EP2903740A2 - One-step biomolecular immobilisation procedure and products thereof - Google Patents

Abstract

The invention relates to a method for covalent immobilisation of a biomolecule to a solid material and products arising therefrom, comprising providing a solid material with an activated surface, preferably by treatment with an oxidising agent, and subsequently contacting said material with a mixture of biomolecule to be immobilised and one or more silane components, preferably present in a pre-mixed binding solution. The immobilisation procedure is particularly useful for immunoassays, enzyme-linked immunosorbent assays (ELISA), surface plasmon resonance immunoassays, microarrays or microfluidic assays.

Description

ONE-STEP BIOMOLECULAR IMMOBILISATION PROCEDURE AND PRODUCTS THEREOF

DESCRIPTION

The invention relates to a method for covalent immobilisation of a biomolecule to a solid material and products arising therefrom, comprising providing a solid material with an activated surface, preferably by treatment with an oxidising agent, and subsequently contacting said material with a mixture of biomolecule to be immobilised and one or more silane components. The

immobilisation procedure is particularly useful for immunoassays, enzyme-linked

immunosorbent assays (ELISA), surface plasmon resonance immunoassays, microarrays or microfluidic assays.

In one embodiment, the invention relates to a highly-sensitive enzyme-linked immunosorbent assay (ELISA), in which the solid phase characterised by the immobilized affinity reagent is developed in significantly reduced time by employing the one-step antibody immobilisation procedure of the present invention. A novel and economic approach based on simple chemical solutions is provided herein, which act as a surface modification-, covalent biomolecule-binding- and dispersion-agent at the same time.

The dilution of a capture antibody in immunodiagnostic kits is prepared in an optimized concentration of this proprietary chemical solution followed by its dispensing on a solid surface, such as in microtitre plate wells, and incubation under ambient conditions. This simple one-step antibody immobilization procedure enables a leach-proof and highly stable covalent binding of antibodies or other proteins or biomolecules, allowing long shelf-life. ELISA based on the present method has much better analytical performance with high sensitivity, short assay duration, and longer shelf-life than the commercially-available conventional ELISAs. The technology has significant commercial potential and is ideal for the development of rapid immunodiagnostic kits. Moreover, the method of the present invention is of tremendous utility for the development of lab-on-a-chip, surface plasmon resonance, microarray, electrochemical, point-of-care, and other immunoassay formats.

BACKGROUND OF THE INVENTION

ELISA is the gold standard of immunodiagnostics in clinical, pharmaceutical and research settings based on its high throughput, selectivity and sensitivity. It has an existing multi-billion dollar and multi-million dollar segmented market in healthcare and industries, and R&D settings, respectively. However, the conventional ELISA requires a lot of time. Therefore, there is a critical need to develop a cost-effective technology for reducing assay duration but without compromising the sensitivity and selectivity of an immunoassay. Recently, the inventors have pioneered the development of multisubstrate-compatible highly-sensitive ELISA procedures that were demonstrated to be many-fold better in terms of analytical performance than the commercial ELISA procedure. However, the developed ELISA procedure mentioned here is more advanced and superior to the previously known procedures. It is the most sensitive and most rapid ELISA format shown to date, which is compatible with several commercial ELISA kits. The technology will lead to the development of next-generation of rapid and highly-sensitive immunodiagnostic kits based on extreme simplicity, cost-effectiveness and superior analytical performance.

Similar, more complicated approaches have been documented in the prior art. WO/2010/044083 and WO/2009/066275 disclose the 1 st generation of multisubstrate-compatible multistep procedures based on the covalent and leach-proof binding of antibodies for ELISA-based immunodiagnostic kits. The methods described therein are however limited by a multistep procedure required for producing the covalent attachment. Typically, a support surface is oxidised, followed by separate treatment with a silane component without additional compounds in the mixture, i.e. without biomolecules. The generation of amine groups on the solid support enables subsequent crosslinking of the biomolecule using standard EDC- (1-Ethyl-3-(3- dimethylaminopropyl)carbodiimid) and sulfo-NHS (Sulfo-N-Hydroxysuccinmide) -based chemistry. These approaches are similarly described in Nature Protocols 6(4), 439-445, 201 1 ; Analytical Chemistry 82(16), 7049-52, 2010; and Diagnostics 2(3), 23-33, 2012; and, Biosensors and Bioelectronics 40(1 ), 297-302, 2013).

WO 2005/080983 discloses a similar method, in particular a method for detecting a ligand in a sample, whereby the affinity substrate is covalently linked to a PDMS surface using sulfo-NHS esters. The PDMS surface is first oxidised using 0₂ plasma, subsequently treated with APTES and finally a bi-functional crosslinker is applied to covalently attach the macromolecule (capture protein).

Zheng et al. (Analyst 137(16) 3800, 2012) discloses a method for covalent attachment of glucose oxidase (GOx) to a glassy carbon electrode comprising pre-treatment with KOH followed by drop-casting of glutasraldehyde-crosslinked GOx, followed by drop-casting of APTES to form GOx-bound carbon electrodes. Utilisation of a pre-mixture comprising a biomolecule to be immobilised and a silane component as a binding solution has been neither suggested nor disclosed in the art.

These 1 st generation developments of the multisubstrate-compatible multistep procedure were further employed for surface plasmon resonance (SPR) based rapid and real-time microfluidic immunoassays (Analyst 136(21 ), 4431-36, 201 1 ). Apart from ELISA and SPR, the 1 st generation technology was also employed for the development of Fc binding proteins- or antibody-bound affinity chromatography columns. These approaches were also limited significantly by complicated procedures requiring multiple steps of treatments with cross-linking agents.

Further developments of the standard technology of the 1 ^st generation have been developed, whereby the procedure was optimized for immobilizing enzymes using more simplified process steps with and without nanomaterials (Talanta, 99, 22-28, 2012; Analyst, 137(16), 3800, 2012). Various potential technologies were developed for the point-of-care electrochemical glucose sensors for diabetic monitoring.

However, the ability to perform similar methods in the absence of an EDC, SNHS, or EDC-like reagent, or other cross-linker, has not been shown previously. In light of the prior art, alternative and simplified methods for the covalent immobilisation of a biomolecule to a solid material are required. The previous methods allow covalent attachment of biomolecules to a solid surface, but are limited by multiple method steps and the use of potentially hazardous chemicals. With additional method steps, the procedures introduce additional variables that ultimately lead to poorer reproducibility. This is a significant

disadvantage when considering the application of the method in the diagnostic and medical research fields.

A significant need remains for improved, reliable and sensitive methods for providing immunoassay reagents, capable of effective covalent immobilization to solid surfaces without requiring long coupling times, multiple treatment and washing steps and unnecessary costs.

SUMMARY OF THE INVENTION

In light of the prior art, the technical problem underlying the invention was the provision of alternative methods for the covalent immobilisation of a biomolecule to a solid material that do not exhibit the disadvantages of those methods known in the art.

This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

Therefore, an object of the invention is to provide a method for covalent immobilisation of a biomolecule to a solid material, comprising

a) contacting said solid material with an oxidising agent to activate the solid material, or providing a preferably pre-activated solid material, wherein said activated solid material comprises carbonyl and/or hydroxyl groups, and

b) contacting said activated solid material with a binding solution, wherein said binding solution comprises a mixture of at least a biomolecule to be immobilised and one or more silane components, and the contents of the binding solution are mixed before contacting with said solid material, thereby producing a covalent attachment between said biomolecule and said solid material via an amide bond.

It was a surprising and unexpected finding that such a simple one-step immobilisation procedure could be carried out. The present invention can exhibit two-steps when required, whereby the immobilisation itself represents the unique "one-step" method, which is surprising in light of the prior art.

The first step of the method relates to either provision of an activated solid surface or activation of the solid surface caused by oxidation of the surface, resulting in provision of carbonyl or hydroxyl groups that subsequently react during the later immobilisation. The first oxidation step is required for various solid surfaces but can optionally be dispensed with, for example when using polystyrene microtiter plates, which are routinely employed for clinical and industrial applications. The term "pre-activated" relates to any solid material that exhibits carbonyl and/or hydroxyl groups on its surface and may therefore be used in step b) as described above.

The methods of the 1 ^st generation described in the prior art all involve multistep preparation of APTES-functionalized platforms and the covalent crosslinking of antibodies or other biomolecules using EDC and Sulfo-NHS. The present invention doesn't involve the pre- preparation of APTES-functionalized platforms or the use of EDC and Sulfo-NHS. All the components of the binding solution, in a preferred embodiment APTES and antibody, are mixed together to form a pre-mixed "one-step" binding solution, which is directly dispensed on the platform (solid surface) resulting in preferably APTES-functionalization and antibody

immobilization in the same step, in one embodiment of just 30 minutes duration. Therefore, the invention is preferably characterised by the use of a pre-mixed binding solution and the obviation of all the unnecessary steps that were involved in previous technologies.

The method of the invention leads to more effective antibody immobilization due to higher functional antibody immobilization density on the solid surface platforms. The method also enables binding of the antibodies in a leach-proof manner. The solution-phase binding of APTES with biomolecule, as occurs in the binding solution as described herein, is much more effective than the binding on the solid phase i.e. APTES-functionalized substrates, as employed in the prior art. The immobilization density of biomolecule (preferably antibody) varies for different substrates. However, analysis has revealed (in the context of real-time binding for surface plasmon resonance) that the immobilization density is improved over the earlier generations of technology.

The results provided herein demonstrate that a mixture of silane component and biomolecule can lead to effective "one-step" immobilisation of the biomolecule without the need for EDC or sulfo-NHS (SNHS) -based chemistry, to provide conditions for a covalent linkage. In one embodiment, the method is carried out in the absence of EDC, the absence of glutaraldehyde and/or the absence of SNHS based linkage chemistries.

The ability to perform similar methods in the absence of an EDC, SNHS, or EDC-like reagent, or other cross-linker, has not been shown previously. However, the present invention demonstrates that the obviation of EDC, NHS and sulfo-NHS chemistries leads to an improved antibody immobilization strategy, where APTES directly binds to the antibodies without the use of heterobifunctional crosslinkers. The method is faster and simpler than those methods described previously and enables improved and more reliable binding between biomolecule and the solid surface, in addition to higher antibody densities. One of the key differentiating features of the invention is the preferably pre-mixed binding solution, comprising a mixture of biomolecule to be immobilised and one or more silane components. It was entirely unknown that such a mixture could react with a pre-treated, or pre-activated solid phase to enable the desired covalent linkage. The invention therefore relates to a method for covalent immobilisation of a biomolecule to a solid material, comprising contacting an activated solid material with a binding solution, comprising a mixture of said biomolecule to be immobilised and one or more silane components. The covalent linkage is preferably an amide bond.

Traditional approaches rely on the induction of amines on the surface of the physical support (or solid material) in a separate step. According to the prior art, initially the hydroxyl groups are generated, subsequently an amine group is created and then separate chemical cross-linkers are applied in a multi-step approach. The "one-step" approach as described herein is a surprising and beneficial approach that simplifies enormously the problems associated previously with solid-surface covalent linkage of biomolecules.

The invention is characterised by a mixture of biomolecule and silane component in the binding solution before contacting with the solid phase. The mixture of biomolecule with silane before contacting to the solid phase is associated with technical advantages, such as reduced precipitation, higher biomolecule densities and improved distribution of the biomolecule.

Previous approaches have disclosed drop-casting either the biomolecule or silane component (such as APTES) onto the surface of the solid phase and subsequently adding the missing component, whether it be biomolecule or APTES. Such approaches are plagued by technical difficulties, such as poor distribution and even precipitation of the biomolecule. Such technical difficulties are particularly evident when using relatively high concentrations of biomolecule, such as described previously in the art, for example in Zheng et al. (Analyst 137(16) 3800, 2012). Described therein is drop-casting of 10 mg/mL GOx followed by drop-casting 4% APTES.

Although immobilisation was achieved, precipitation of the GOx is evident during drop-casting. Glutaraldehyde cross-linking was also required in Zheng et al., in order to achieve effective immobilisation. The pre-mixing of APTES and affinity reagent (biomolecule) as per the present invention enables even distribution of APTES and biomolecule and is possible without precipitation of the biomolecule.

The present invention involves the preparation of one-step solution that involves the mixing of preferably capture antibodies with APTES prior to contact with the solid material. APTES is then used as a dispersion, immobilization and surface functionalization agent simultaneously. The amino groups of APTES start binding to the carboxyl groups of the biomolecule (for example antibody) immediately after mixing. This occurs in solution before application to the solid surface. After dispensing on the solid substrate, the same reaction continues along with a new reaction, wherein the alkoxy groups on APTES start binding to the hydroxyl (carbonyl) groups on the substrate. This step wise pre-preparation of the binding solution represents a unique and surprisingly good set of reactive conditions for solid phase immobilisation.

