WO2017037408A1

WO2017037408A1 - Method for antigen detection

Info

Publication number: WO2017037408A1
Application number: PCT/GB2016/000160
Authority: WO
Inventors: Zoe PIKREMENOU; Mark Trehane Drayson
Original assignee: The University Of Birmingham
Priority date: 2015-09-04
Filing date: 2016-09-05
Publication date: 2017-03-09
Also published as: GB201515693D0

Abstract

Nanoparticles functionalized with one of an antigen and an antibody specific for the antigen, and a luminescent metal complex which may be a FRET donor, are provided. An assay for determining the presence and/or concentration of the antigen in a sample comprises mixing the functionalized nanoparticles with a FRET acceptor conjugated to the other of the antigen and the antibody specific for the antigen, measuring a first fluorescence signal of the mixture, adding a quantity of the sample to the mixture, and measuring a second fluorescence signal of the mixture. Any difference between the first and second fluorescence signals is indicative of the presence of the antigen in the sample. The assay finds use in the detection and/or quantification of light chains in a sample, which can be used in detecting or monitoring monoclonal gammopathies.

Description

Method for antigen detection

Field of the invention

The present invention relates to a method for detecting the presence of an antigen in a sample, using fluorescence resonance energy transfer (FRET). More particularly, the present invention relates to a FRET-based immunoassay for determining the presence and/or concentration of light chains in a sample.

Background to the invention

Immunoglobulin molecules are made up of two identical heavy and two identical light chain polypeptide molecules. There are five types of heavy chains: gamma, alpha, mu, delta, and epsilon; and two types of light chains: kappa (κ) and lambda (λ). Both the heavy and light chain molecules are produced within the plasma cell and then assembled into an immunoglobulin molecule, forming the 5 classes of immunoglobulins: (Ig)-lgG, IgA, IgM, IgD, and IgE, each of which is constructed with either κ or A light chain molecules.

Normal and abnormal plasma cells produce more light chains than heavy chains, and these excess light chains are released into the bloodstream. These unbound, or free, light chains (FLCs) are typically cleared rapidly and metabolized by the kidneys. Increases in serum free light chains may occur due to decreased renal clearance, increased polyclonal immunoglobulin production, or monoclonal gammopathies.

Monoclonal gammopathy is a disorder caused by abnormal proliferation of a single clone of plasma cells. Monoclonal gammopathies may be present in a wide spectrum of diseases, that include malignancies of plasma cells or B lymphocytes (multiple myeloma (MM), macroglobulinaemia, plasmacytoma, B-cell lymphoma), disorders involving monoclonal proteins of abnormal structure (primary systemic amyloidosis (AL), light chain deposition disease (LCDD), cryoglobulinaemia), and apparently benign, premalignant conditions (monoclonal gammopathy of undetermined significance (MGUS), asymptomatic MM). While the specific clinical diagnosis is dependent on a number of clinical features, as well as other laboratory assessments, the presence of the monoclonal gammopathy is a laboratory diagnosis. For example, an excess of FLCs in a sample may be indicative of a malignant plasma cell disease such as multiple myeloma. Although there are existing assays available that allow for the measurement of light chains within serum, there remains a need for improved assays with increased sensitivity and/or accuracy, which can be used for the detection of diseases such as multiple myeloma. The earlier the disease is detected, the greater the opportunity a patient has to receive treatment, and the greater their chances of survival.

The present invention has been devised with these issues in mind. Summary of the invention

According to a first aspect of the invention, there is provided an assay for determining the presence and/or concentration of an antigen in a sample, the assay comprising:

- providing nanoparticles functionalized with:

(i) one of the antigen and an antibody specific for the antigen; and

(ii) a FRET donor constituted by a luminescent metal complex;

- mixing the functionalized nanoparticles with a FRET acceptor conjugated to the other of the antigen and the antibody specific for the antigen, and measuring a first fluorescence signal of the mixture; and

- adding a quantity of the sample to the mixture, and measuring a second fluorescence signal of the mixture;

wherein any difference between the first and second fluorescence signals is indicative of the presence of the antigen in the sample.

