. coupling in 100% aqueous medium with micelles versus coupling in a semi-organic DMSO/water medium with the copolymer initially dissolved in DMSO, on the gain in sensitivity of the ELISA test. The condition for protein/copolymer coupling in DMSO/aqueous buffer medium has routinely been reported in the literature (Allard et al., Bioconjugate Chem. 2001, 12, 972-979, etc.).

[0181] The polymer-antigen couplings were carried out either in solution in an Eppendorf tube or on an ELISA microplate after immobilization of the micelle or the copolymer.

[0182] Carrying Out the Couplings

[0183] A. Preparation of Micelle-S100B Conjugates

[0184] The micelles of copolymer PLA-b-P(NAS-co-NVP) were prepared as previously reported in the section “ELISA study on the detection of anti-p24 antibody”. They were initially at a concentration of 5.21 mg/mL, with a size (before coupling) of 56 nm and a PI=0.1.

[0185] The solution of S100B protein was diluted to a concentration of 0.6 mg/mL in PBS 1×.

[0186] The coupling protocol, identical to that carried out for the protein p24, is described in Table 4 below.

TABLE-US-00004 TABLE 4 Vol, in Vol, in Vol, μL, of μL, of [S100] [copo] PBS 0.6 g/L S100 micelles (mg/mL) (mg/mL) micelles-S100 0 150 150 0.3 2.605 micelles alone ref 150 0 150 0 2.605
Incubation for 18 hours at ambient temperature.

[0187] B. Preparation of Polymer-S100B Conjugates in DMSO

[0188] The coupling protocol is described in Table 5 below.

TABLE-US-00005 TABLE 5 Vol, in Vol, in Vol, in Vol, μL, of μL, of μL, of [S100] [copo] PBS 0.6 g/L S100 DMSO copo sol (mg/mL) (mg/mL) copo- 135 150 0 15 0.3 2.605 S100 S100 135 150 15 0 0.3 0 ref copo ref 285 0 0 15 0 2.605
Incubation for 18 hours at ambient temperature.

[0189] Characterization of Couplings

Assay of Residual Amine Functions Remaining on Protein after Coupling (% Modification of Amines)

[0190] The assay was carried out as reported previously for the protein p24. Following coupling, the percentage of modified amines was approximately 100%.

[0191] S100B ELISA

[0192] 5 different conditions, shown diagrammatically in FIG. 8, were evaluated:

[0193] 1. The reference S100B immobilized directly on the microtitration plate (S100B ref);

[0194] 2. The copolymers in the form of micelles alone immobilized on the plate in a first step (micelles alone ref), then plate coupling, on immobilized micelles, of the S100B protein (micelles then S100);

[0195] 3. The copolymers in 5% DMSO alone, non-micellar, immobilized on the plate in a first step (copo ref) then plate coupling, onto immobilized copolymer, of the protein S100B (copolymer then S100);

[0196] 4. The immobilization of micelle-S100B conjugates carried out in an Eppendorf tube (micelles+S100); and

[0197] 5. The immobilization of polymer in 5% DMSO+S100B conjugates carried out in an Eppendorf tube (copolymer+S100).

Carrying Out the ELISA

[0198] 100 μL/well of single micelles (1st step condition 2) or of copolymer alone (1st step condition 3) diluted in 74 μg/mL of water were distributed into a 96-well microplate (Nunc Maxisorp F96). The microplate was incubated for 12 h at ambient temperature with stirring, in order to obtain adsorption, then emptied. In order to carry out couplings under dilute conditions, 100 μL/well of a 5 μg/mL solution of S100B was added directly to the microplate into the wells that had been incubated with the micelles alone or the copolymer alone. Furthermore, 100 μL/well of S100B in an amount of 5 μg/mL (condition 1, S100B reference) or of micelles-S100B conjugate in an amount of 74 μg/mL of micelles and 5 μg/ml of S100B (condition 4), or indeed of copolymer-S100B conjugate in an amount of 74 μg/mL of non-micellar copolymer and 5 μg/mL of S100B (condition 5) were distributed into the empty wells. The microplate was incubated for 12 additional hours at ambient temperature, with stirring, in order to obtain adsorption (conditions 1, 4, 5) or coupling (conditions 2, 3). The microplate was emptied; next, three TBS (Tris buffered saline)-Tween®-20 0.05% washes were carried out. The wells were saturated by adding 300 μL/well of passivation buffer (0.2 M Tris buffer, pH 6.2) containing a protein or peptide type saturation agent. The microplate was incubated for 1 h at 37° C., followed by 3 washes with TBS. An anti-S100B antibody (clone 8D5) in the form of Fab′ and coupled with alkaline phosphatase was distributed (100 μL/well, concentration from 0.2 μg/mL to 1.2 μg/mL), incubated for 1 hour at 37° C., followed by washing 3 times with TBS. Finally, 100 μL/well of the substrate p-nitrophenyl phosphate was added. The colorimetric signal was read at 405 nm on a microplate reader.

[0199] FIG. 9A represents the ELISA results for the various types of immobilization that were carried out. Immobilization of a micelles+S100B conjugate provided more signal than the immobilized S100B alone without increasing the background noise. The signal-to-noise ratio is thus improved when micelles are used to immobilize the protein S100B (FIG. 9B). The immobilization of polymer+S100B obtained from coupling in a DMSO/water medium, though, only induced a very low sensitivity compared with the free S100B. Finally, the fact of coupling the protein at another time to the micelles (100% aqueous buffer) or the copolymer (DMSO/aqueous buffer medium) already immobilized on the solid phase did not improve the sensitivity compared with free S100B.

Conclusion

[0200] Coupling the antigen to the copolymer in the form of micelles can improve the sensitivity of detection compared with a free S100B system, in contrast to the same coupling on the copolymer in the non-micellar form (semi-organic DMSO/water conditions, polymer initially dissolved in DMSO), or compared with prior coupling of the antigen to a solid phase modified by the micelles or the same, but non-micellar, copolymer.