In a preferred embodiment of the invention, the concentration of capture antibodies used in the immunoassays is relatively small. The use of low antibody concentrations and subsequent strong signals in ELISA, Biacore or SPR assays represents a particularly surprising and beneficial finding. Low concentrations of antibody demands therefore smaller amounts of such an expensive reagent, making preparation of immunoassay reagents more affordable and simple to construct. The present method enables reliable immobilization at high efficiencies and with low antibody concentrations. In a preferred embodiment, the method of the invention is characterised in that the biomolecule is an antibody, antibody fragment, a recombinant protein, Fc-binding protein (protein A, protein G, protein A G), streptavidin, or recombinant proteins or fragment thereof, and is present in the binding solution at a concentration of 0.01 to 100 μg/ml. In a further preferred embodiment, the biomolecule is present in the binding solution at a concentration of 0.1 to 50 μg/ml, preferably at a concentration of 0.1 to 20 μg/ml, or 1 to 10 μg/ml. In another embodiment, the concentration of antibody or other affinity reagent is below 500, 100, 50, 20, or below 10 μg/ml. In a preferred embodiment, the method of the present invention is characterised in that said biomolecule is a protein, nucleic acid, lectin, polysaccharide or lipid molecule. Essentially any biomolecule is suitable for covalent linkage due to the presence of the required chemical moieties present in biologically produced molecules. The broad application of the method for any given biomolecule is a surprising and useful development of the prior art. When the biomolecule is a protein, said protein is preferably an antibody, antibody fragment, a

recombinant protein, Fc-binding protein (protein A, protein G, protein A/G), streptavidin, or recombinant proteins or fragments thereof. Considering the immunoassay applications of the method, any given biomolecule related to the immunological field could be applied in the assay, including cytokines, antibodies, antigens, peptide, antibodies or antibody fragments.

The solid material of the invention can essentially be any solid material, such as a support, a support surface, a solid phase, made of any compound that can form a solid phase. In one embodiment, the method of the present invention is characterised in that said solid material comprises of a synthetic polymer; agarose; silica-based material, such as glass, bioglass, silica monoliths or porous silica; silicon and silicon derivative based substrates; metal-coated surfaces, such as gold-and silver-coated surfaces; nanoparticle-/nanocomposite-coated surfaces; surfaces coated with thin oxide films, such as Si0₂, ZnO, Zx0₂, Al₂0₃, NdGa0₃, La01 , Ti0₂, LSAT, MgAI₂0₄; glassy carbon; screen-printed carbon electrodes; cellulose; cellulose acetate; nanocrystalline cellulose; and/or chitosan.

It was a surprising realisation that the "one-step" procedure is not limited to particular solid support surfaces. The solid material of the invention can be any kind of natural or synthetic solid substrate. The oxidation agent has the ability to prepare or activate the solid surface. The subsequent application of the biomolecule/silane mixture can be seen as a completely unpredictable and surprising development, whereby any activated surface has the ability to react and covalently link the biomolecule. The first oxidation step is required for various solid surfaces that require the activating first step. The oxidation step of the method can optionally be dispensed with, for example when using polystyrene microtiter plates, which already exhibit a sufficiently activated surface for application of the silane-biomolecule binding solution.

In one embodiment, the method of the present invention is characterised in that said synthetic polymer comprises polystyrene (PS), polypropylene (PP), polyethylene PE), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC), water-resistant photopolymer, such as Watershed® XC 1 1 122, cyclic olefin copolymer such as TOPAS® or a cyclo-olefin polymer such as Zeonor® and Zeonex®.

In a preferred embodiment, the method of the present invention is characterised in that

a) the surface area of the solid material is increased by using as solid material a structure with increased surface area such as beads, nanoparticles, porous structures, nanomaterials, such as carbon nanotubes, graphene, and/or quantum dots, nanocomposites and/or magnetic or paramagnetic beads, b) the binding sites of the solid material are increased using polymers with multiple binding sites or dendrimers, or c) a combination thereof.

In a preferred embodiment, the method of the present invention is characterised in that the surface area of the solid material is increased by using as solid material Graphene nano platelets (GNPs), preferably of a diameter of 0.1 to 100 μηη, more preferably 1 to 10 μηη, such as 5 μηη

In a preferred embodiment, the method of the present invention is characterised in that said oxidizing agent is a hydroxide-containing solution, piranha solution, oxygen plasma treatment or corona discharge treatment.

In one embodiment, the method of the present invention is characterised in that said hydroxide- containing solution comprises of sodium hydroxide, potassium hydroxide and/or ammonium hydroxide, preferably potassium hydroxide, whereby said hydroxide is potassium hydroxide and is preferably present at a concentration of 0.1 to 10%, preferably 0.5 to 5%, more preferably 1 %.

The particular preferred concentrations of the oxidising solutions are associated with

unexpected effects, whereby the relatively low concentrations of hydroxide mentioned herein provide reliable covalent linkage of the biomolecule at a density that enables useful application in immunoassay procedures.

In one embodiment, the method of the present invention is characterised in that said silane component is 3-aminopropyltriethoxysilane (APTES), (3-aminopropyl)-trimethoxysilane

(APTMS), (3-mercaptpropyl)trimethoxysilane (MPTMS) and/or 3-glycidoxypropyltriethoxysilane (GOPTS), whereby said binding solution comprises preferably of APTES at a concentration of 0.1 to 10%, preferably 0.2 to 5%, more preferably 0.25% to 2%, such as 0.25%, 0.5% or 1 %.

It was entirely surprising that the mixture of biomolecule and silane component would enable a covalent linkage. The particular preferred concentrations of the silane component are associated with unexpected effects, whereby the relatively low concentrations of silane component mentioned herein provides reliable covalent linkage of the biomolecule at a density that enables useful application in immunoassay procedures.

In one embodiment, the method is characterised in that the biomolecule is an antibody, antibody fragment, a recombinant protein, Fc-binding protein (protein A, protein G, protein A G), streptavidin, or recombinant proteins or fragment thereof, and is present in the binding solution at a concentration below 500 μg/ml, preferably between 0.01 to 100 μg/ml. In one embodiment, the method is characterised in that the biomolecule is an antibody, antibody fragment, a recombinant protein, Fc-binding protein (protein A, protein G, protein A G), streptavidin, or recombinant proteins or fragment thereof, and is present in the binding solution at a

concentration of 0.1 to 50 μg/ml, preferably at a concentration of 1 to 10 μg/ml. These concentrations of antibody surprisingly enable immobilisation at higher densities than is possible using the methods described in the art

In a preferred embodiment, the method of the present invention is characterised in that said cross-linking agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), EDC-/V- hydroxysuccinimide (NHS), EDC-sulfoNHS, glutaraldehyde and/or 1 ,4,-phenylene

diisothiocyanate (PDITC) are not employed in the method of the invention.

In a preferred embodiment, the method of the present invention is characterised in that step a) of claim 1 is carried out via incubation of solid material and hydroxide-containing solution for at least 1 minute, preferably 1 to 60 minutes, more preferably 2 to 30 minutes and especially preferred for 10 minutes, optionally followed by washing said solid material with water 1 to 10 times, preferably 5 times , and

step b) of claim 1 is carried out via incubation of activated solid material and binding solution for at least 1 minute, preferably 1 to 120 minutes, more preferably 15 to 60 minutes and especially preferred for 30 minutes, optionally followed by washing said solid material with a wash buffer, comprising preferably phosphate buffered saline (PBS), 1 to 10 times, preferably 5 times.

A further aspect of the invention relates to a solid material obtainable or obtained via the method described herein, comprising

a) a solid material selected from the group consisting of a synthetic polymer; agarose; silica-based material, such as glass, bioglass, silica monoliths or porous silica; silicon and silicon derivative based substrates; metal-coated surfaces, such as gold-and silver- coated surfaces; nanoparticle-/nanocomposite-coated surfaces; surfaces coated with thin oxide films, such as Si0₂, ZnO, Zx0₂, Al₂0₃, NdGa0₃, La01 , Ti0₂, LSAT, MgAI₂0₄; glassy carbon; screen-printed carbon electrodes; cellulose; cellulose acetate;

nanocrystalline cellulose; and/or chitosan, and/or

b) a biomolecule covalently attached to the material via an amide bond, whereby the

biomolecule is selected from the group consisting of protein, nucleic acid, lectin, polysaccharide or lipid molecule.

In light of table 3, provided below, the solid material of the present invention exhibits a higher molecular density of immobilised biomolecule than the solid materials produced via the methods described in the art. This represents a significant advantage when providing very sensitive assays, intended to detect very low numbers of analyte in a sample. The immobilised biomolecule density provided by the method of the present invention has not been achieved by any other method described in the art. The density value as such may therefore, in one embodiment, be used as a differentiating factor distinguishing the solid materials of the present invention from those of the prior art. The solid materials of the present invention may therefore be used in biosensors, for detecting analytes in environmental samples, such as soil or water samples, or medical samples, for example body fluids.

In one embodiment, the solid material is characterised in that the surface of said solid material exhibits a mass density of biomolecule (ng/cm² of solid material surface), preferably antibody, antibody fragment, a recombinant protein, Fc-binding protein (protein A, protein G, protein A G), streptavidin, or recombinant proteins or fragments thereof, of greater than 175 ng/cm², preferably between 175 and 250 ng/cm², more preferably between 175 and 200 ng/cm², for example 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 195 or 200 ng/cm². These values represent improvements over the densities achieved via the methods of the prior art. In one embodiment, the solid material is characterised in that the surface of said solid material exhibits a molecular density per Dalton (molecules/cm²/Dalton) of antibody or antibody fragment of greater than 4.61 x 10⁹ molecules/cm²/Dalton, preferably between 4.61 x 10⁹ and 6 x 10⁹, more preferably between 4.61 x 10⁹ and 5 x 10⁹, for example 4.75 x 10⁹, 4.8 x 10⁹, 4.87 x 10⁹, 4.9 x 10⁹, 4.95 x 10⁹ molecules/cm²/Dalton. As can be observed from Table 3, application of the present method comprising immobilisation of anti-HFA antibody lead in a preferred embodiment to a mass density of 182.6 ± 1 .0 (ng/cm²), which - in light of a molecular mass of the 150 KDa antibody used - corresponds to a molecular density of 7.3 x 10¹¹ (molecules/cm²). According to the embodiment of the example, the number of molecules present per Dalton (molecular density/antibody mass; in this case 7.3 x 10¹¹ /150) is 4.87 x 10⁹. Considering that antibodies or antibody fragments of various sizes may be applied in the present method, the molecular density can be calculated as molecular density per Dalton. This value allows for the varying size of the antibody with respect to the mass density. For example, a smaller antibody could be present at the same mass density, but with a higher molecular density due to the smaller size of the antibody and corresponding lessening of constraints on surface density of the molecule during immobilisation.

As described above, the solution-phase binding of APTES with biomolecule, as occurs in the binding solution as described herein, is much more effective than the binding on the solid phase i.e. APTES-functionalized substrates, as employed in the prior art. As shown below (in the context of real-time binding for surface plasmon resonance), the immobilization density is improved over the earlier generations of technology. This increased density represents an advantage in that the solid surfaces are more sensitive to low amounts of analyte in solution, thereby enabling more sensitive tests for any given analyte capable of binding the biomolecule attached to the solid surface. The higher density also enables the immobilised biomolecule to withstand abrasion and extensive washing procedures, whilst maintaining strong signals in whichever analytical setup (eg ELISA) is applied.

In a preferred embodiment, the solid material of the present invention is characterised in that the material is in the form of a slide, microtitre plate, incubation chamber, micro- or nanofluidic channel or device, bead or immunoaffinity column. These physical forms of the solid material mentioned herein are preferred embodiments and do not represent limiting embodiments of the invention.

A further aspect of the invention relates to a kit comprising means for carrying out the method as described herein, comprising

an oxidising agent, preferably as described herein,

- a silane component, preferably as described herein, and

a solid material selected from the group consisting of a synthetic polymer;

agarose; silica-based material, such as glass, bioglass, silica monoliths or porous silica; silicon and silicon derivative based substrates; metal-coated surfaces, such as gold-and silver-coated surfaces; nanoparticle-/nanocomposite-coated surfaces; surfaces coated with thin oxide films, such as Si0₂, ZnO, Zx0₂, Al₂0₃, NdGa0₃, La01 , Ti0₂, LSAT, MgAI₂0₄; glassy carbon; screen-printed carbon electrodes; cellulose; cellulose acetate; nanocrystalline cellulose; and/or chitosan, and optionally

a biomolecule covalently attached to the material via an amide bond, whereby the biomolecule is selected from the group consisting of protein, nucleic acid, lectin, polysaccharide or lipid molecule.

