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WO2009066275A1 - Procédé d'immobilisation de molécules biologiques sur un support et produits de celui-ci - Google Patents

Procédé d'immobilisation de molécules biologiques sur un support et produits de celui-ci Download PDF

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Publication number
WO2009066275A1
WO2009066275A1 PCT/IE2008/000112 IE2008000112W WO2009066275A1 WO 2009066275 A1 WO2009066275 A1 WO 2009066275A1 IE 2008000112 W IE2008000112 W IE 2008000112W WO 2009066275 A1 WO2009066275 A1 WO 2009066275A1
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Prior art keywords
protein
support
substrate
binding
biomolecule
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Kumar Vashist Sandeep
Holthofer Harry
Leister Kirk
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Dublin City University
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Dublin City University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—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

Definitions

  • This invention relates to a method of immobilising biological molecules to a support and products thereof.
  • the method relates to immobilising biological molecules to a support through covalent binding.
  • affinity chromatography exploits the specific binding of a protein such as an enzyme for its ligand such as an inhibitor of the enzyme.
  • the ligand can be immobilised on an insoluble support and packed into a chromatography column, a mixture of biological molecules including the enzyme of interest is applied to the column and as only the enzyme has a specific binding affinity for the ligand, the enzyme becomes bound to the column while the remaining biological molecules pass through the column.
  • the enzyme can then be eluted from the column in a highly pure form.
  • Biological molecules immobilised on a support have wide ranging applications in fields such as scientific research, diagnostics, drug discovery, clinical trials and the like.
  • antibodies immobilised on solid supports such as, microtitre plates or affinity chromatography supports are well known and companies such as BIACORE®, Sigma Aldrich® and Biomat® S.N.C produce chemical or protein functionalised generic chips for antibody immobilization that can be utilised in techniques such as high throughput immunoassays, antibody screening, immunodiagnostics and the like.
  • a large majority of biological molecules are immobilised on solid supports using a simple adsorption process. However, this can result in a non-homogenous layer of absorbed biological molecules being formed on the surface of a support. A non-homogeneous layer of an absorbed biological molecule will introduce errors and non-reproducibility of results into experiments.
  • the simple adsorption of antibodies onto a support can result in the antibodies being immobilised in a random orientation which can result in a loss of specific binding activity of the immobilised antibodies.
  • F c binding proteins display high affinity for the constant fragment (F c ) region of antibodies and have been used to bind antibodies in an oriented and site directed manner. F c binding proteins exhibit a strong affinity towards gold and bind to polystyrene by adsorption. Many companies including Biomat® S.N.C have exploited the simple adsorption of F c binding proteins to polystyrene to make functionalised F c binding protein platforms for immunoassays. But the simple adsorption based immobilisation has the disadvantage that the functional activity of the F c binding protein degenerates with time as it is not very stable.
  • the invention provides a support comprising a substrate wherein a surface of the substrate comprises functional amino or aldehyde groups for covalent binding to a biomolecule.
  • the substrate may be selected from one or more of: polystyrene, gold, polylysine, silicon and silicon derivatives, porous silicon, polysilicon, quartz, Zeonex, Zeonor, glass, polymethylmethacrylate (PMMA), polycarbonate, cellulose acetate, agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2- hydro-ethyl-methacrylate (HEMA).
  • PMMA polymethylmethacrylate
  • PMMA polycarbonate
  • cellulose acetate agarose
  • sepharose agarose
  • cellulose polyacrylamide
  • trisacryl sepacryl
  • ultragel AcA azlactone
  • methacrylamide polymer Eupergit
  • HEMA 2- hydro-ethyl-methacrylate
  • the protein may be selected from one or more of: an F c binding protein, an antibody, a lectin, a chaperone protein, an adhesion protein and a ligand.
  • the protein may be an affinity protein.
  • the biomolecule may be a protein
  • the F c binding protein may be selected from one or more of: Protein A/G, Protein A and Protein G.
  • the support may further comprise an antibody bound to the F c binding protein.
  • the antibody may be one or more selected from: anti-HBsAg, anti -troponin I, anti-troponin T, anti-myoglobin, anti-PSA (prostate specific antigen), anti- creatine kinase (CK-MB), anti-insulin, anti-PAP (prostate acid phosphatase), anti-hCG (human chorionic gonadotropin), anti-leptin, anti-Legionella antigen, anti-Rotavirus, anti-verotoxin, anti-adenovirus, anti-Pf LDH, anti-digoxin, anti- b2 -microglobulin, anti-T3, anti-T4, anti-TSH, anti-C reactive peptide (CRP) and anti-C peptide.
  • anti-HBsAg anti -troponin I, anti-troponin T, anti-myoglobin, anti-PSA (prostate specific antigen), anti- creatine kinase (CK-MB), anti-insulin, anti-
  • the invention further provides for an antibody functionalised biochip comprising a support substrate and an antibody attached to an Fc binding protein wherein the amino or carboxyl group of the Fc binding protein is covalently attached to a surface of the substrate.
  • the Fc binding protein may be covalently attached to a surface of the substrate through the carboxyl group.
  • the invention further provides a method of covalently binding a biomolecule to a support substrate comprising the steps of: (a) providing a support substrate;
  • the biomolecule may be covalently bound to the support substrate through a carboxyl group.
  • the method may further comprise the step of cross linking the induced amine groups with glutaraldehyde prior to step (e) to form aldehyde groups.
  • the method may further comprise the step of:
  • the biomolecule may be covalently bound to the support substrate through an amino group.