Fluorescence Resonance Energy Transfer (also known as Forster Resonance Energy Transfer) is a mechanism of energy transfer from a donor chromophore in an excited state to a ground- state acceptor chromophore via a non-radiative dipole-dipole coupling. The efficiency of energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor, making FRET an extremely sensitive probe for detecting whether two chromophores are within close proximity. FRET has therefore been used to investigate molecular interactions. The transfer of energy leads to a reduction in the donor's fluorescence intensity and excited state lifetime. FRET may also result in an increase in the fluorescence of the acceptor if the acceptor molecule is itself fluorescent. A pair of molecules that interact via FRET may be referred to as a donor-acceptor pair. It will be appreciated that the donor need not be a fluorescent compound, and that luminescent compounds behave in the same way as fluorescent compounds in regard to their emission.

The nanoparticles are functionalized with the FRET donor and either the antigen or the antibody specific for the antigen. By "functionalized with" it will be understood that the FRET donor and the antigen or antibody are attached to the nanoparticle (and thus are separate to the nanoparticle), rather than forming a part of the nanoparticle itself or being embedded within the nanoparticle. The FRET donor and antigen or antibody may be attached (e.g. chemically attached) to the surface of the nanoparticle. Thus the FRET donor and antigen or antibody molecules effectively coat the nanoparticle.

The present invention provides a FRET-based competitive immunoassay for the detection of antigens. In the absence of free antigens in the sample, the FRET acceptor is brought into close proximity to the FRET donor by virtue of the antibody binding to the antigen, which is either coated on the nanoparticle or conjugated to the FRET acceptor. Energy transfer between the FRET donor-acceptor pair effectively quenches the fluorescence of the donor. When the sample is added, free antigens present in the sample compete for the antibody binding sites, thereby reducing the binding of the antibody to the antigen coated on the nanoparticle or conjugated to the FRET acceptor, and reducing the number of donor-acceptor pairs within sufficiently close proximity for FRET to occur. As a result, the fluorescence signal of the FRET donor decreases, the decrease being proportional to the amount of free antigen present in the sample. The assay can therefore be used for the detection and quantification of a target antigen in a sample.

Conveniently, the assay of the present invention circumvents the need for lengthy incubation- wash-incubation-wash sequences which are common to many immunodiagnostic assays, making the detection and quantification of antigens more easily transferable to the clinic. Additionally, providing a nanoparticle coated with several metal complexes as well as the antigen or antibody is much more efficient than chemically labelling the antibody or antigen proteins themselves.

Furthermore, the use of nanoparticles coated with a FRET donor and either the antigen or the antibody specific for the antigen has been found to increase the sensitivity of the assay compared to existing assays for the detection of light chains. The nanoparticles enable a more efficient interaction of the FRET donor-acceptor pairs, since they carry a plurality of FRET donor complexes. Therefore, antigen-antibody binding brings the FRET acceptor into close proximity to several FRET donor complexes with which the acceptor can interact. The functionalized nanoparticles therefore increase the probability of FRET interaction as well as increasing the difference in signal observed by reducing the distance between the donors and acceptors.

In some embodiments, the nanoparticles are functionalized with the antigen, and the FRET acceptor is conjugated to the antibody specific for the antigen.

In some embodiments the nanoparticles are further coated with one or more additional substances such as polymers, proteins, surfactants or stabilisers. In some embodiments, the nanoparticles are additionally coated with a surfactant, e.g. an anionic surfactant such as Zonyl® FSA.

In some embodiments, the functionalized nanoparticle is water-soluble.

In some embodiments, the assay is a homogeneous assay. As is known in the art, a "homogeneous assay" is an assay which does not require separation of the antibody-antigen complex (e.g. using washing steps). This is in contrast to a heterogeneous assay which requires separation of the complex, for example using a solid phase (e.g. beads or magnetic particles). In some embodiments, all of the assay components (i.e. the functionalized nanoparticles, the FRET acceptor conjugate, and any free antigens present in the sample) are in solution or aqueous suspension.