Such kits may be brought onto the market for end-users to apply the inventive method described herein in the lab immediately before carrying out the method as described. The kits of the invention enable a technician, medical or diagnostic practitioner to generate solid phases comprising reliably covalently attached biomolecules, such as antibodies or other proteins, immediately before analysis. This leads to more sensitive results, as the biomolecule has not been stored or shipped for long periods of time before analysis. The kit of the invention is therefore a preferred embodiment and enables a skilled practitioner to enjoy the benefits of the method described herein.

In further embodiments of the invention, the kit may comprise of oxidising agents and silane components as described herein. For example, the oxidising agent may be present in the kit in the form of a solution that comprises of sodium hydroxide, potassium hydroxide and/or ammonium hydroxide, preferably potassium hydroxide, whereby said hydroxide is potassium hydroxide and is preferably present at a concentration of 0.1 to 10%, preferably 0.5 to 5%, more preferably 1 %. The silane component of the kit is preferably 3-aminopropyltriethoxysilane (APTES), (3-aminopropyl)-trimethoxysilane (APTMS), (3-mercaptpropyl)trimethoxysilane (MPTMS) and/or 3-glycidoxypropyltriethoxysilane (GOPTS), whereby said silane component comprises preferably of APTES at a concentration of 0.1 to 10%, preferably 0.2 to 5%, more preferably 0.25% to 2%, such as 0.25%, 0.5% or 1 %.

The kit of the invention is preferably provided as a single unit, enclosed in one packaging unit, comprising preferably the reagents needed for carrying out the method of the present invention. The silane component is preferably provided as a solution comprising APTES in the optimum concentration for creation of a binding solution with the required biomolecule. The kit may also comprise additional elements used in the method, for example covering films, dishes or trays for incubation, pipette tips, or other laboratory items.

A further aspect of the invention relates to an immunoassay comprising a method, use of a method or use of a solid material as described herein, preferably selected from an enzyme- linked immunosorbent assay (ELISA), a surface plasmon resonance immunoassay, for example in a BIAcore format, a microarray method, a reflectometric interference spectroscopy (RlfS) method, an electrochemical immunoassay, a microfluidic immunoassay, and/or a centrifugal microfluidic method, for example in a LabDisk platform format. These various immunoassays are examples of concrete experimental, diagnostic or medical setups that may make use of the method as described herein.

A further aspect of the invention relates to an ELISA method, comprising a) production of a solid material, preferably a microtitre plate, according to the method of the preceding claims, comprising covalently immobilised antibody, whereby the antibody is capable of binding a molecule to be detected, or

use of a solid material as described herein,

b) blocking non-bound sites of said solid material with a blocking reagent, preferably a protein-containing solution, such as bovine serum albumin (BSA),

c) incubation of said solid material with a solution comprising the molecule to be detected, such as a patient sample or diagnostic sample, thereby immobilising said molecule to be detected to said solid material via the immobilised antibody of a),

d) detection of said immobilised molecule to be detected, preferably via

i. incubation of a solution comprising a biotinylated antibody of the same or essentially the same affinity as the antibody of step a) with the complex of step c), and

ii. incubation of the complex from i. with a solution comprising

streptavidin-coupled horseradish peroxidise (SA-HRP), followed by iii. detection of HRP signal, preferably via TMB substrate assay and measurement of optical density.

In one embodiment, the ELISA method as described herein is characterised in that

said immobilised antibody of step a) is capable of specifically binding C-reactive protein (CRP), which is the molecule to be detected in the solution of step c).

C-reactive protein (CRP) is protein found in the blood, the levels of which rise in response to inflammation (i.e. C-reactive protein is an acute-phase protein). CRP is used mainly as a marker of inflammation. Apart from liver failure, there are few known factors that interfere with CRP production. Measuring and charting CRP values can prove useful in determining disease progress or the effectiveness of treatments. Blood, usually collected in a serum-separating tube, is analysed in a medical laboratory or at the point of care. Various analytical methods are available for CRP determination, such as ELISA, immunoturbidimetry, rapid immunodiffusion, and visual agglutination. Normal concentration in healthy human serum is usually lower than 10 mg/L, slightly increasing with aging. Higher levels are found in late pregnant women, mild inflammation and viral infections (10-40 mg/L), active inflammation, bacterial infection (40-200 mg/L), severe bacterial infections and burns (>200 mg/L). CRP tests may also be used in testing for cancer or cardiovascular disease. In one embodiment, the invention relates to the use of a method, solid phase or kit of the present invention in the diagnosis of any CRP-associated or related disease. The present invention enables an extremely and unusually sensitive test for CRP levels from a biological fluid of a patient, due to the fresh generation of immunoassay kit components with antibody that has not been stored after physical attachment to a solid surface.

In one embodiment, the ELISA method as described herein is characterised in that said immobilised antibody of step a) is capable of specifically binding human fetuin A (HFA), which is the molecule to be detected in the solution of step c). In one embodiment, the ELISA method as described herein is characterised in that said immobilised antibody of step a) is capable of specifically binding human Lipocalin-2 (LCN2), which is the molecule to be detected in the solution of step c).

DETAILED DESCRIPTION OF THE INVENTION

The developed technology leads to a sensitive and rapid ELISA without increasing the cost, complexity or hands-on time, which is ideal for clinical, industrial and research settings. It completely eliminates the storage of antibody-bound ELISA plates as the 1-step antibody immobilization procedure takes just 30 min. This will eliminate the storage cost and will further increase the analytical performance and reproducibility of immunoassays.

Key technology differentiating features of the present invention in comparison to the prior art:

• Allows the development of more rapid and more sensitive ELISA-based immunodiagnostic kits.

• Completely eliminates the storage of pre-bound antibody-coated ELISA plates as the

proprietary 1-step capture antibody immobilisation takes only 30 min.

· Leads to cost-effective immunodiagnosis by eliminating the requirement of costly pre- bound antibody-coated plates that need to be refrigerated during storage and delivery

• Works for all sandwich and direct immunoassay formats i.e. chemiluminescent,

fluorescent, optical etc.

• Works on all types of substrates including inert substrates i.e. multisubstrate-compatible. · Provides a generic technology that can be further employed in various

immunobiosensors/immunoassay formats such as those based on microarrays, surface plasmon resonance (SPR), quartz crystal microbalance, electrochemistry, microfluidics, lab-on-a-chip, etc.

• The one-step immobilization procedure works for various biomolecules such as proteins, lectins, nucleic acids and others.

The invention can be used in the following areas of application:

Based on the huge market of ELISA-based immunodiagnostic kits [multi-billion US$ in clinical settings; and, multi-million US$ in research and development (R&D) (Frost & Sullivan report (2008) N 151 -55)], the customized development of these kits using the 1-step antibody immobilization procedure described herein will be the immediate application. As the present method provides significantly reduced immunoassay duration with better analytical performance, but without increasing the cost, there will be significant market interest. Moreover, it will lead to greater cost savings as it will completely obviate the storage of antibody-bound diagnostic kits.

The second major application is the development of surface plasmon resonance-based real-time microfluidic immunoassays (such as GE Healthcare's), which has witnessed exponential market growth in the last decade. It is now an established standard and approved technology in all industries' R&D, technical operations and QC settings apart from its widespread use in the clinical and academic R&D settings. The third application is microfluidics-based immunodiagnostic kits, where the technology will in fact be much preferred due to its 1-step procedure, as it will eliminate the number of process steps. The 1-step antibody immobilization-based localized coatings in defined chambers, cavities or inside channels will be beneficial and easy to use compared to state of the art antibody coatings. The invention can be used to develop immunoassays for one or several analytes, where the analysis could be done along a particular channel, in separated channels, or on a microarray. Additionally, the APTES in the 1 -step coating can be used for steering liquid onto given positions in the microfluidic channel/device by using the phase guides or pinning structures that allow an exact positioning of the solution within the microfluidic channel/device. The technology is also useful for the 1 -step development of leach-proof Fc-binding proteins- or antibody-bound immunoaffinity columns, which are used in all biopharmaceuticals in the manufacturing of drugs/biologies. The advantage of the developed technology is that the APTES-functionalization and the biomolecular binding can be performed simultaneously in 1 - step on various materials i.e. agarose, silica monoliths/porous silicon/derivatives, nanocrystalline cellulose, chitosan, nanoparticles, nanomaterials (carbon nanotubes, graphene, and quantum dots), nanocomposites, polymers, dendrimers and magnetic/paramagnetic beads.

The technology is also of use in bioanalytical applications, biosensors, diagnostics and assay development for the signal enhancement. The signal enhancement can be done based on the increase in surface area (using nanomaterials, beads, nanoparticles, porous materials etc.) or by increasing the number of binding sites (using polymers, dendrimers, etc.) or by a combination of both.

The technology is also highly useful along with our previously developed proprietary

multisubstrate-compatible modified microtiter plate format, where it will enable the cheaper preconfirmation of immunoassays or bioanalytical applications on various biochip substrates for different assay formats.

This technology is also ideal for the one-step immobilization of antibodies on electrodes for the development of antibody-based electrochemical biosensors.

The invented technology can also be applied for the 1-step immobilization of other biomolecules such as lectins, Fc binding proteins, single domain antibodies, antibody fragments, and nucleic acids, which have free carboxyl groups for biomolecular conjugation.

The invention will also be useful for generating large surfaces or areas coated with antibodies, which is of special interest for array replication. The functionalised copy surfaces are needed to enable copies of microarrays. The copy surfaces, equivalent to the copied paper by Xerox, bear antibodies, functional proteins or molecules, which interact in a defined way with proteins (like biotin, streptavidin, nitrilotriacetic acid (NTA)). The state of the art generation of the copy surfaces is a multi-step process with at least three different steps i.e. activation, first

functionalization with silanes and a second functionalization with proteins or molecules. In some cases, additional activation steps between silane and protein have to be provided. With respect to the existing state of the art techniques, the 1-step immobilization of antibodies or functional binders like biotin or NTA-groups is a clear improvement in terms of ease of use, costs and time consumption.

Acronyms and Definitions

FIGURES

The figures provided herein represent examples of particular embodiments of the invention and are not intended to limit the scope of the invention. The figures are to be considered as providing a further description of possible and potentially preferred embodiments that enhance the technical support of one or more non-limiting embodiments.

Figure 1. Schematic of the developed 1-step antibody immobilization strategy for CRP sandwich ELISA.

Figure 2. One-step antibody immobilization-based highly-sensitive and rapid sandwich IA for human C-reactive protein (CRP). (A) Detection of HFA concentrations in PBS buffer and CRP concentrations spiked in diluted human plasma and diluted whole blood, (B) Experimental process controls, (C) Comparison of the developed ELISA with the commercially-available conventional ELISA, (D) Correlation of the developed ELISA with the commercially-available conventional ELISA, and (E) Stability of anti-CRP Ab-bound MTPs (stored in 0.1 M PBS, pH 7.4 at 4°C) for 8 weeks. All experiments were done in triplicate with the error bars representing standard deviation.

Figure 3. Optimization of (A) APTES concentration and (B) incubation time for the developed 1- step antibody immobilization-based CRP ELISA. The APTES concentration used in (A) is the final concentration of APTES after mixing 1 :1 (v/v) with capture anti- CRP antibody. All experiments were done in triplicate and the error bars represent standard deviation. Figure 4. Direct ELISA for HRP based on the developed 1-step antibody immobilization strategy; our previously developed multi-step covalent antibody immobilization strategy (Nature Protocols 6(4), 23-33, 201 1 ), and the convention passive adsorption-based procedure as employed in commercial ELISA kits. All experiments were done in triplicate. The error bars represent standard deviation.

Figure 5. The percentage change in reflectivity due to the 1-step immobilization of anti-CRP capture antibodies on piranha-treated surface plasmon resonance gold chip using the flow-rate of 10 μΙ_Ληίη.

Figure 6. Highly-sensitive rapid sandwich IA for human fetuin A. (A) Schematic of the IA procedure, (B) Comparison of the developed IA with our previously developed and commercial IA procedures, (C) Experimental process controls, (D) Detection of HFA concentrations spiked in diluted human plasma and whole blood, and (E) Stability of anti-HFA Ab-bound MTPs (stored in 0.1 M phosphate-buffered saline (PBS), pH 7.4 at 4°C). All experiments were done in triplicate and the error bars represent standard deviation.