  • the substrate may be selected from one or more of: polystyrene, gold, polylysine, silicon and silicon derivatives, porous silicon, polysilicon, quartz, Zeonex, Zeonor, glass, polymethylmethacrylate (PMMA), polycarbonate, cellulose acetate, agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2- hydro-ethyl-methacrylate (HEMA).
  • PMMA polymethylmethacrylate
  • PMMA polycarbonate
  • cellulose acetate agarose
  • sepharose agarose
  • cellulose polyacrylamide
  • trisacryl sepacryl
  • ultragel AcA azlactone
  • methacrylamide polymer Eupergit
  • HEMA 2- hydro-ethyl-methacrylate
  • the biomolecule may be a protein.
  • the protein may be an affinity protein.
  • the protein may be selected from one or more of; an Fc binding protein, an antibody, a lectin, a chaperone protein, an adhesion protein and a ligand.
  • the F c binding protein may be selected from one or more of: Protein A/G, Protein
  • the method may further comprise the step of binding an antibody to the F c binding protein.
  • the antibody may be one or more selected from: anti-HBsAg, anti-troponin I, anti-troponin T, anti-myoglobin, anti-PSA (prostate specific antigen), anti- creatine kinase (CK-MB), anti-insulin, anti-PAP (prostate acid phosphatase), anti-hCG (human chorionic gonadotropin), anti-leptin, anti-Legionella antigen, anti-Rotavirus, anti-verotoxin, anti-adenovirus, anti-Pf LDH, anti-digoxin, anti- b2-microglobulin, anti-T3, anti-T4, anti-TSH, anti-C reactive peptide (CRP) and anti-C peptide.
  • anti-HBsAg anti-troponin I
  • anti-troponin T anti-myoglobin
  • anti-PSA prostate specific antigen
  • anti-CK-MB anti-insulin
  • anti-PAP prostate acid phosphatase
  • the step of creating hydroxyl groups on the support may comprise the steps of: - treating the substrate with a basic solution;
  • the basic solution may be a solution of potassium hydroxide or sodium hydroxide.
  • the step of inducing amines on the support substrate may comprise the steps of: incubating the hydroxyl group functionalised support with an amine solution; and drying the support;
  • the amine solution may be selected from: 3-aminopropyl triethoxy silane or 3- aminopropyl trimethoxy silane.
  • the support may be dried in a vacuumed desiccator placed in an oven.
  • the step of covalently binding a biomolecule to the support substrate may comprise the steps of: forming a cross-linking solution; mixing the biomolecule with the cross-linking solution; incubating the support with the biomolecule - cross-linking solution mixture; and quenching the cross-linking solution.
  • the cross-linking solution may comprise 1 -ethyl 3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC) and Sulfo-N-Hydroxysuccinmide.
  • the quencher may be 2-mercaptoethanol.
  • the step of binding an antibody to the F c binding protein may comprise the steps of: blocking non-specific protein binding sites on the base substrate; and incubating the support with an antibody solution.
  • the non-specific protein binding sites may be blocked with a bovine serum albumin solution, such as a 1% solution of bovine serum albumin.
  • the invention further provides isolation and purification comprising an affinity biomolecule covalently bound to a surface of the support wherein the affinity biomolecule is bound to the support through a terminal amino or carboxyl group.
  • the support may be selected from one or more of: agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2-hydro-ethyl-methacrylate (HEMA).
  • the support may be in the form of a chromatography column.
  • the affinity biomolecule may be a protein.
  • the protein may be selected from one or more of: an Fc binding protein, an antibody, a lectin, a chaperone protein, an adhesion protein, and a ligand.
  • o Covalent binding (cross-linking) of biological molecules to a surface of a substrate provides a very strong linkage of the biological molecule to the substrate;
  • o Biological molecules can be bound to a wide range of supports including supports with an inert or a reactive surface; o Biological molecules can be bound to optically transparent supports; o Functional amino or aldehyde groups are induced on the surface of a support itself; o Antibodies are bound strongly to a surface of a substrate without any modification to the functionality of the antibody; and o Oriented and site directed attachment of antibodies to a support when using an F c binding protein as the variable F ab region is free to bind antigens.
  • Fig. 1 is a schematic of a method for immobilising antibodies to a support substrate via an F c binding protein. This immobilisation strategy is based on the covalent crosslinking of a carboxyl group of an F c binding protein to the solid substrate.
  • Fig. 2 is a schematic of an alternative method for immobilising antibodies to a support substrate via an F c binding protein. This strategy is based on the covalent crosslinking of an amino group of an F c binding protein to the solid substrate;
  • Fig. 3 is a schematic of a fluorescence detection system in which SF is a spatial filter assembly, M1-M3 are planar mirrors, CL is a collimating lens, DC is a dichroic beam splitter, and FL is a focusing lens system;
  • Fig. 4A is a series of fluorescent images showing the decrease in fluorescent intensity when decreasing concentrations of goat anti-mouse IgG-Cy5 labelled antibody were detected by mouse IgG bound to Zeonex® substrate using the
  • EDC-sulfo NHS and protein A based immobilisation strategy The concentration of antibody in ⁇ g/ml is given at the top of each image. The exposure time was 10 seconds for all concentrations.