It is known that for FRET to occur the absorption or excitation spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor. The degree to which they overlap is known as the spectral overlap integral. Suitable FRET donor-acceptor pairs are known to those skilled in the art, or can be easily determined by determining the spectral overlap of a given donor with a potential acceptor compound using standard techniques. For example, suitable acceptors for a donor complex comprising an Eu ion can be selected based on the spectral overlap of their absorption spectra in the range of Eu emission (580 nm-720 nm).

In some embodiments wherein the luminescent metal complex is an Eu complex, the FRET acceptor is Alexafluor 647 (AF647) dye. It is known that there is a large spectral overlap between the dye absorbance and Eu(lll) emission. Eu(lll) is a known FRET donor. Other FRET acceptors include allophycocyanin.

In some embodiments, the method may comprise incubating the mixture after mixing the functionalized nanoparticles with the FRET acceptor conjugated to the other of the antigen and the antibody specific for the antigen, and prior to measuring the first fluorescence signal of the mixture. Alternatively or additionally, the mixture may be incubated after the addition of the sample, prior to measuring the second fluorescence signal of the mixture. During the incubation(s) the antigen and antibody molecules interact according to their concentrations and affinities for each other. The incubation time(s) may be selected to be longer than the time it takes for these interactions to reach equilibrium.

In some embodiments, the mixture is incubated for a period of time of from 1 seconds to 30 minutes, from 10 seconds to 15 minutes, or from 30 seconds to 10 minutes.

As used herein, 'measuring the fluorescence signal' may comprise measuring the intensity and/or duration (i.e. lifetime) of emission. It will be appreciated that prior to measuring the signal, the mixture is first excited by exposing the mixture to an external light source. The exposure to light excites the donor, causing the donor to emit light at a different wavelength. It will be appreciated that the wavelength of light used for excitation of the mixture will depend on the nature of the metal complex.

It will also be appreciated that the assay is carried out in a liquid medium, such as a suitable buffer (e.g. PBS). For example, functionalized nanoparticles in suspension may be mixed with a solution or suspension of the FRET acceptor conjugate. It will be further appreciated that suitable concentrations of the functionalized nanoparticles and the FRET acceptor conjugate may depend on factors such as the loading of the nanoparticles. The assay conditions can be optimized by those skilled in the art.

In some embodiments, the assay is for determining the concentration of the antigen in a sample, wherein the difference between the first and second fluorescence signals is proportional to the concentration of the antigen in the sample. To determine the concentration of the antigen, the change in fluorescence signal between the first and second measurements may be compared to a reference, for example a calibration curve generated using a series of standard solutions containing different concentrations of the antigen. The calibration curve may correlate the antigen concentration with the percentage change in the fluorescence signal.

In some embodiments, the antigen is kappa or lambda light chains. In these embodiments, the antibody is specific for kappa or lambda light chains, respectively.

In some embodiments, the antibody is specific for kappa or lambda light chains whether the light chains are free or bound to a heavy chain, i.e. as part of a whole immunoglobulin. In other embodiments, the antibody is specific for free light chains (i.e. the antibody binds only to free kappa or lambda light chains). It may be useful to separately determine the concentrations of total light chains (i.e. bound and free), for both lambda and kappa, and also free light chains for both lambda and kappa.

The sample may be obtained from a human or other animal subject. The sample may be blood, urine or serum.

The present invention therefore provides an assay for detecting and/or quantifying light chains in a sample, which finds particular use in detecting or monitoring monoclonal gammopathies.

Thus, according to a second aspect of the invention, there is provided a method for detecting or monitoring a monoclonal gammopathy, the method comprising the assay according to the first aspect of the invention, wherein the antigen is kappa or lambda light chains.