Figure 7. Optimization of (A) APTES concentration and (B) incubation time for the developed 1- step antibody immobilization-based HFA ELISA. The APTES concentration used in (A) is the final concentration of APTES after mixing 1 :1 (v/v) with capture anti-HFA antibody. All experiments were done in triplicate and the error bars represent standard deviation.

Figure 8. One-step kinetics-based HFA ELISA using 1 -step antibody immobilization procedure. (A) Schematic of bioanalytical procedure, (B) Detection of HFA in buffer and HFA spiked in diluted human serum and whole blood, (C) Specific detection of HFA with respect to various experimental controls. Optimization of (D) incubation time and (E) number of washings in the developed IA. (F) Stability of capture anti-HFA antibody-bound MTPs stored in 0.1 M PBS, pH 7.4 at 4°C.

Figure 9. Developed Surface Plasmon Resonance (SPR)-based immunoassay (IA) for human fetuin A (HFA). (A) Schematic of bioanalytical procedure, (B) Comparison of the developed IA with our previously developed and commercial IA formats, (C) Detection of HFA in various sample matrices, (D) Sensorgram for the detection of various HFA concentrations in HEPES buffered saline (HBS) buffer, (E) consecutive HFA lAs on the same anti-HFA antibody-bound SPR chip for detecting 5 ng mL^"1 HFA, when it was regenerated after each IA by treatment with glycine-HCI (10 mM, pH 2.0), and (F) Stability of anti-HFA antibody-bound SPR chip stored at 4°C, as determined by the detection of 5 ng mL^"1 HFA.

Figure 10. Schematic of the developed 1-step antibody immobilization-based human lipocalin-2 (LCN2) sandwich ELISA using graphene nano platelets (GNPs).

Figure 11. (A) Human LCN2 immunoassays using the developed GNPs-based, previously developed (without GNPs) and conventional sandwich immunoassay procedures. (B) Specific human LCN2 detection with respect to various experimental process controls. (C) Detection of human LCN2 in buffer, whole blood, serum and plasma by the developed GNPs-based immunoassay. (D) Stability of the anti-LCN2 antibody-bound MTPs when stored in 0.1 M PBS, pH 7.4 at 4 °C for 8 weeks. All experiments were done in triplicate, while the error bars represent standard deviation.

Figure 12. (A) Optimization of APTES concentration used for the dispersion of GNPs in the developed GNPs-based immunoassay procedure. (B) Optimization of GNPs-functionalization time using the optimized APTES concentration. (C) Demonstration of the developed GNPs- based immunoassay procedure to perform human LCN2 sandwich immunoassays on various commercially-relevant substrates i.e. polystyrene (PS), poly(methyl methacrylate) (PMMA), Zeonex™ (Znx), Zeonor™ (Znr), polycarbonate (PC) and cellulose acetate (CA). All

experiments were done in triplicate, while the error bars represent standard deviation.

Figure 13. Correlation of developed 1-step antibody immobilization-based sandwich ELISA using GNPs with the commercial sandwich ELISA kit for detecting carious concentrations of human LCN2 spiked in diluted human plasma. All experiments were done in triplicate, while the error bars represent standard deviation.

EXAMPLES

The examples provided herein represent practical support for particular embodiments of the invention and are not intended to limit the scope of the invention. The examples are to be considered as providing a further description of possible and potentially preferred embodiments that demonstrate the relevant technical working of one or more non-limiting embodiments.

Experimental Example 1 : Antibody immobilization based sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for the determination of C-reactive protein (CRP).

In a preferred embodiment, the method comprises the steps of: generation of hydroxyl groups on substrate by KOH pre-treatment; one-step covalent binding of capture anti-human CRP antibody (dispersed in 3-aminopropyltriethoxysilane (APTES)) to the microtiter plate (MTP); blocking the non-specific protein binding sites by BSA; detection of CRP in samples; binding of biotinylated anti-human CRP antibody; binding of streptavidin-horseradish peroxidise (SA-HRP); TMB substrate assay; stopping the reaction with 2N sulphuric acid; and, measuring the optical density at 450 nm (with reference at 570 nm). See Figure 1 for an overview.

C-reactive protein (CRP), a 1 18 kDa pentameric protein consisting of five non-covalently bonded and non-glycosylated identical subunits of 206 amino acids each, was first identified in 1930 by William S. Tillet and Thomas Francis in the sera of patients acutely infected with pneumococcal pneumonia. It is a member of a class of acute-phase reactants that mediates innate and adaptive immunity. It is an early indicator of infectious or inflammatory conditions and is usually elevated in patients with neonatal sepsis, meningitis and occult bacteremia (J. Basic Appl. Sci. 9, 496-499, 2013). CRP is produced by hepatocytes in response to a variety of inflammatory cytokines, such as interleukin (IL)-6, IL-1 and tumor necrosis factor alpha, in case of infection or tissue inflammation. The CRP may also be elevated in urinary tract infections, pancreatitis, pneumonia and pelvic inflammatory disease. The American Heart Association/Center for Disease Control guidelines in 2010 identified CRP as the best inflammatory marker for use in clinical practice. CRP has been demonstrated to be an independent strongest predictor of cardiovascular events such as heart attacks, ischemic stroke, coronary artery disease, and acute myocardial infarction. Recently, it has also been demonstrated to be an independent predictor for the development of diabetes in men. CRP is also a marker for atherosclerotic cardiovascular risk. CRP levels are important indicators of cardiac tolerance and are thus associated with cardiorespiratory fitness. CRP and IL-6 play important roles in the growth and progression of malignant tumors such as esophageal cancer. The hsCRP levels and other markers may predict the development of dementia, including that in the Alzheimer's disease. It can predict long-term cardiovascular risk in individuals without any prior evidence of

cardiovascular disease. The repeated CRP measurements in an acute setting provide clinicians valuable information to establish the correct disease diagnosis and to refrain the unnecessary use of antibiotics.

The CRP levels are significantly important for the diagnosis of neonatal sepsis as it is the best indicator of neonatal sepsis. The precise and early diagnosis of infected neonates, which is hindered at the moment due to unreliable clinical signs and absence of good diagnostic tests, is critical. The serial measurements on 2nd and 3rd days are more informative in comparison to a single CRP measurement as there are physiological changes in CRP levels in neonates in the initial days. Although enzyme-linked immunosorbent assay (ELISA) has always been the gold standard for the detection and quantification of CRP, a wide range of analytical techniques and assay formats have also been devised. The CRP levels in the normal human serum are usually less than 10 mg/L (10 μg/mL). The median physiological serum concentration of CRP in humans is 0.8 mg/L. However, they can reach up to 350-400 mg/L in disease states. The CRP levels increase in the first 6-8 h with peak at -48 h. Thereafter, it falls when the inflammatory stimulus is removed with a half-life of 18 h. The CRP levels are in the ranges of 10-40 mg/L, 40-200 mg/L, and > 200 mg/L in cases of mild inflammation and viral infections, active inflammation and bacterial infections, and severe bacterial infections and burns, respectively. The CRP levels greater than the cut-off point of 3 mg/L are associated with an increased risk of occlusive arterial disease, especially acute coronary syndrome.

The two different CRP concentration ranges, normal (0.2-480 mg/L) and high sensitivity (0.08-80 mg/L), need to be detected in neonatal sepsis. The CRP levels greater than the cut-off point of 5 mg/L are indicative of neonatal sepsis. Initially, the high sensitivity CRP assay is performed. But if the CRP levels are >80 mg/L, the normal CRP assay is also done.

Materials

Phosphate buffered saline (Cat.# 18912-014; PBS, pH 7.4) was procured from Invitrogen, while Tween 20 and Nunc microwell 96 well polystyrene plate (Cat.# 12-565-31 1 ) were purchased from Carl Roth GmbH and Fisher Scientific, respectively. Potassium hydroxide (KOH) and 3- APTES were obtained from Sigma-Aldrich. The human CRP Duoset kit's (DY1707) components, i.e. anti-human CRP capture antibody, recombinant human CRP and biotinylated anti-human CRP detection antibody, were procured from RnD Systems, USA. 3,3 ,5,5 - tetramethylbenzidine (TMB) substrate, stop solution, bovine-serum albumin (BSA) and streptavidin-conjugated horseradish peroxidase (SA-HRP) were bought from Sigma-Aldrich, Germany. The human whole blood (Cat.# 232754, HQ-Chex Level 2) was procured from Streck, USA, while CRP-free human serum (Cat.# 8CFS) was purchased from HyTest Ltd., Finland. The autoclave was from Systec GmbH, Germany, while the MTP reader used was Perkin Elmer Wallac VICTOR 1420 Multilabel Counter. All buffers and solutions were prepared in autoclaved ultrapure water-DNase and RNase free (Cat. # 10977; Gibco, Germany). The binding and washing buffers employed for the developed CRP ELISA were PBS with 0.1 % BSA and PBS with 0.05% Tween 20 (PBST), respectively. The working aliquots of commercial lyophilized human CRP were made in 20 mM Tris-HCI, pH 8.0 with 0.1 % BSA (as mentioned in the product brochure), while the CRP spiking was done in diluted human whole blood and serum (diluted in the binding buffer). The dilutions of KOH and 3-APTES were made in DIW. In the developed ELISA, the DIW and PBST washings were done with 300 μΙ_ of the respective solutions, while 100 μΙ_ was taken for each of the various solutions i.e. 1 % KOH, capture anti-CRP antibody solution (where anti-CRP Ab was mixed with 1 % APTES in the ratio of 1 :1 (v/v)), CRP, biotinylated anti-CRP detection antibody, HRP-conjugated streptavidin, and TMB substrate.

One-step antibody immobilization procedure

The capture anti-CRP antibody (5 μg/mL in PBS) was mixed with 1 % APTES in the ratio of 1 :1 (v/v). This resulted in its final concentration of 2.5 μg/mL in 0.5% APTES, which was added to the MTP wells and incubated for 30 min at RT. The anti-HFA antibody-bound wells were then washed five times with PBS. The above-mentioned procedure works for normal polystyrene- based MTP. However, for other substrates that can be employed using our modified MTP format, the MTP wells should be pretreated with 1 .0% (w/v) KOH at RT for 10 min followed by five DIW washes. The KOH-pretreatment generates the desired hydroxyl groups, which is required for the 1-step antibody immobilization on various substrates.

Method steps in detail

100 μΙ_ of 1 % KOH was provided to each well of the normal Nunc microtiter plate (MTP) and left incubated for 10 min. The KOH-pretreated MTP was then washed 5 times with 300 μΙ_ of ultrapure water.

100 μΙ_ of 5 μg/mL of capture anti-CRP antibody (prepared in 1 % APTES) was then dispensed to each of the desired MTP's wells that are required for the CRP assay.

The MTP was incubated for 30 min at room temperature (RT) after covering with parafilm (sealing films may also be used).

- The Ab-coated MTP was washed 5 times with 300 μΙ_ of washing buffer (PBS).

- The Ab-coated MTP was then blocked with 300 μΙ_ of 5% BSA by incubating for 1 h at RT.

The BSA-blocked Ab-coated MTP was washed 5 times with 300 μΙ_ of washing buffer.

The BSA-blocked Ab-coated MTP was then provided with varying concentrations of CRP in triplicate and left incubated at RT for 1 h.

- The CRP-bound MTP was washed 5 times with 300 μΙ_ of washing buffer.

The CRP-bound MTP was then provided with 100 μΙ_ of detection antibody, i.e. biotinylated anti-CRP antibody pre-bound to SA-HRP, and incubated for 1 h at RT. (Note: 0.17 μg/mL each of biotinylated anti-human CRP antibody and 1 :3000 diluted streptavidin-HRP were mixed in the ratio of 1 : 1 and incubated for 20 min just before usage.)

- The detection Ab-bound MTP was washed 5 times with 300 μΙ_ of washing buffer. The detection Ab-bound MTP was then incubated with 100 μΙ_ of TMB substrate and left incubated for 17 min at RT.

The enzyme-substrate reaction was stopped by adding 50 μΙ_ of the stop solution. (2 N H₂S0₄ can also be used as the stop solution.)

- The absorbance of the final solution was measured at the specific wavelength of 450 nm and the reference wavelength of 570 nm. The results were mentioned as 450-570 nm.