  • Fig. 4B shows the qualitative assay curve between the captured fluorescent signal intensity and the respective concentration of goat anti-mouse IgG-Cy5 labelled antibody;
  • Fig. 5 is the SPR response curve demonstrating the: binding of mouse IgG to protein A which has been covalently bound through carboxyl groups to a Zeonex® coated surface of a gold SPR chip; blocking excess mouse IgG binding sites on the protein A coated substrate with 1% BSA; binding of goat anti-mouse IgG to mouse IgG; and regeneration of the protein A coated surface by 30 mM HCl;
  • Fig. 6 A to F show the surface view as assessed by Atomic Force Microscopy
  • Fig 6A is the AFM image (of view size, 5 x 5 ⁇ m) of the surface of a blank Zeonex® substrate after surface cleaning step (Step (b));
  • Fig 6B is the AFM image (5 x 5 ⁇ m) of the surface after 3-APTES treatment (Step (d));
  • Fig 6C is the AFM image (5 x 5 ⁇ m) of the surface of Protein A covalently bound to 3-APTES by EDC and sulfo NHS through carboxy groups (Step (e));
  • Fig 6D is the AFM image (5 x 5 ⁇ m) of the surface of Mouse IgG bound to Protein A;
  • Fig 6E is the AFM image (2 x 2 ⁇ m) of the surface of Mouse IgG bound to Protein A; and
  • Fig 6F is the 3-dimensional view of the mouse IgG bound surface (5 x 5 ⁇ m) analyzed by AFM
  • Fig. 7 A and B are graphs showing data for mouse IgG - anti mouse IgG assays when mouse IgG was immobilised via F c binding proteins on a 96-well polystyrene ELISA plate.
  • Fig. 7A shows the results of an assay when the F c binding proteins have been covalent bound via their carboxyl groups
  • Fig. 7B shows the results of an assay when the F c binding proteins have been covalently bound via their amino groups
  • Fig. 8 is a graph showing the results of an assay when an antibody (mouse IgG) has been directly bound covalently to a surface of a substrate via its carboxyl group (EDC-SNHS) or amino group (Glutaraldehyde).
  • the invention provides a method which overcomes the need for gold coating of substrates in order to covalently attach biological molecules to a surface of a support.
  • the invention provides a method for covalent binding of biological molecules to a surface of a support through an NH 2 or COOH group of the biological molecules.
  • the methods described herein can be used for immobilising proteins to a surface of a support. As almost all proteins have a carboxyl terminal having a COOH group and an amino terminal having an NH 2 group, any of these proteins can be covalently bound to a functionalised surface of a support.
  • biomolecules also have a carboxyl terminal having a COOH group and/or an amino terminal having an NH 2 group, and thus they can also be covalently bound to a functionalised surface of a support.
  • the method of the invention can be used for immobilising a variety of biological molecules to a support.
  • the antibodies can be directly or indirectly (via an F c binding protein) bound to a support.
  • the method provides an immobilisation strategy for covalently binding biological molecules to a surface of commercially relevant substrates (supports) via amine or aldehyde functional isation of the supports.
  • a wide range of supports including supports having an inert or reactive surface, can be functionalised using the methods described herein.
  • biological molecules can be covalently bound to a wide range of commercial substrates via their amino or carboxyl groups.
  • the immobilistation methods described herein allow for biological molecules to be covalently bound onto a surface of an optically transparent substrate.
  • the method exploits the widely known benefits of oriented and site directed immobilization of antibodies using F c binding proteins along with the additional benefit of long-term stability provided by covalent immobilization of F c binding proteins to a substrate.
  • F c binding proteins for example protein A/G, protein A or protein G, orientate the immobilized antibodies.
  • the F c binding proteins bind to the constant F c region of antibodies thereby keeping the antigen binding sites on the variable F ab region of the antibodies free to bind to antigens.
  • Protein A/G is a recombinant protein made commercially by gene fusion of the F c -binding domains of protein A and protein G. Protein A/G binds with strong affinity to nearly all the antibodies being employed widely for immunodiagnostics and biosensors (such as all human IgG subclasses, IgA, IgE, IgM and to a lesser extent IgD) at pH 5-8.
  • affinity biomolecules such as affinity proteins for example lectins, chaperones, adhesion proteins, ligands for enzymes, antibodies, Fc binding proteins and the like can be immobilised on a support.
  • the biomolecule immobilised support can be used for isolation or purification methods. For example, downstream purification of a biological molecule using various suface affinities with improved orientation or immobilisation strategies to achieve increased capacity, selectivity and specificity.
  • biomolecules can be immobilised to a variety of chromatographic supports for example: agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2-hydro-ethyl-methacrylate (HEMA) and the like.
  • chromatographic supports for example: agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2-hydro-ethyl-methacrylate (HEMA) and the like.
  • affinity chromatography and immuno-affinity chromatography applications the biomolecule immobilised support can be packed in a chromatography column.
  • F c binding proteins i.e. Protein A, protein G and protein A/G based columns have been used industrially in the biological, bioprocess, biopharmaceutical and diagnostics industries as well as in research laboratories for the purification of antibodies and proteins due to their ability to specifically select the antibody or protein of interest.
  • Fc binding protein based columns such as Nab Protein A protein columns, Nab Protein G protein columns and Nab Protein A/G protein columns (by Thermo Scientific previously Pierce); Nunc ⁇ ProPur® Antibody Purification Spin Columns based on Protein A and Protein G (by Fisher Scientific); EZview Red Protein A gel and EZview
  • F c binding proteins can be immobilised on the surface of a variety of substrate supports (including both reactive and inert supports) by inducing amine or aldehyde groups on the surface of the support.