The monoclonal gammopathies may be associated with diseases or disorders including malignancies of plasma cells or B lymphocytes (multiple myeloma (MM), macroglobulinaemia, plasmacytoma, B-cell lymphoma), disorders involving monoclonal proteins of abnormal structure (primary systemic amyloidosis (AL), light chain deposition disease (LCDD), cryoglobulinaemia), and apparently benign, premalignant conditions (monoclonal gammopathy of undetermined significance (MGUS), asymptomatic MM).

In some embodiments, the monoclonal gammopathy is associated with a malignant plasma cell disease selected from multiple myeloma, AL amyloidosis, solitary plasmacytoma, extramedullary plasmacytoma, multiple solitary plasmacytoma, plasma cell leukaemia, Waldenstrom's macroglobulinaemia, B-cell non-Hogdkin lymphomas, B-cell chronic lymphocytic leukaemia or MGUS.

An excess of kappa and/or lambda light chains (FLCs and/or total light chains), in a sample obtained from a subject may be indicative of a monoclonal gammopathy. A subject may therefore be identified as having a monoclonal gammopathy if the concentration of total and/or free kappa and/or lambda light chains is increased relative to a reference level, or if the ratio of kappa:lambda (total and/or free) is increased or decreased relative to a reference level. The reference level may be obtained from a control sample taken from a healthy subject, or it may be a published literature value representative of normal light chain levels.

The method of the second aspect of the invention may therefore further comprise comparing the change in fluorescence signal (i.e. the difference between the first and second fluorescence signal), or the concentration of kappa and/or lambda light chains (total and/or free) determined therefrom, to a reference level.

According to a third aspect of the invention, there is provided a nanoparticle functionalized with:

- kappa or lambda light chains or an antibody specific for either kappa or lambda light chains and

- a luminescent metal complex.

Also provided is a composition comprising nanoparticles in accordance with the third aspect of the invention.

The composition may be a solution or suspension. A solution of the functionalized nanoparticles may have a concentration of at least 0.1 nM, at least 0.5 nM, at least 1 nM or at least 5 nM.

The composition may comprise one or more additional substances such as polymers, solvents, biological fluids, proteins, surfactants and stabilisers. In some embodiments, the composition comprises polyacrylamide, an anionic surfactant (e.g. a fluorinated surfactant such as Zonyl® FSA), bovine serum albumin (BSA) and/or serum (e.g. human serum or foetal calf serum). Polyacrylamide enables the nanoparticles to be dried and resuspended. In some embodiments, the composition comprises polyacrylamide. The polyacrylamide may be prepared as a solution (e.g. 1 mg/ml aqueous solution), which is added to the composition comprising the nanoparticles in the amount required (e.g. 0.1 to 1% v/v).

In some embodiments, the composition comprises an anionic surfactant (e.g. Zonyl® FSA). The concentration of the anionic surfactant may be from 0.01 to 0.1%.

In some embodiments, the composition comprises serum (e.g. human serum or foetal calf serum). The concentration of the serum may be from 0.2 % to 2% (v/v).

In some embodiments, the composition comprises bovine serum albumin (BSA). The BSA may be prepared as a solution (e.g. 1 mg/ml aqueous solution), which is added to the composition comprising the nanoparticles in the amount required (e.g. 0.1 to 1% v/v).

According to a fourth aspect of the invention, there is provided a kit comprising:

- a first component comprising nanoparticles in accordance with the third aspect of the invention, wherein the luminescent metal complex constitutes a FRET donor; and

- a second component comprising a FRET acceptor conjugated to an antibody specific for the light chains with which the nanoparticle is functionalized.

The first and second components may be a solution or suspension.

The kit may additionally comprise suitable buffers, reagents, apparatus, reaction vessels and/or an instruction manual for carrying out an assay in accordance with the first aspect of the invention.

The following statements are applicable to all aspects of the present invention, as appropriate.

Nanoparticles are defined as particles having a maximum diameter of between 1 and 500 nanometres. The nanoparticles may be from 3 to 300 nm, from 4 to 100 nm, from 5 to 50 nm or from 7 to 20 nm, or from 8 to 15 nm, for example 10 or 13 nm.