Determination of Analytical Characteristics

The results are plotted using the four-parameter logistic-based standard curve analysis, where O.D. values₄₅onm - 570nm (Mean ± S.D.) are plotted versus the log of CRP concentrations on the X-axis. The SigmaPlot (or OriginLab) software-derived analytical parameters i.e. min, max, slope and EC₅₀ are taken from the software analysis report. The calculations for mean, standard deviation and percentage coefficient of variance (%CV) are performed by the mathematical functions of MS Excel 2010. The inter-day variability of the assay is calculated from five assay repeats in triplicate on five consecutive days. The intra-day variability of the assay is calculated from five assay repeats in triplicate within a day. The dynamic range of the assay starts from the first point on the lower sigmoidal and ends at the last point before saturation on the upper sigmoidal. The linear range of the assay covers the assay points in the sigmoidal curve that show linearity, i.e. first linear point after the lower sigmoidal and the last linear point before the start of upper sigmoidal. The specificity of the assay will be tested for a particular concentration that is near the EC50 in the presence of interferences that are usually prevalent in the actual sample matrix. This will be done at the pathophysiological levels of the interferences. The assay will be tested on the diluted commercial human serum spiked with various concentrations of CRP.

Developed CRP ELISA

The anti-CRP antibody-bound MTP wells were blocked with 1 % (v/v) BSA (diluted in 0.1 M PBS, pH 7.4) for 30 min at RT and subsequently washed five times with PBS. The antibody-coated MTP wells were then incubated with CRP (varying concentration; 3.9-4000 pg ml_^"1) for 1 h at RT, and, subsequently washed five times with PBS. Thereafter, biotinylated anti-CRP detection antibody (0.17 μg ml_^"1) was provided to all MTP wells and incubated for 1 h at RT followed by five PBS washes. Subsequently, HRP-conjugated streptavidin, at a dilution of 1 :3000, was added to all MTP wells and incubated for 20 min at RT followed by five PBS washes. The TMB substrate was then added (as per manufacturer's guidelines) and the enzyme-substrate reaction was stopped after 17 min by adding 50 μΙ_ of 2N H₂S0₄. The absorbance was measured at 450 nm with reference at 570 nm. All experiments were carried out in triplicate with zero ng/mL CRP (in 0.1 M PBS, pH 7.4 with 0.1 % BSA) as control, whose absorbance was subtracted from all assay values. The conventional sandwich ELISA was performed as per the manufacturer's guidelines provided in the product information sheet without any modification. Various experimental process controls were also employed to determine the efficiency of BSA blocking; non-specific interactions of BSA with CRP, biotinylated anti-CRP antibody and SA-HRP; and, non-specific interaction of capture anti-CRP antibody with biotinylated anti-CRP antibody. All datasets were subjected to standard curve analysis using SigmaPlot software, version 1 1 .2. The EC50, R² and hillslope values were determined from the report generated by the software during standard curve analysis based on the four-parameter logistic function. The analytical sensitivity, limit of detection (LOD), and the intra- and inter-day variability were determined by the standard procedures, as specified in our previous reports (Anal. Chem. 82, 7049-7052, 2010; Nat. Protoc. 6, 439-445, 2011; Biosens. Bioelectron. 40, 297-302, 2013; Analyst 136, 4431-4435, 2011).

Results

Data is shown in Figures 2 and 3. The precise determination of human C-reactive protein (CRP) is essential in neonatal sepsis, cardiovascular diseases, meningitis and infectious/inflammatory conditions. We have developed 1 -step antibody immobilization-based rapid immunodiagnostic (ID) kit for CRP, which is analytically superior to the commercial CRP sandwich ELISA kit and our previously developed procedures (Anal. Chem. 82, 7049-7052, 2010; Nat. Protoc. 6, 439- 445, 2011; Biosens. Bioelectron. 40, 297-302, 2013). The 1-step antibody immobilization procedure is multisubstrate-compatible and leads to the leach-proof binding of antibodies. It involves just 30 min incubation of antibody solution [anti-CRP capture antibody and 1 % 3- aminopropyltriethoxysilane (APTES) mixed 1 :1 (v/v)] on the microtiter plate (MTP). The subsequent process steps are similar to that in the commercial kit. APTES acts both as dilution agent for antibodies and as surface functionalization agent for MTP. The developed ID kit has significantly reduced the sandwich immunoassay duration from 19 h (in commercial kit) to 4 hours with better analytical performance. It has dynamic range of 3.9-4000 pg/mL with linearity between 125-2000 pg/mL (Fig. 2B), which can detect the entire pathophysiological range of human CRP in serum (0.08-480 μg/mL) after appropriate sample dilution. The limit of detection, half-maximal effective concentration, inter-day and intra-day variability were 28 pg/mL, 827 pg/mL, 1 .2-10.1 %, 0.1 -8.9%, respectively. The developed CRP ELISA detected CRP in buffer and human serum (Fig. 2A) without any interference from involved process steps and CRP assay components (Fig. 2B). It was superior to the conventional ELISA (Fig. 2C) in terms of increased detection range, improved sensitivity, significantly reduced overall immunoassay duration and cost-effectiveness. 0.5% (v/v) APTES (concentration after mixing with anti-CRP antibody) was found to be the optimized final concentration of APTES (Fig. 3A), while 0.5 h was the optimized duration for 1-step antibody immobilization (Fig. 3B). The developed ELISA determines precisely CRP as it had perfect correlation with the commercial kit (Fig. 2D). The antibody-bound MTPs, stored at 4°C in 0.1 M phosphate-buffered saline (PBS), pH 7.4, were found to be highly stable as there was no decrease in their functional activity even after 8 weeks. Therefore, it can be reliably employed for the detection of CRP in clinical diagnostics.

Direct ELISA for HRP

Comparative analysis between the developed 1 -step antibody immobilization strategy of the present invention, our previously developed multi-step covalent antibody immobilization strategy (Nature Protocols 6(4), 23-33, 201 1 ) and the convention passive adsorption-based procedure as employed in commercial ELISA kits was carried out, as demonstrated in Figure 4. The developed 1-step antibody immobilization strategy was employed for the development of direct ELISA for horseradish peroxidase (HRP) (Fig. 4). 0.5% (v/v) APTES and 0.5 h were the optimized final concentration and duration, respectively, for the 1 -step antibody immobilization. The developed ELISA was the most sensitive ELISA format in comparison to our previously developed multistep procedure (based on covalent antibody immobilization) (Nature Protocols 6(4), 23-33, 201 1 ) and the conventional passive adsorption-based ELISA (as employed in commercial kit). The overall duration of conventional ELISA was about 18 h. It was reduced to 4.5 h by our previously developed covalent ELISA using multistep procedure on APTES- functionalized MTP (Nature Protocols 6(4), 23-33, 201 1 ). However, the developed ELISA based on 1 -step antibody immobilization has reduced the overall duration to just 2 h. It has significantly reduced the assay cost by decreasing the complexity and completely obviating the need of prebound antibody-coated MTPs. Moreover, the procedure is multisubstrate-compatible and can be employed for developing immunoassay on various substrates.

Experimental Example 2: Antibody immobilization-based human fetuin A ELISA

Materials

Phosphate buffered saline (PBS, 0.1 M, pH 7.4), 3,3 ,5, 5 -tetramethylbenzidine (TMB) substrate kit and bovine-serum albumin (BSA) were purchased from Thermo Scientific, while potassium hydroxide (KOH) and 3-APTES were obtained from Sigma-Aldrich. The human plasma and whole blood were procured from Biological Specialty Corp., USA and Streck, USA, respectively. The HFA/AHSG Duoset kit, with all HFA assay components, was procured from RnD Systems, USA. The capture antibody used was mouse anti-HFA, while detection was achieved through the use of biotinylated goat anti-HFA antibody and streptavidin-conjugated horseradish peroxidase (HRP). All assay components were reconstituted in 0.1 M PBS, pH 7.4 with 1 % (v/v) BSA. Buffers and solutions were prepared in Milli-Q deionised water (DIW). The dilutions of all HFA assay components and BSA were made in 0.1 M PBS, pH 7.4, whereas KOH and 3- APTES were diluted in DIW. The HFA spiked samples were prepared by mixing various concentrations of HFA in diluted human plasma and whole blood. The HFA dilutions were made in BSA-preblocked glass vials, prepared by incubating with 1 % (w/v) BSA for 30 min, to minimize analyte loss due to non-specific adsorption on sample tube surfaces and/or altered immunogenicity (Analyst 136, 1406-1411, 2011). In the developed ELISA, the DIW and PBS washings were done with 300 μί of the respective solutions, while 100 μί was taken for each of the various solutions i.e. 1 % KOH, capture anti-HFA antibody solution (where anti-HFA Ab was mixed with 1 % APTES in the ratio of 1 :1 (v/v)), HFA, biotinylated anti-HFA detection antibody, HRP-conjugated streptavidin, and TMB substrate. The assay temperature was maintained at 37°C using a thermostat from Labnet International Inc., USA, while the absorbance was measured by Tecan Infinite M200 Pro microplate reader from Tecan GmbH, Austria.

Poly(methyl methacrylate) (PMMA), polystyrene (PS) and Zeonex™ (Znx) slides were purchased from Microfluidic Chip Shop GmbH, Jena, Germany; polycarbonate (PC) and cellulose acetate (CA) were from VTT, Finland; and, Zeonor™ (Znr) was procured from Zeon Chemicals, Germany. The pressure-sensitive adhesive (PSA) and bottomless 96-well ELISA plates were bought from Adhesive Research, Ireland and Greiner Labortechnik, Germany, respectively.

One-step antibody immobilization procedure The capture anti-HFA antibody (8 μg/mL in PBS) was mixed with 1 % APTES in the ratio of 1 :1 (v/v). Thereafter, this capture anti-HFA antibody solution, having final concentration of 4 μg/mL in 0.5% APTES, was added to the MTP wells and incubated for 30 min at RT. The anti-HFA antibody-bound wells were then washed five times with PBS.

The procedure works for normal polystyrene-based MTP, but for other substrates (as employed in our modified MTP format) the MTP wells are pretreated with 1.0% (w/v) KOH at RT for 10 min followed by five DIW washes. The KOH-pretreatment generates the desired hydroxyl groups, which is required for the 1-step antibody immobilization on various substrates.

Developed HFA ELISA

The anti-HFA antibody-bound MTP wells were blocked with 1 % (v/v) BSA (diluted in 0.1 M PBS, pH 7.4) for 30 min at 37°C and subsequently washed five times with PBS. The antibody-coated MTP wells were then incubated with HFA (varying concentration; 4.9 pg ml_^"1-20 ng ml_^"1) for 1 h at 37°C, and, subsequently washed five times with PBS. Thereafter, biotinylated anti-HFA detection antibody (200 ng/mL) was provided to all MTP wells and incubated for 1 h at 37°C followed by five PBS washes. Subsequently, HRP-conjugated streptavidin, at a dilution of 1 :200, was added to all MTP wells and incubated for 20 min at 37°C followed by five PBS washes. The TMB substrate was then added (as per manufacturer's guidelines) and the enzyme-substrate reaction was stopped after 20 min by adding 50 μΙ_ of 2N H₂S0₄. The absorbance was measured at 450 nm with reference at 540 nm. All experiments were carried out in triplicate with zero ng/mL HFA (in 0.1 M PBS, pH 7.4) as control, whose absorbance was subtracted from all assay values. The conventional sandwich ELISA was performed as per the manufacturer's guidelines provided in the product information sheet without any modification. Various experimental process controls were also employed to determine the efficiency of BSA blocking; non-specific interactions of BSA with HFA, biotinylated anti-HFA antibody and SA-HRP; and, non-specific interaction of capture anti-HFA antibody with biotinylated anti-HFA antibody. All datasets were subjected to standard curve analysis using SigmaPlot software, version 1 1 .2. The EC50, R² and hillslope values were determined from the report generated by the software during standard curve analysis based on the four-parameter logistic function. The analytical sensitivity, limit of detection (LOD), and the intra- and inter-day variability were determined by the standard procedures, as specified in our previous reports (Anal. Chem. 82, 7049-7052, 2010; Nat. Protoc. 6, 439-445, 201 1 ; Biosens. Bioelectron. 40, 297-302, 2013; Analyst 136, 4431-4435, 201

Results

Data is shown in Figure 6 and 7. A highly-sensitive rapid sandwich immunoassay (IA) was developed for the detection of human fetuin A (HFA), which is a specific biomarker for hepatocellular carcinoma and atherosclerosis, and associated with arthritis, cardiovascular diseases, malaria, diabetes and metabolism-associated syndrome. It employs the one-step antibody (Ab) immobilization procedure, where the anti-HFA Ab, mixed with 1 % (v/v) 3- aminopropyltriethoxysilane (APTES) in the ratio of 1 :1 (v/v), were dispensed to 96-well microtiter plate (MTP) wells and left incubated for 30 min, which leads to the leach-proof binding of capture anti-HFA Ab to the MTP (Fig. 6A). The developed IA has significantly reduced overall immunoassay duration, many-fold higher sensitivity, reduced complexity and lower cost than the conventional and our previously-developed IA procedures (Anal. Chem. 82, 7049-7052, 2010; Nat. Protoc. 6, 439-445, 2011; Biosens. Bioelectron. 40, 297-302, 2013) (Fig. 6B). It detects HFA in the dynamic range of 4.9-20,000 pg ml_^"1 with the limit of detection and analytical sensitivity of 7 pg ml_^"1 and 10 pg ml_^"1. The intra- and inter-day variability were 1 .2-8.5 and 2.1- 10.2, respectively, while the EC₅₀ was 2.6 ng ml_^"1. The developed IA had no interference with the immunological reagents (Fig. 6C) and correlated well with the commercial kit. It detects HFA concentrations spiked in complex patient sample matrices i.e. diluted whole blood and plasma (Fig. 6D). The Ab-bound MTPs, stored at 4°C in 0.1 M phosphate-buffered saline (PBS), pH 7.4, were found to be highly stable as there was no decrease in their functional activity even after 6 weeks. Therefore, the developed IA can be reliably employed in clinical, industrial and other bioanalytical settings. It has tremendous potential for the development of highly-sensitive in vitro diagnostic kits and biosensors for numerous disease biomarkers and analytes.