  • the support could be resin supports such as conventional chromatographic resins for example agarose/sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, eupergit (methacrylamide polymer), HEMA (2-hydroethyl methacrylate) being used in the form of a chromatography column. Chromatography columns made from Fc immobilised supports of this type can be used for biosensor, bioprocesses and bioanalytical applications. Further, the amino or carboxyl terminal of any particular IgG type antibody can be bound to the substrate supports directly or indirectly using intermediate proteins i.e. Fc binding proteins.
  • biomolecule is covalently bound to the surface of a support. Immobilisation of biomolecules by covalent binding will result in a homogenous layer of bound biomolecules as each functional group (EDC-SNHS or glutaraldehyde) induced on the surface of the support will form a covalent bond with one terminal NH 2 or COOH group of each biomolecule therefore the induced functional groups will bind to the biomolecules in a 1 : 1 ratio, thereby eliminating the aggregation of biomolecules on the surface. As such, a reproducible, homogeneous layer of biomolecules are immobilised on the surface of the support.
  • EDC-SNHS or glutaraldehyde glutaraldehyde
  • these supports are particularly well suited for use in industrial scale applications, in which large volumes of solutions will be applied to the supports, as the biomolecule will remain chemically tethered to the support for the duration of the protocol.
  • the supports Given the strong covalent bond formed between the surface of the support and the biomolecule, the supports can be reused as the biomolecule does not leach from the surface of the support.
  • the method involves cleaning a bioanalytical surface of a support, chemically creating hydroxyl groups on the cleaned surface and thereafter, inducing amine groups on the surface for example by employing 3-aminopropyl triethoxy silane (3-APTES) or 3-aminopropyl trimethoxy silane.
  • 3-APTES 3-aminopropyl triethoxy silane
  • 3-aminopropyl trimethoxy silane 3-aminopropyl trimethoxy silane
  • Example 1 Immobilising biomolecules to a surface of a substrate
  • the invention relates to a method of covalently immobilising biomolecules such as proteins to a surface of support via their terminal amino or carboxy groups.
  • the method comprises the following steps:
  • the support substrate can be selected from any known support suitable for use with biomolecules.
  • the support may have a reactive or an inert surface.
  • the support may be optically opaque.
  • the support may be selected from: polystyrene, gold, polylysine, silicon and silicon derivatives, porous silicon, polysilicon, quartz, Zeonex, Zeonor, glass, polymethylmethacrylate (PMMA), polycarbonate, cellulose acetate, chromatography resins such as agarose, sepharose, cellulose, polyacrylamide, trisacryl, sepacryl, ultragel AcA, azlactone, methacrylamide polymer (Eupergit) and 2-hydro-ethyl-methacrylate (HEMA).
  • the choice of support may depend on the end use application for the support.
  • the surface cleaning procedure varies based on the substrate to be used.
  • Inert and/or polymeric supports Treat support with absolute ethanol for at least 10 min and rinse five times with deionized water.
  • the polylysine coated supports already have amine groups present on their surface. So in this case, there is no need for functionalising the surface of the support (step (c)) or for APTES treatment to induce amine groups (step (d)).
  • step (c) the surface is cleaned by treatment with absolute ethanol for about ten minutes.
  • amine groups on polylysine are activated by treatment with 0.1 M 2-[N-morpholino] ethane sulfonic acid (MES) buffer, pH 4.7 for at least 5 min. Biomolecules can then be covalently bound to the surface
  • MES 2-[N-morpholino] ethane sulfonic acid
  • the glass substrate was treated for 1 h in boiling piranha solution (70% sulphuric acid (H 2 SO 4 ): 30% hydrogen peroxide (H 2 O 2 ) (at a v/v ratio of 3:1) respectively).
  • the glass substrates were removed from the cleaning solution, rinsed with high purity water (Ultrapure Milli-Q Reagent Water System Millipore), then washed in ethanol three times followed by washing in high purity water five times. Finally, the cleaned glass substrates are dried in a stream of nitrogen.
  • the bare silicon substrate was immersed into a mixture of HCl: Methanol (1 :1, v/v) at room temperature for 30 minutes and then rinsed five times with deionized water. It was immersed into concentrated sulphuric acid at room temperature for 30 minutes and then rinsed well with deionized water five times to remove all sulphuric acid residues from the substrate. The substrate was boiled for 30 minutes in boiling water and then dried in air.
  • the surface of the substrate is functionalised by treating the cleaned substrate with 1% potassium hydroxide (KOH) for 10 min and rinsed five times with deionized water. Thereafter, the support is placed in an Oxygen plasma (Harrick Scientific Corporation) for about 3 min to generate hydroxyl groups on the surface of the support.
  • KOH potassium hydroxide
  • Hydroxyl groups are generated on the surface based on the oxidation of surfaces only. Most of the substrates have got hydrocarbon chains, silicon or metals etc. on their surface, which after oxidation leads to the formation of hydroxyl groups attached to them. Thus, for the support to bear hydroxyl groups, the surface of the support must be capable of oxidation. Hydroxyl groups have even been generated on inert substrates such as Zeonor® and Zeonex® polymers using this method.
  • Step (d) Induction of amine groups
  • APTES 3-aminopropyl triethoxysilane
  • 3-APTES made in deionized water
  • hydroxyl group functionalised support placed inside a petridish in the fume hood. Thereafter, place the covered petri dish housing the support inside a desiccator and apply a vacuum by suction. Place the desiccator in the oven at 80° C for at least 6 hours. Remove the support from the oven and place at room temp for 20 min. Finally rinse the support with deionized water five times.
  • Step (e) (i) Covalent binding of carboxyl groups of biomolecules to the induced amine groups on a surface of the functionalised support
  • the carboxyl groups of biomolecules are covalently bound to the amine groups on the surface of the support using the procedure stated below.