Experimental Example 3: Antibody immobilization-based rapid HFA ELISA using 1 -step kinetics

Materials

Phosphate buffered saline (PBS, 0.1 M, pH 7.4), 3,3 ,5, 5 -tetramethylbenzidine (TMB) substrate kit and bovine-serum albumin (BSA) were purchased from Thermo Scientific, while potassium hydroxide (KOH) and 3-APTES were obtained from Sigma-Aldrich. The human plasma and whole blood were procured from Biological Specialty Corp., USA and Streck, USA, respectively. The HFA/AHSG Duoset kit, with all HFA assay components, was procured from RnD Systems, USA. The capture antibody used was mouse anti-HFA, while detection was achieved through the use of biotinylated goat anti-HFA antibody and streptavidin-conjugated horseradish peroxidase (HRP). All assay components were reconstituted in 0.1 M PBS, pH 7.4 with 1 % (v/v) BSA. Buffers and solutions were prepared in Milli-Q deionised water (DIW). The dilutions of all HFA assay components and BSA were made in 0.1 M PBS, pH 7.4, whereas KOH and 3- APTES were diluted in DIW. The HFA spiked samples were prepared by mixing various concentrations of HFA in diluted human plasma and whole blood. The HFA dilutions were made in BSA-preblocked glass vials, prepared by incubating with 1 % (w/v) BSA for 30 min, to minimize analyte loss due to non-specific adsorption on sample tube surfaces and/or altered immunogenicity (Analyst 136, 1406-1411, 2011). In the developed ELISA, the DIW and PBS washings were done with 300 μΙ_ of the respective solutions, while 100 μΙ_ was taken for each of the various solutions i.e. 1 % KOH, capture anti-HFA antibody solution (where anti-HFA Ab was mixed with 1 % APTES in the ratio of 1 :1 (v/v)), HFA, biotinylated anti-HFA detection antibody pre-bound to HRP-conjugated streptavidin, and TMB substrate. The biotinylated anti-hCRP detection antibody (200 ng ml.^"1) was incubated in the ratio of 1 :1 (v/v) with SA-HRP (diluted

1 :200), which led to the formation of biotinylated anti-HFA detection antibody pre-bound to HRP- conjugated streptavidin. The assay temperature was maintained at 37°C using a thermostat from Labnet International Inc., USA, while the absorbance was measured by Tecan Infinite M200 Pro microplate reader from Tecan GmbH, Austria.

One-step antibody immobilization procedure The capture anti-HFA antibody (8 μg/mL in PBS) was mixed with 1 % APTES in the ratio of 1 :1 (v/v). Thereafter, this capture anti-HFA antibody solution, having final concentration of 4 μ9ΛηΙ_ in 0.5% APTES, was added to the MTP wells and incubated for 30 min at RT. The anti-HFA antibody-bound wells were then washed five times with PBS.

Developed 1-step kinetics-based HFA ELISA

The anti-HFA antibody-bound MTP wells were blocked with 1 % (v/v) BSA (diluted in 0.1 M PBS, pH 7.4) for 30 min at 37°C and subsequently washed five times with PBS. The pre-blocked MTPs can be stored in 0.1 M PBS, pH 7.4 at 4°C for up to 2 months. They were then provided with biotinylated anti-hCRP detection antibody pre-bound to SA-HRP and HFA (varying concentrations; 0.1 1-283 ng ml_^"1) in buffer, diluted human serum and whole blood. The MTPs were left incubated at 37°C for 15 min and then washed twice with PBS. The TMB substrate was then added (as per manufacturer's guidelines) and the enzyme-substrate reaction was stopped after 14 min by adding 50 μΙ_ of 2N H₂S0₄. The absorbance was measured at 450 nm with reference at 540 nm. All experiments were carried out in triplicate with zero ng/mL HFA (in 0.1 M PBS, pH 7.4) as control, whose absorbance was subtracted from all assay values. The conventional sandwich ELISA was performed as per the manufacturer's guidelines provided in the product information sheet without any modification. Various experimental process controls were also employed to determine the efficiency of BSA blocking; the non-specific interactions of BSA with HFA, biotinylated anti-HFA antibody and SA-HRP; and, the non-specific interaction of capture anti-HFA antibody with biotinylated anti-HFA antibody. All datasets were subjected to standard curve analysis using SigmaPlot software, version 1 1.2. The EC50, R² and hillslope values were determined from the report generated by the software during standard curve analysis based on the four-parameter logistic function. The analytical sensitivity, limit of detection (LOD), and the intra- and inter-day variability were determined by the standard procedures, as specified in our previous reports (Anal. Chem. 82, 7049-7052, 2010; Nat. Protoc. 6, 439-445, 2011; Biosens. Bioelectron. 40, 297-302, 2013; Analyst 136, 4431-4435, 2011).

Results

Data is shown in Figure 8. A novel 1 -step kinetics-based immunoassay (IA) was developed for the rapid detection of human fetuin A (HFA) in just 30 min. It involves the preliminary preparation of anti-HFA antibody (Ab)-bound and bovine serum albumin (BSA)-blocked 96-well microtiter plate (MTP) that can be effectively stored at 4°C in 0.1 M PBS, pH 7.4 for extended duration. The IA (Fig. 8A) involves the sequential dispensing of biotinylated anti-HFA detection Ab pre- conjugated to streptavidin-labeled horse radish peroxidase and analyte sample. The MTP was incubated for 15 min at room temperature, which leads to the formation of sandwich immune complexes, and then washed twice to take out the excess unbound reagents. Finally, the enzyme-substrate reaction was performed by providing the TMB substrate, stopping the reaction after 14 min, and measuring absorbance at 450 nm with reference at 540 nm. The leach-proof Ab-bound MTP was prepared by proprietary 1-step Ab-immobilization strategy, where anti-HFA Ab, mixed with 1 % (v/v) 3-aminopropyltriethoxysilane (APTES) in the ratio of 1 :1 (v/v), was dispensed into MTP wells and incubated for 30 min. The developed IA is the most rapid HFA IA, which has 12- and 7-fold reduced IA duration than the conventional and our previously- developed procedures (Anal. Chem. 82, 7049-7052, 2010; Nat. Protoc. 6, 439-445, 2011), respectively, when Ab-bound and BSA-blocked MTPs were used in all formats. It detects 0.1- 283 ng ml_^"1 of HFA with limit of detection, analytical sensitivity and EC₅₀ of 0.3 ng ml_^"1, 1 ng ml_^"1 and 24.2 ng ml_^"1, respectively. The intra- and inter-day variability were 1.8-7.3 and 2.4- 12.1 , respectively. The developed IA detects HFA-spiked in diluted human serum and whole blood (Fig. 8B). It has no interference with IA components (Fig. 8C); optimized for incubation time (Fig. 8D) and number of washings (Fig. 8E); and, correlates well with the commercial IA with percentage recoveries between 91 -108. The anti-HFA antibody (Ab)-bound and bovine serum albumin (BSA)-blocked MTPs can be effectively stored at 4°C in 0.1 M PBS, pH 7.4 for 8 weeks without any decrease in functional activity (Fig. 8F).

Experimental Example 4: Antibody immobilization-based surface plasmon resonance immunoassay for HFA

Immobilisation for SPR- preliminary assessment

The surface plasmon resonance (SPR)-based real-time and rapid microfluidic 1 -step anti-CRP antibody immobilization on piranha-treated Au SPR chip was performed employing the flow rate of 10 μΙ_Ληίη (Fig. 5). It clearly demonstrates that the developed 1-step antibody immobilization strategy leads to the leach-proof binding of antibodies to the SPR gold chip. The initial experiments have been conducted on a prototype of SPR instrument. However, the final CRP immunoassay along with its complete optimization and analytical characterization as described below is performed on the commercially-available BIAcore SPR systems from GE Healthcare.

Reagents and Materials

EDC, SNHS and 2-(/V-morpholino)ethane sulfonic acid (MES, pH 4.7) were purchased from Thermo Scientific. 3-APTES (purity 98%, w/v), Tween 20, H₂0₂ (30%, v/v) and H₂S0₄ (97.5%, v/v) were procured from Sigma-Aldrich. The human Fetuin A AHSG kit with all the necessary components was obtained from R&D Systems Inc., USA. All buffers and solutions were prepared with 18ΜΩ Milli-Q ultrapure water (UPW) filtered through a 2-μηη filter. Surface Plasmon Resonance (SPR) was performed on BIAcore 3000 from GE Healthcare, Uppsala, Sweden. The SIA kit (BR-1004-05) (containing SPR Au chips), carboxymethyl dextran (CMD)- functionalized Au chips, ethanolamine hydrochloride (1 M, pH 8.5), HBS-EP (0.01 M 4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) pH 7.4, 0.15 M NaCI, 3 mM EDTA, 0.005% v/v surfactant P20) and Glycine-HCI (10 mM, pH 2.0) were purchased from GE Healthcare.

The SPR Au chip was assembled according to the instructions supplied by the manufacturer. HBS-EP was used as the running buffer for BIAcore. All the sample dilutions were made in the running buffer. The dilutions of HFA were made in BSA-preblocked glass vials, prepared by incubating with 1 % (w/v) BSA for 30 min, to minimize the sample loss due to non-specific adsorption on sample tube surfaces and/or effects due to altered immunogenicity (Analyst 136, 1406-1411, 2011).

Developed 1-step antibody immobilization procedure The Au chip was cleaned by treatment with 90 μΙ_ of 1 % (w/v) KOH for 5 min followed by extensive washing with UPW. The capture anti-HFA antibody (200 μg/mL in HBS) was mixed with 1 % APTES in the ratio of 1 :1 (v/v). Thereafter, this 90 μΙ_ of this capture anti-HFA antibody solution, having final concentration of 100 μg/mL in 0.5% APTES, was provided to the Au chip and incubated for 30 min at RT. The anti-HFA antibody-bound Au SPR chip was then washed extensively with HBS.

Previously developed covalent antibody immobilization procedure

Surface cleaning of SI A Au chip and APTES functionalization

The Au chip was cleaned with piranha etch [60 μΙ_ of H₂S0₄ (97.5%, v/v): 30 μΙ_ of H₂0₂ (30%, v/v)] for two minutes followed by extensive washing with UPW. The chip was then incubated with 100 μΙ_ of 2% (v/v) APTES for 1 h at room temperature (RT) in a fume hood followed by five washes with UPW.

EDC activation of anti-HFA antibody

Anti-HFA antibody (990μΙ_ of 100 μg/ml in HBS) was incubated at room temperature for 15 min with 10 μΙ_ of cross-linking solution containing EDC (4 mg/mL) and sulfo-NHS (1 1 mg/mL) in 0.1 M MES buffer, pH 4.7. The procedure led to the activation of carboxyl groups on anti-HFA antibody with EDC. The EDC-activated anti-HFA antibody was captured on the APTES- functionalized Au chip (previously described) for the covalent immobilization strategy.