  • the biomolecule may be a protein such as an F c binding protein, or an antibody a lectin, a chaperone protein, an adhesion protein, or a ligand for a protein such as an inhibitor of an enzyme.
  • Cross linking solution In an eppendorf tube, add 0.4 mg 1 -ethyl 3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC) and 1.1 mg Sulfo-N-Hydroxysuccinmide (Sulfo NHS) and dissolve in 100 ⁇ l of 0.1 M MES, pH 4.7.
  • EDC 1-ethyl 3-(3-dimethylaminopropyl)- carbodiimide hydrochloride
  • Sulfo NHS Sulfo-N-Hydroxysuccinmide
  • Biomolecule solution In a second eppendorf tube, add 10 ⁇ l of the above cross linking solution and add
  • BSA bovine serum albumin
  • Step (e) (ii) Covalent binding of amino groups of biomolecules to a surface of a functionalised support
  • an additional step has to be performed after the amine groups have been induced on the surface of the support.
  • the induced amine groups are cross linked to glutaraldehyde to generate functional aldehyde groups that can be covalently attached to amino groups of biomolecules.
  • the biomolecule may be an F c binding protein, or an antibody, lectin, a chaperone protein an adhesion protein, or a ligand for a protein such as an inhibitor of an enzyme.
  • Glutaraldehyde is a homobifuntional crosslinker and can therefore bind to two amino groups:- one amino group of the amine fuctionalised substrate surface and one amino group of a biomolecule thereby covalently attaching a biomolecule to the substrate.
  • the support is incubated with 1% BSA (in PBS, pH 7.4) for about 1 hour 30 mins at room temperature and then washed five times with PBS.
  • BSA in PBS, pH 7.4
  • an antibody can be immobilised to the covalently bound F c binding protein in a specific and site orientated manner using the following protocol. Add an antibody diluted in PBS, for example mouse IgG at a concentration of 12 ⁇ g/ml, to the functionalised support and incubate overnight at 4° C. Wash the antibody functionalised substrate five times with PBS.
  • PBS for example mouse IgG at a concentration of 12 ⁇ g/ml
  • the characterisation of immobilized antibodies on a Zeonex® support was performed using a fluorescent detection system, atomic force microscopy (AFM), surface plasmon resonance (SPR) and enzyme linked immunosorbent assay
  • a fluorescence detection system (depicted schematically in Fig. 3) was designed and built in-house using a range of commercially available components to enable the measurement of the signal emitted by the fluorophores bound to immobilised antibodies.
  • This system comprised of a He-Ne laser excitation source, Sharpvision charge coupled device (CCD) camera (IDT, USA) and a range of "off the shelf optical components supplied by Edmund Optics, USA.
  • the system was used to examine the binding of anti-mouse IgG-Cy5 labelled antibody to the mouse IgG bound to a Zeonex® substrate via Protein A.
  • the assay was carried out using a proprietary cone shaped substrate (fabricated in Zeonex®) having enhanced fluorescence signal collection efficiency.
  • Fig. 4 illustrates the successful immobilisation of anti-mouse IgG-Cy5 labelled antibody to the mouse IgG bound Zeonex® substrate.
  • the results, as illustrated in Fig. 4, show the fluorescent immunoassay where a range of serially decreasing concentrations of goat anti-mouse IgG-Cy5 labelled were detected by the mouse IgG bound Zeonex® substrates correlating with the decrease in fluorescent intensity at each step.
  • This experiment clearly demonstrated the successful employment of the antibody immobilisation strategy for fluorescence based immunoassays.
  • Atomic force microscopy was performed to image the surface of a support during different stages of the antibody immobilization process.
  • Triangular silicon nitride cantilevers (type E) with nominal spring constant 0.1 N/m (Veeco, USA) having a nominal thickness of 0.6 ⁇ m were used for imaging by PicoPlus AFM system (Agilent Technologies, USA). 8 x 8 ⁇ m AFM scanner was employed. The cantilevers were washed in ethanol and deionized water and dried at 70° C for 30 minutes.
  • FIG. 6 shows the AFM based surface analysis of the uniformity of the surface at different stages in the antibody immobilization procedure on the Zeonex® substrate.
  • Fig. 6A shows the surface view as analyzed by AFM in non-contact mode of blank Zeonex® substrate after surface cleaning step.
  • Fig 6B shows the surface analysis after 3-APTES treatment step.
  • Fig 6C shows the Protein A coated surface, where the carboxyl group of protein A is crosslinked to 3-APTES by EDC and sulfo NHS.
  • Fig 6D and E show the Mouse
  • Protein A and Mouse IgG at various stages of the immobilization procedure without the formation of any clumps or aggregates.
  • Zeonex® in solution form dissolved in cyclohexane was spin-coated on the SPR chips by the procedure mentioned below.
  • Zeonex® pellets for injection moulding were initially dissolved in cyclohexane (2 g/1). The mixture was left overnight to dissolve and thereafter sonicated for 40 minutes before use to ensure complete dissolution.
  • Zeonex® coated gold SPR chips were then attached to the SPR chip supports and protein A was covalently attached to the Zeonex® surface of the coated SPR chip.
  • the protein A coated SPR chip was then inserted in the Biacore 3000 set up and the immobilization of mouse IgG was carried out by continuous flow of mouse IgG (0.12 mg/ml) over the protein A coated SPR chip for 30 min.
  • the non-specific protein binding sites on the SPR chip were blocked by continuous flow of 1% blocker BSA solution in PBS, pH 7.4 for 1 min.
  • Goat anti-mouse IgG was then detected and bound by continuous flow of goat anti-mouse IgG (120 ⁇ g/ml) for 30 min.
  • the protein A coated SPR surface was regenerated by continuous flow of 30 mM HCl for 7 min.
  • Fig. 5 shows that the various steps in the antibody immobilization procedure worked well on the protein A coated gold SPR chip, as analyzed in real time by
  • Mouse IgG was immobilised on the protein A coated gold SPR chip by continuous flow of mouse IgG (0.12 mg/ml) for 30 min at a flow rate of 5 ⁇ l/min.
  • Enzyme Linked Immunosorbent Assay ELISA was performed on 6 mm x 6 mm cut solid substrate chips of Zeonex®,
  • Mouse IgG was bound to the substrates using the immobilisation method described above. Thereafter, the immunosensing solid substrates were provided with goat anti-mouse IgG-horse radish peroxidase (HRP) labelled (1.21 ⁇ g/ml) and incubated at RT for 1 hour. The goat anti-mouse IgG-HRP bound solid substrates were then washed five times with PBS, pH 7.4 and placed in fabricated Teflon wells (diameter 10 mm) with the biomolecular functionalized side facing upwards.
  • HRP goat anti-mouse IgG-horse radish peroxidase
  • TMB 3, 3', 5, 5'- tetramethylbenzidine
  • 96-well ELISA plates meant for fluorescent ELISA were used as supports as they had a thin layer of plastic film forming the base of the wells. These ELISA plates were initially blocked with BSA by incubation at room temperature for one and a half hours. Thereafter, the thin bottoms of the three columns of the ELISA plate (having four wells each) with a spacing of one column between them were removed using a steel punch to stamp through the well and remove the base. Each column of the bottomless wells was then attached to the flat surfaces of Zeonex®, Zeonor®. PMMA, polycarbonate, cellulose acetate, polystyrene and glass.
  • Example 1 Thereafter, immobilization procedure of Example 1 was used to compare the immobilization of antibodies on different substrates. For this assay, there was no need to use Teflon cells as the surface area of the bottom of the ELISA wells being employed was exactly the same as for the standard wells.
  • the ELISA performed on various solid substrates demonstrated the successful immobilization of antibodies on a surface of the substrate and therefore the applicability of the immobilisation method for immunodiagnostic and biosensor applications.
  • mice IgG and anti mouse IgG assay were performed using the devised protein immobilisation strategies on the flat bottom 96-well polystyrene ELISA plates from Nunc as described below. All experiments were done in triplicate.
  • the polystyrene wells in the 96-well ELISA plate were cleaned by treating with absolute ethanol for 10 min and then washing five times with 300 ⁇ l of deionized water (DIW).
  • DIW deionized water
  • the surface of the 96-well ELISA plate was functionalized by treating with 100 ⁇ l of 1% potassium hydroxide (KOH) for 10 min and then washing five times with 300 ⁇ l of DIW. Thereafter, the ELISA plate was placed in the Oxygen plasma for 3 minutes to generate hydroxyl groups on the surface.
  • KOH potassium hydroxide
  • the hydroxyl group functionalized 96-well ELISA plate was provided with 100 ⁇ l of 2% 3-APTES (in DIW) per well at room temperature inside the fume hood. Thereafter, the ELISA plate was placed inside the glass desiccator and a vacuum was created inside the desiccator prior to placing it in an oven at about 80° C for about 6 hours. After 6 hours, the desiccator was removed from the oven, placed at room temperature for 20 min to cool down and then washed five times with 300 ⁇ l of DIW.
  • Step (e) Crosslinking of induced amine groups on 96-well ELISA plate to the carboxyl group on F c binding proteins
  • EDC and 1.1 mg suifo NHS were added and dissolved in 100 ⁇ l of 0.1 M MES. pH 4.7.
  • an F c binding protein such as Protein A, Protein A/G, or Protein G (IO ⁇ g/ml in PBS) was prepared and 10 ⁇ l of the crosslinking solution from the first eppendorf tube was added to the second eppendorf tube. The mixture was left for 15 min at room temperature. 1.4 ⁇ l of 2-mercaptoethanol (20 mM) was then added to quench the excess unbound EDC. Finally, 100 ⁇ l of this crosslinking solution i.e. EDC-sulfo NHS-F C binding protein was added to each well of the amine functionalized ELISA plate wells and incubated at room temperature for 2 hours. Wells were then washed five times with 300 ⁇ l of PBS.
  • Mouse IgG was immobilized on to F c binding protein functionalized 96-well ELISA plate by adding 100 ⁇ l of mouse IgG (12.1 ⁇ g/ml in PBS) to each modified microtiter plate well and leaving it overnight at 4° C. Thereafter, the modified microtiter plate wells were washed five times with 300 ⁇ l of PBS. Blocking of the base substrate in ELISA plate wells
  • the ELISA plate wells were incubated with 1% BSA (in PBS, pH 7.4) for 1 hour and 30 min at room temperature to block non-specific protein binding sites on the base substrate and then washed five times with 300 ⁇ l of PBS.
  • BSA in PBS, pH 7.4
  • TMB substrate solution was made by mixing equal amounts of TMB solution
  • the polystyrene wells in the 96-well ELISA plate were cleaned by treating with absolute ethanol for 10 min and then washing five times with 300 ⁇ l of deionized water (DIW).