Antibody immobilization procedure

For the covalent immobilization strategy, 50 μΙ_ of EDC-activated antibody (100 μg/mL) was injected over all the four flow cells of an APTES-functionalized Au chip at a flow rate of 10 μΙ_Ληίη and the baseline was allowed to stabilize. BSA (20 μΙ_ of 1 % (w/v)) was then used for blocking.

Conventional covalent immobilization of anti-HFA antibody on CM5-dextran chip

A CM5 dextran-functionalized Au chip was docked into BIAcore 3000 and primed. Pre- concentration studies were performed in order to obtain the optimum pH of the sodium acetate buffer for use with anti-HFA antibody immobilization (pH range of 4.0-5.0). Further activation was performed at a pH of 4.2 (optimum). Afterwards, the CM5-dextran chip was activated by injecting a 50 μΙ_ solution containing 200 μg of EDC and 550 μg of SNHS in 0.1 M MES buffer, pH 4.7, through all the flow cells at a flow rate of 10 μΙ_Ληίη. Thereafter, 50 μΙ_ of anti-HFA antibody (100 μg/mL) was injected over all the four flow cells of an APTES-functionalized Au chip at a flow rate of 10 μΙ_Ληίη and the baseline was allowed to stabilize. The chip was blocked initially by providing consecutively 20 μΙ_ of 1 M ethanolamine hydrochloride, pH 8.5 followed by 20 μΙ_ of 1 % (w/v) BSA at a flow rate of 10 L/min. Ethanolamine hydrochloride (1 M), pH 8.5 blocks the unreacted ester groups in the CM5-dextran matrix, while BSA blocks the non-specific binding sites on the chip surface.

HFA detection

Fifty microlitres of the dilution buffer (10mM HBS, pH 7.4) was passed through all the flow cells before HFA capture and the resultant changes in the SPR response units (RU) for each of the four flow cells were recorded. Fifty microlitres of HFA at seven different dilutions (0.3, 0.6, 1 .2, 2.5, 5.0, 10.0 and 20.0 ng/mL) were then passed through the flow cells. Subsequently, the RU values obtained for the blanks were subtracted from the RU values obtained for captured HFA of the corresponding flow cells. Once the SPR chip is used for HFA immunoassay, it can be reused by effectively regenerating it to anti-HFA antibody-bound SPR chip by treatment with 20 μΙ_ of 10 mM glycine-HCI, pH 2.0.

The SPR-based HFA detection curves were plotted with SigmaPlot software, version 1 1 .2 using four parameter logistic fit. The EC50, R² and hillslope values were determined from the report generated by the software during standard curve analysis based on the four-parameter logistic function.

Results

Data is shown in Figure 9. We have developed a highly-sensitive, rapid and label-free surface plasmon resonance (SPR)-based immunoassay (IA) for human fetuin A (HFA), which is a specific biomarker for hepatocellular carcinoma and atherosclerosis. It employs one-step procedure for the leach-proof immobilization of anti-HFA capture antibody (Ab) on gold SPR chip in 30 min (Fig. 9A). The Ab, diluted in 1 % (v/v) 3-aminopropyltriethoxysilane (APTES), was dispensed on KOH-treated chip, left incubated for 30 min, and then washed with HEPES- buffered saline (HBS, 10 mM, pH 7.4). All subsequent steps, i.e. blocking, HFA detection and regeneration, were performed in Biacore 3000 at a flow rate of 10 μΙ_Ληίη using HBS as running buffer. The HFA concentrations were made in bovine serum albumin-preblocked sample vials to prevent analyte loss (Analyst 136, 1406-1411, 2011). The developed IA is superior to our previously developed IA and the commercial carboxymethyl (CM5) dextran SPR chip-based IA (Analyst 136, 4431-4435, 2011) in terms of enhanced sensitivity, increased dynamic range, reduced duration, lesser number of steps and low cost (Fig. 9B). The enhanced sensitivity is due to the higher Ab immobilization density. The developed IA detects 0.3-20 ng/mL of HFA spiked in diluted human whole blood and plasma with limit of detection and analytical sensitivity of 0.4 and 0.7; and 0.3 and 0.6, respectively (Fig. 9C,D). The Ab-bound SPR chip, regenerated after each lA with glycine-HCI (10 mM, pH 2.0), was used reproducibly for 35 consecutive HFA lAs without any decrease in its functional activity (Fig. 9E). It can be effectively stored at 4°C for prolonged duration as there was only 18% decrease in its functional activity after 4 months. The developed IA has high precision as it has perfect correlation with the commercial sandwich ELISA kit. Therefore, it can be reliably employed in healthcare, industrial and bioanalytical settings for the detection of other disease biomarkers and analytes.

Experimental Example 5: Antibody immobilization-based HFA ELISA using graphene

Materials

Phosphate buffered saline (PBS, 0.1 M, pH 7.4), 3,3 ,5, 5 -tetramethylbenzidine (TMB) substrate kit and bovine-serum albumin (BSA) were purchased from Thermo Scientific, USA. Potassium hydroxide (KOH) and 3-APTES were obtained from Sigma-Aldrich. Graphene nano platelets (GNPs; diameter 5 μηη) were purchased from Cheap Tubes, USA. The human LCN2 Duoset kit, containing all sandwich ELISA components, i.e. anti-human LCN2 capture antibody, biotinylated anti-human LCN2 detection antibody and streptavidin-conjugated horseradish peroxidase (SA- HRP), was procured from RnD Systems, USA. PBS (0.1 M, pH 7.4) with 1 % (v/v) BSA was used for reconstituting all assay components; PBST (PBS with 0.05% Tween 20) was used as washing buffer; while ultrapure water (UPW) (18 ΜΩ, Direct Q, Millipore) was used for preparing buffers, KOH and 3-APTES. The human whole blood, serum and plasma were purchased from Streck (USA), HyTest Ltd. (Finland) and Biological Specialty Corp. (USA), respectively. The human LCN2-spiked samples were prepared by mixing various concentrations of human LCN2 in 1 :1000 diluted human plasma, serum and whole blood. One mg of GNPs was mixed with 1 ml_ of 0.25% APTES and dispersed by keeping in an ultrasonic bath for 1 h before it is used in the developed immunoassay. The dilutions of human LCN2 were made in BSA-preblocked sample vials in order to minimize the analyte loss due to non-specific binding on sample tube surfaces (Analyst 136, 1406-1411, 2011). In the developed ELISA, the UPW and PBST washings were done with 300 μί of the respective solutions, while 100 μί was taken for each of the various solutions i.e. 1 % KOH, capture anti-human LCN2 antibody solution (where anti- human LCN2 antibody was mixed with GNPs in 0.25% APTES in the ratio of 1 :1 (v/v)), human LCN2, biotinylated anti-human LCN2 detection antibody, HRP-conjugated streptavidin, and TMB substrate. The assay temperature was maintained at 37°C using a thermostat obtained from Labnet International Inc., USA, while the absorbance was measured by Tecan Infinite M200 Pro microplate reader from Tecan GmbH, Austria.

Polystyrene (PS), poly(methyl methacrylate) (PMMA) and Zeonex were procured from

Microfluidic Chip Shop GmbH, Jena, Germany; polycarbonate (PC) and cellulose acetate were from VTT, Finland; and, Zeonor was purchased from Zeon Chemicals, Germany. The pressure sensitive adhesive and bottomless 96-well MTP were bought from Adhesive Research, Ireland and Greiner Labortechnik, Germany, respectively.

One-step antibody immobilization procedure

The capture anti-human LCN2 antibody (4 μg/mL) was mixed with GNPs (1 mg/mL) in 0.25% APTES in the ratio of 1 :1 (v/v). Thereafter, this capture anti-human LCN2 antibody solution, having final concentration of 2 μg/mL antibody, 0.5 mg/mL GNPs and 0.125% APTES, was added to the MTP wells and incubated for 30 min at RT. The anti-HFA antibody-bound GNPs- functionalized wells were then washed six times with PBST.

The procedure works for normal polystyrene-based MTP, but for other substrates (as employed in our modified MTP format) the MTP wells are pretreated with 1.0% (w/v) KOH at RT for 10 min followed by six DIW washes. The KOH-pretreatment generates the desired hydroxyl groups, which is required for the 1-step antibody immobilization on various substrates.

Sandwich immunoassay procedure

The non-specific binding sites on anti-human LCN2 antibody-bound MTP were blocked by treating with 1 % (v/v) BSA for 30 min at 37°C followed by six PBST washes. Subsequently, varying concentrations of human LCN2 (0.6-5120 pg/mL) were incubated in the anti-human LCN2-coated MTP for 1 h at 37°C followed by six PBST washes. Thereafter, biotinylated anti- human LCN2 detection antibody (100 ng/mL) was added, incubated for 1 h at 37°C, and then washed six times with PBST. This was followed by adding SA-HRP (diluted 1 :200) to each MTP well, incubating for 20 min at 37°C and washing six times with PBST. Subsequently, the TMB substrate was added (as per the manufacturer's guidelines) and the enzyme-substrate reaction was stopped after 20 min by adding 50 μΙ_ of 2N H₂S0₄. The absorbance was measured at 450 nm with reference at 540 nm. All the experiments were done in triplicate with zero ng/mL human LCN2 (in 0.1 M PBS, pH 7.4 with 1 % BSA) as control, whose absorbance was subtracted from all the assay values. The conventional sandwich ELISA procedure was followed as per the manufacturer's guidelines provided in the product information sheet without any modification. Various experimental process controls were also employed in order to check the efficiency of BSA blocking; the non-specific interactions of BSA with LCN2, biotinylated anti-LCN2 antibody and SA-HRP; and the non-specific interaction of capture anti-LCN2 antibody with biotinylated anti-LCN2 antibody. All datasets obtained from the developed and conventional human LCN2 sandwich ELISAs were subjected to standard curve analysis using Sigma Plot software, version 1 1.2. The EC50, R² and hillslope values were determined from the report generated by the software during standard curve analysis based on the four-parameter logistic function. The analytical sensitivity, limit of detection (LOD), and the intra- and inter-day variability were determined by the standard procedures, as specified in our previous reports (Anal. Chem. 82, 7049-7052, 2010; Nat. Protoc. 6, 439-445, 2011; Biosens. Bioelectron. 40, 297-302, 2013;

Analyst 136, 4431-4435, 2011).

Table 1. A comparative analysis of developed 1-step antibody immobilization-based human lipocalin-2 sandwich ELISA using GNPs with various commercial-available sandwich ELISA kits and assay formats.

Manufacturer Antibody LOD* Refer to

immobilization

technique

Developed GNPs-based Chemical crosslinking 0.6 pg/mL Reported here assay

Covalent assay Chemical crosslinking 2.5 pg/mL Vashist et al., Procedia

Chem 6, 141-148, 2012

Passive assay Passive adsorption 40 pg/mL Vashist et al., Procedia

Chem 6, 141-148, 2012

R&D Systems, Inc. Passive adsorption 78 pg/mL www.rndsystems.com

Boster Biological Passive adsorption 10 pg/mL www.immunoleader.com Technology Co., Ltd.

Antibody and Passive adsorption 0.4 ng/mL www.antibody.hku.hk Immunoassay Services

Meso Scale Diagnostics Passive adsorption 2.9 pg/mL www.mesoscale.com BioPorto Diagnostics Passive adsorption 0.2 ng/mL www.bioporto.com

CycLex Co., Ltd Passive adsorption 26.7 pg/mL www.cyclex.co.jp

BioVendor Passive adsorption 20 pg/mL www.biovendor.com

Argutus Medical Passive adsorption 0.4 ng/mL www.argutusmed.com

*the lowest concentration detected by the assay.

Table 2. Comparison of developed GNPs-based immunoassay with our previously developed and conventional immunoassays.