  • DIW deionized water
  • the surface of the 96-well ELISA plate was functionalized by treating with 100 ⁇ l of 1% potassium hydroxide (KOH) for 10 min and then washing five times with 300 ⁇ l of DIW. Thereafter, the ELlSA plate was placed in the Oxygen plasma for 3 minutes to generate hydroxyl groups on the surface.
  • KOH potassium hydroxide
  • the hydroxyl group functionalized 96-well ELISA plate was provided with 100 ⁇ l of 2% 3-APTES (in DIW) per well at room temperature inside the fume hood. Thereafter, the ELISA plate was placed inside the glass desiccator and a vacuum was created inside the desiccator prior to placing it in an oven at about 80° C for about 6 hours. After 6 hours, the desiccator was removed from the oven, placed at room temperature for 20 min to cool down and then washed five times with 300 ⁇ l of DIW.
  • Fc binding protein solution i.e. Protein A, protein G or protein A/G (10 ug/ml) was added to the aldehyde activated ELISA plate wells and incubated at room temperature for 2 hours. Wells were then washed five times with 300 ⁇ l of PBS. This led to F c binding proteins bound surface.
  • Blocking of the base substrate in ELISA plate wells The ELISA plate wells were incubated with 1% BSA (in PBS, pH 7.4) for 1 hour and 30 min at room temperature to block non-specific binding sites on the base substrate and then washed five times with 300 ⁇ l of PBS.
  • BSA in PBS, pH 7.4
  • Mouse IgG was immobilized on to F c binding protein functionalized 96-well ELISA plate by adding 100 ⁇ l of mouse IgG (12.1 ⁇ g/ml in PBS) to each modified microtiter plate well and leaving it overnight at 4° C. Thereafter, the modified microtiter plate wells were washed five times with 300 ⁇ l of PBS.
  • TMB substrate solution was made by mixing equal amounts of TMB solution (0.4 g/L) and Peroxide solution (containing 0.02 % hydrogen peroxide in citric acid buffer) as per the instructions of the TMB substrate kit from Pierce. 100 ⁇ l of this TMB substrate solution was added to each ELISA plate well used in the immunoassay. The peroxidase enzyme, in the presence of H 2 O 2 , catalyses the oxidation of colorless TMB substrate to a blue colored product. After a fixed reaction time (30 min), the reaction was stopped with 100 ⁇ l of IN H 2 SO 4 and the absorbance of the solution was measured at 450 nm with reference at 650 nm.
  • Fig. 7A and B demonstrate that F c binding proteins can be bound in a functionally oriented manner by crosslinking their carboxyl as well as amino groups. Thus, both these strategies for binding proteins can be effectively used for binding capture antibodies in immunoassays as demonstrated here.
  • the strategy employing EDC-sulfoNHS for binding the carboxyl group of F c binding proteins appears to be better compared to the strategy employing glutaraldehyde for binding the amino group of F c binding proteins as shown in Fig. 7 A and B.
  • Example 4 Direct binding of biomolecules to a surface of a substrate
  • Example 1 the immobilisation strategy of Example 1 was employed to directly immobilise a mouse IgG antibody to a surface of a substrate by covalent bonding through either amino or carboxyl groups. It will be apparent that this methodology can be used to bind any biomolecule having a terminal amino or carboxyl group to the surface of a support.
  • mouse IgG can be directly bound to the substrate by covalent means without employing any F c binding protein.
  • Two immobilisation strategies were employed for direct mouse IgG binding, one based on EDC-sulfo NHS for binding the carboxyl group of mouse IgG and another based on glutaraldehyde for binding the amino group of mouse IgG, .
  • the immobilisation strategies described herein can be employed to create an antibody functionalised biochip.
  • the immobilisation strategies can covalently bind proteins to a wide range of support substrates
  • the biochip can comprise any one of a number of supports. The support can be selected depending on the experimental procedures to be used with the chip.
  • F c binding proteins can be covalently bound to the surface of a biochip for functionalisation with antibodies for use in drug targeting, antibody screening, immune response, immunodiagnostics, biosciences, charge transfer and nanobiotechnology applications.
  • the biochip can contain one or more antibody for example one or more antibody selected from anti-HBsAg, anti-troponin I, anti-troponin T, anti-myoglobin, anti-
  • PSA anti-creatine kinase
  • CK-MB anti-insulin
  • anti-PAP prostate acid phosphate
  • anti-hCG human chorionic gonadotropin
  • anti-leptin anti- Legionella antigen
  • anti-Rotavirus anti-verotoxin
  • anti-adenovirus anti-Pf LDH
  • anti-digoxin anti-b2 -microglobulin
  • anti-T3, anti-T4 anti-TSH
  • CRP C reactive peptide
  • the biochip is more stable and can be employed for the binding of capture antibody many times resulting in a reusable chip.
  • many immunoassays can be performed on the same chip resulting in cost effectiveness compared to chips having passively adsorbed F c binding proteins that can only be used only a few times. If multiple assays are performed on the passively adsorbed F c binding proteins, the bound F c binding proteins are washed away due to a leaching effect which will result in a decrease in assay sensibility and a decrease in the reproducibility of the assay.

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Abstract

L'invention porte sur un support comprenant un substrat muni d'une surface du substrat comprenant des groupes amino ou aldéhyde fonctionnels, pour la liaison covalente d'une biomolécule. La biomolécule peut être une protéine telle qu'une protéine d'affinité. L'invention porte également sur un procédé pour la liaison covalente d'une biomolécule à la surface d'un support par son groupe amino ou carboxyle. Le procédé consiste à disposer un substrat de support; à nettoyer une surface d'un substrat de support; à fonctionnaliser le substrat de support par création chimique de groupes hydroxyle sur la surface nettoyée du substrat de support; à induire des amines sur la surface du substrat de support; et à établir une liaison covalente d'une biomolécule au substrat de support. La biomolécule peut être une protéine telle qu'une protéine d'affinité.
PCT/IE2008/000112 2007-11-22 2008-11-24 Procédé d'immobilisation de molécules biologiques sur un support et produits de celui-ci Ceased WO2009066275A1 (fr)

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ITTO20100577A1 (it) * 2010-07-05 2012-01-06 Univ Degli Studi Genova Supporto planare avente una superficie ultrapiatta e dispositivo per la rivelazione di antigeni comprendente detto supporto planare
CN102507671A (zh) * 2011-10-11 2012-06-20 中国科学院长春应用化学研究所 一种多孔硅生物芯片及其制备方法
KR101227686B1 (ko) * 2010-11-01 2013-01-29 서강대학교산학협력단 다공성 검출센서 및 그 제조방법
WO2014056896A2 (fr) 2012-10-08 2014-04-17 Albert-Ludwigs-Universität Freiburg Procédure d'immobilisation biomoléculaire en une étape et produits en découlant
WO2017009869A1 (fr) * 2015-07-14 2017-01-19 Nanosniff Technologies Pvt. Ltd. Procédé d'immobilisation d'une ou plusieurs biomolécule(s) réceptrices sur une ou plusieurs surface(s) solide(s)
CN106841633A (zh) * 2016-12-27 2017-06-13 郑乐民 在荧光微球表面定向包被抗体的方法及其在糖化载脂蛋白a1检测中的用途
CN108163802A (zh) * 2017-12-06 2018-06-15 李瑞萱 一种抗原检测材料及其制备方法和应用
CN110297084A (zh) * 2019-07-09 2019-10-01 清华大学天津高端装备研究院 一种生物芯片的抗体高效固定方法
CN112098639A (zh) * 2020-09-21 2020-12-18 天津医科大学 以氧化石墨烯为载体的二抗的合成及应用
CN112904015A (zh) * 2019-11-19 2021-06-04 王復民 蛋白质传感器及其制造方法
CN114829620A (zh) * 2019-12-23 2022-07-29 雷希斯特尔股份公司 生物物体和非生物物体例如细菌细胞向表面例如悬臂的附着
CN115824980A (zh) * 2022-11-26 2023-03-21 山东乾乾若医疗科技有限公司 一种检测尿液中IgG蛋白的微环谐振器及其制备方法与应用

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WO2012004721A1 (fr) * 2010-07-05 2012-01-12 Universita' Degli Studi Di Genova Support plan ayant une surface ultraplate et dispositif de détection d'antigène comprenant ledit support plan
US8932991B2 (en) 2010-07-05 2015-01-13 Universita' Degli Studi de Genova Planar support having an ultraflat surface and a device for detecting antigens comprising said planar support
ITTO20100577A1 (it) * 2010-07-05 2012-01-06 Univ Degli Studi Genova Supporto planare avente una superficie ultrapiatta e dispositivo per la rivelazione di antigeni comprendente detto supporto planare
KR101227686B1 (ko) * 2010-11-01 2013-01-29 서강대학교산학협력단 다공성 검출센서 및 그 제조방법
CN102507671A (zh) * 2011-10-11 2012-06-20 中国科学院长春应用化学研究所 一种多孔硅生物芯片及其制备方法
CN102507671B (zh) * 2011-10-11 2014-01-08 中国科学院长春应用化学研究所 一种多孔硅生物芯片及其制备方法
WO2014056896A2 (fr) 2012-10-08 2014-04-17 Albert-Ludwigs-Universität Freiburg Procédure d'immobilisation biomoléculaire en une étape et produits en découlant
US10329396B2 (en) * 2015-07-14 2019-06-25 Nanosniff Technologies Pvt. Ltd. Process for immobilizing one or more receptor biomolecules on one or more solid surfaces
WO2017009869A1 (fr) * 2015-07-14 2017-01-19 Nanosniff Technologies Pvt. Ltd. Procédé d'immobilisation d'une ou plusieurs biomolécule(s) réceptrices sur une ou plusieurs surface(s) solide(s)
CN106841633A (zh) * 2016-12-27 2017-06-13 郑乐民 在荧光微球表面定向包被抗体的方法及其在糖化载脂蛋白a1检测中的用途
CN108163802A (zh) * 2017-12-06 2018-06-15 李瑞萱 一种抗原检测材料及其制备方法和应用
CN110297084A (zh) * 2019-07-09 2019-10-01 清华大学天津高端装备研究院 一种生物芯片的抗体高效固定方法
CN112904015A (zh) * 2019-11-19 2021-06-04 王復民 蛋白质传感器及其制造方法
CN114829620A (zh) * 2019-12-23 2022-07-29 雷希斯特尔股份公司 生物物体和非生物物体例如细菌细胞向表面例如悬臂的附着
CN112098639A (zh) * 2020-09-21 2020-12-18 天津医科大学 以氧化石墨烯为载体的二抗的合成及应用
CN112098639B (zh) * 2020-09-21 2024-01-02 天津医科大学 以氧化石墨烯为载体的二抗的合成及应用
CN115824980A (zh) * 2022-11-26 2023-03-21 山东乾乾若医疗科技有限公司 一种检测尿液中IgG蛋白的微环谐振器及其制备方法与应用

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