Developed Previously Conventional

Developed

(GNPs-based) (without GNPs)

Detection Range (pg/mL) 0.6-5120 2.5-5120 40-5120

LOD (pg/mL) 0.6 3 44

Analytical Sensitivity 0.9 7 80

(pg/mL)

ECso (pg/mL) 41 1 624 821

% CV

Intra-day (n=6) 1.2-1 1.5 1 .1-1 1 .8 1.6-17.2

Inter-day (n=6) 1.6-12.9 1 .9-14.5 2.4-18.1

Assay duration (hours) ~4 ~6 -20

Assays on various Yes Yes No

substrates

Results

Data is shown in Figures 10 to 13. We have developed a highly sensitive immunoassay using graphene nano platelets (GNPs) for the rapid detection of human Lipocalin-2 (LCN2) in plasma, serum and whole blood (Fig. 10 and 1 1 ). It has the dynamic range, linear range, limit of detection and analytical sensitivity of 0.6-5120 pg/mL, 80-2560 pg/mL, 0.6 pg/mL, and 0.9 pg/mL, respectively. It is the most sensitive assay for the detection of LCN2 (Table 1 ), which has 80-fold higher analytical sensitivity and 3-fold lesser immunoassay duration than the

commercially-available sandwich enzyme linked immunosorbent assay (ELISA) kit (Table 2). The functionalization of microtiter plate (MTP) with GNPs, dispersed in 3- aminopropyltriethoxysilane (APTES), provided the increased surface area that leads to higher immobilization density of capture antibodies. Moreover, the generation of free amino groups on GNPs by APTES enables the leach-proof covalent crosslinking of anti-human LCN2 capture antibody. The anti-LCN2 antibody-bound MTPs were highly stable as they did not show any significant decrease in their functional activity when stored at 4°C in 0.1 M PBS for 8 weeks (Fig. 1 1 D). The optimum concentration of APTES was found to be 0.25% (before mixing with capture antibody) (Fig. 12), while the optimum incubation time was 30 min (Fig. 12B). The developed immunoassay was multisubstrate-compatible (Fig. 12C) and correlated well with the

conventional ELISA (Fig. 13), thereby demonstrating its high precision and potential utility for highly sensitive analyte detection in industrial and clinical settings.

Analysis of Biomolecule Density

Table 3. Determination of molecular densities of immobilized anti-HFA antibody and detected amount of HFA when different SPR immunoassay formats, based on various antibody immobilization strategies, were employed.

ARU: Change in resonance units (RU) caused by binding

*Calculated using the commonly used conversion factor i.e. 1000 RU=100 ng/cm².

**Calculated by [Mass density (ng/cm²) / Molecular weight (in ng)]. Molecular weight of anti-HFA antibody and HFA were 150 kDa and 43.5 kDa, respectively. In order to calculate molecular weight in SI units, the conversion factor 1 kDa = 1000 Da = 1000 g was used. The molecular weight of anti- HFA antibody and HFA are 24.9x10^"11ng and 7.0x10 ^~11ng, respectively.

***Calculations were performed for the detection of 5 ng/mL of HFA i.e. the concentration just above the EC₅₀.

Moreover, various CRP immunoassay formats, based on microarrays, centrifugal LabDisk platforms and RlfS, can be developed using the 1 -step antibody immobilization strategy. Va other biomolecules and biochip substrates can also be used apart from the use of different silanes. Additionally, further experimentation shows that the preferred embodiments of the invention provide surprising and unexpected effects, thereby solving the problem of the

invention in a non-obvious fashion.

Claims

1 . Method for covalent immobilisation of a biomolecule to a solid material, comprising a) contacting said solid material with an oxidising agent to activate the solid

material, or providing a pre-activated solid material, wherein said activated solid material comprises carbonyl and/or hydroxyl groups, and b) contacting said activated solid material with a binding solution, wherein said binding solution comprises a mixture of at least a biomolecule to be immobilised and one or more silane components, and the contents of the binding solution are mixed before contacting with said solid material, thereby producing a covalent attachment between said biomolecule and said solid material via an amide bond.

2. Method according to any one of the preceding claims, characterised in that

said biomolecule is a protein, nucleic acid, lectin, polysaccharide or lipid molecule.

3. Method according the preceding claims, characterised in that

said protein is an antibody, antibody fragment, a recombinant protein, Fc-binding protein (protein A, protein G, protein A/G), streptavidin, or recombinant proteins or fragments thereof.

4. Method according to any one of the preceding claims, characterised in that

said solid material comprises a synthetic polymer; agarose; silica-based material, such as glass, bioglass, silica monoliths or porous silica; silicon and silicon derivative based substrates; metal-coated surfaces, such as gold-and silver-coated surfaces;

nanoparticle-/nanocomposite-coated surfaces; surfaces coated with thin oxide films, such as Si0₂, ZnO, Zx0₂, Al₂0₃, NdGa0₃, La01 , Ti0₂, LSAT, MgAI₂0₄; glassy carbon; screen-printed carbon electrodes; cellulose; cellulose acetate; nanocrystalline cellulose; and/or chitosan.

5. Method according to the preceding claim, characterised in that

said synthetic polymer comprises polystyrene (PS), polypropylene (PP), polyethylene PE), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC), water-resistant photopolymer, cyclic olefin copolymer or a cyclo-olefin polymer.

6. Method according to any one of the preceding claims, characterised in that a) the surface area of the solid material is increased by using as solid material a structure with increased surface area such as beads, nanoparticles, porous structures, nanomaterials, such as carbon nanotubes, graphene, and/or quantum dots, nanocomposites and/or magnetic or paramagnetic beads, b) the binding sites of the solid material are increased using polymers with multiple binding sites or dendrimers, or c) a combination thereof.

7. Method according to the preceding claim, characterised in that

the surface area of the solid material is increased by using as solid material graphene nano platelets (GNPs), preferably of a diameter of 0.1 to 100 μηη, more preferably 1 to 10 μηη, such as 5 μηη.

8. Method according to any one of the preceding claims, characterised in that

said oxidizing agent is a hydroxide-containing solution, piranha solution, oxygen plasma treatment or corona discharge treatment.

9. Method according to the preceding claim, characterised in that

said hydroxide-containing solution comprises of sodium hydroxide, potassium hydroxide and/or ammonium hydroxide, preferably potassium hydroxide.

10. Method according to the preceding claim, characterised in that

said hydroxide-containing solution comprises of potassium hydroxide present at a concentration of 0.1 to 10%.

1 1. Method according to the preceding claim, characterised in that

said hydroxide-containing solution comprises of potassium hydroxide present at a concentration of 0.5 to 5%.

12. Method according to any one of the preceding claims, characterised in that

said silane component is 3-aminopropyltriethoxysilane (APTES), (3-aminopropyl)- trimethoxysilane (APTMS), (3-mercaptpropyl)trimethoxysilane (MPTMS) and/or 3- glycidoxypropyltriethoxysilane (GOPTS).

13. Method according to the preceding claim, characterised in that

said binding solution comprises APTES at a concentration of 0.1 to 10%.

14. Method according to the preceding claim, characterised in that

said binding solution comprises APTES at a concentration of 0.2 to 5%, more preferably 0.25 to 2%.

15. Method according to the preceding claim, characterised in that

said binding solution comprises APTES at a concentration of 0.25%, 0.5% or 1 %.

16. Method according to any one of the preceding claims, characterised in that

the biomolecule is an antibody, antibody fragment, a recombinant protein, Fc-binding protein (protein A, protein G, protein A/G), streptavidin, or recombinant proteins or fragment thereof, and is present in the binding solution at a concentration below 500 μg/ml, preferably between 0.01 to 100 μg/ml.

17. Method according to the preceding claim, characterised in that

the biomolecule is present in the binding solution at a concentration of 0.1 to 50 μg/ml, preferably at a concentration of 1 to 10 μg/ml.

18. Method according to any one of the preceding claims, characterised in that

the cross-linking agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), EDC-/V- hydroxysuccinimide (NHS), EDC-sulfoNHS, glutaraldehyde and/or 1 ,4,-phenylene diisothiocyanate (PDITC) are not employed in said method.

19. Method according to any one of the preceding claims, characterised in that

step a) of claim 1 is carried out via incubation of solid material and hydroxide-containing solution for at least 1 minute, preferably 1 to 60 minutes, more preferably 2 to 30 minutes and especially preferred for 10 minutes, optionally followed by washing said solid material with water 1 to 10 times, preferably 5 times , and/or

step b) of claim 1 is carried out via incubation of activated solid material and binding solution for at least 1 minute, preferably 1 to 120 minutes, more preferably 15 to 60 minutes and especially preferred for 30 minutes, optionally followed by washing said solid material with a wash buffer, comprising preferably phosphate buffered saline (PBS), 1 to 10 times, preferably 5 times.

20. Solid material obtainable via the method of any one of the preceding claims, comprising a) a solid material selected from the group consisting of a synthetic polymer;

agarose; silica-based material, such as glass, bioglass, silica monoliths or porous silica; silicon and silicon derivative based substrates; metal-coated surfaces, such as gold-and silver-coated surfaces; nanoparticle-/nanocomposite- coated surfaces; surfaces coated with thin oxide films, such as Si0₂, ZnO, Zx0₂, Al₂0₃, NdGa0₃, La01 , Ti0₂, LSAT, MgAI₂0₄; glassy carbon; screen-printed carbon electrodes; cellulose; cellulose acetate; nanocrystalline cellulose; and/or chitosan, and b) a biomolecule covalently attached to the material via an amide bond, whereby the biomolecule is selected from the group consisting of protein, nucleic acid, lectin, polysaccharide or lipid molecule.

21. Solid material according to the preceding claim, characterised in that

the surface of said solid material exhibits a mass density of biomolecule (ng/cm² of solid material surface), preferably antibody, antibody fragment, a recombinant protein, Fc- binding protein (protein A, protein G, protein A G), streptavidin, or recombinant proteins or fragments thereof, of greater than 175 ng/cm², preferably between 175 and 250 ng/cm², more preferably between 175 and 200 ng/cm².

Solid material according to claims 20 or 21 , characterised in that

the surface of said solid material exhibits a molecular density per Dalton

(molecules/cm²/Dalton) of antibody or antibody fragment of greater than 4.61 x 10⁹ molecules/cm²/Dalton, preferably between 4.61 x 10⁹ and 6 x 10⁹, more preferably between 4.61 x 10⁹ and 5 x 10⁹ molecules/cm²/Dalton.

Solid material according to the preceding claim, characterised in that

the material is in the form of a slide, microtitre plate, incubation chamber, micro- or nanofluidic channel or device or immunoaffinity column.

Kit comprising means for carrying out the method of the preceding claims, comprising an oxidising agent according to claims 8 to 1 1 , a silane component according to claims 12 to 15, and a solid material selected from the group consisting of a synthetic polymer;

agarose; silica-based material, such as glass, bioglass, silica monoliths or porous silica; silicon and silicon derivative based substrates; metal-coated surfaces, such as gold-and silver-coated surfaces; nanoparticle-/nanocomposite-coated surfaces; surfaces coated with thin oxide films, such as Si0₂, ZnO, Zx0₂, Al₂0₃, NdGa0₃, La01 , Ti0₂, LSAT, MgAI₂0₄; glassy carbon; screen-printed carbon electrodes; cellulose; cellulose acetate; nanocrystalline cellulose; and/or chitosan, and optionally a biomolecule to be covalently attached to the material via an amide bond, whereby the biomolecule is selected from the group consisting of protein, nucleic acid, lectin, polysaccharide or lipid molecule.

Immunoassay comprising a method, use of a method, use of a kit or use of a solid material according to any one of the preceding claims,

preferably selected from an enzyme-linked immunosorbent assay (ELISA), a surface plasmon resonance immunoassay, for example in a BIAcore format, a microarray method, a reflectometric interference spectroscopy (RIfS) method, an electrochemical immunoassay, a microfluidic immunoassay, and/or a centrifugal microfluidic method, for example in a LabDisk platform format.

ELISA method according to the preceding claim, comprising a) production of a solid material, preferably a microtitre plate, according to the method of the preceding claims, comprising covalently immobilised antibody, whereby the antibody is capable of binding a molecule to be detected, or use of a solid material according to the preceding claims 20 to 23, b) blocking non-bound sites of said solid material with a blocking reagent,

preferably a protein-containing solution, such as bovine serum albumin (BSA), c) incubation of said solid material with a solution comprising the molecule to be detected, such as a patient sample or diagnostic sample, thereby binding said molecule to be detected to said solid material via the immobilised antibody of a), d) detection of said immobilised molecule to be detected, preferably via i. incubation of a solution comprising a biotinylated antibody of the same or essentially the same affinity as the antibody of step a) with the complex of step c), and ii. incubation of the complex from i. with a solution comprising streptavidin- coupled horseradish peroxidise (SA-HRP), followed by iii. detection of HRP signal, preferably via TMB substrate assay and

measurement of optical density.

27. ELISA method according to the preceding claim, characterised in that

said immobilised antibody of step a) is capable of specifically binding C-reactive protein (CRP), which is the molecule to be detected in the solution of step c).

28. ELISA method according to any one of the preceding claims, characterised in that

said immobilised antibody of step a) is capable of specifically binding human fetuin A (HFA), which is the molecule to be detected in the solution of step c).

29. ELISA method according to any one of the preceding claims, characterised in that

said immobilised antibody of step a) is capable of specifically binding human Lipocalin-2 (LCN2), which is the molecule to be detected in the solution of step c).