As used herein "loading" is defined as the total number of metal complex molecules or antigens/antibodies with which each nanoparticle is functionalized. The loading is to some extent dependent on the size of the particle. For particles smaller than 30 nm, the metal complex loading may be at least 100, at least 500, at least 1000, at least 2000 or even at least 4000. For particles smaller than 30 nm, the metal complex loading is no greater than 100,000, no greater than 10,000 or no greater than 7000. For particles larger than 30nm but smaller than 60nm, the loading may be at least 2000, at least 7000, at least 10,000, at least 20,000 or even at least 30,000. For particles larger than 60nm, the loading may be at least 40,000, at least 60,000, at least 80,000 or even at least 100,000.

In some embodiments, the antigen or antibody loading is at least one, at least 5, at least 10 or at least 50. In some embodiments, the antigen or antibody loading is from 1 to 100, from 2 to 50 or from 5 to 30, e.g. about 10.

The nanoparticles may be made of any suitable material, for example metals, polymers or biomaterials. In some embodiments, the nanoparticles are made from metal, such as a noble metal. In other embodiments, the nanoparticles are made from a non-metal such as Si0₂.

As used herein, noble metal has its usual meaning to refer to the elements in Groups 10 and 11 of the periodic table of elements. In some embodiments, the noble metal is selected from palladium, silver, platinum and gold. In some embodiments, the noble metal is selected from platinum and gold. In some further embodiments, the noble metal is gold.

The luminescent metal complex may be selected from complexes of any transition metal (i.e. d- block), p-block or rare earth metal.

In some embodiments, the rare earth metal is a lanthanide (Ln). In some embodiments, the rare earth metal is europium (Eu), neodymium (Nd), ytterbium (Yb), erbium (Er) or terbium (Tb). In some further embodiments, the rare earth metal is Eu or Tb. The Eu lifetime (microseconds) is particularly well-suited to FRET-based immunoassays.

In some embodiments, the metal which forms the nanoparticle is doped. For example, doped Eu nanoparticles can be used to upconvert light. This allows the donor to be excited, for example, using near infra-red light, resulting in the emission of visible light. Nanoparticles made from lanthanide metals that are capable of upconverting light in this way are referred to as upconverting lanthanide nanoparticles. Thus, in some embodiments the nanoparticle is an upconverting lanthanide nanoparticle.

In some embodiments, the transition metal is selected from iridium (Ir), ruthenium (Ru), zinc (Zn), nickel (Ni), osmium (Os), iron (Fe), manganese (Mn), molybdenum (Mb) or rhenium (Re).

In some embodiments, the luminescent metal complex comprises at least one ligand comprising:

- at least one donor atom for coordinating to the metal ion;

- at least one linker for binding to the nanoparticle; and

- optionally, at least one aromatic sensitizer.

The luminescent metal complex may comprise at least two, at least three or at least four or more ligands.

Typically, more than one donor atom is required to coordinate to the metal ion. It will be appreciated that, for a single metal ion, all of the donor atoms may be provided by a single ligand comprising a number of donor atoms, or multiple ligands may coordinate to the metal ion, each ligand providing one or more donor atoms.

In some embodiments, the linker comprises a thiol group. The presence of the thiol group enables the complex to bind to the surface of gold nanoparticles. In some embodiments, the complex comprises at least one ligand having at least two thiol groups.

In some embodiments, the linker comprises a silyl group. The presence of the silyl group enables the complex to bind to the surface of silica nanoparticles. In some embodiments, the complex comprises at least one ligand having at least two silyl groups.

The sensitizer comprises at least one aromatic group. The sensitizer absorbs light and transfers energy to the metal ion. A sensitizer is typically required unless the nanoparticle is an upconverting lanthanide nanoparticle. In some embodiments, the sensitizer is a quinoline group. The ligand may comprise one or two or more quinoline groups. In some embodiments, the ligand comprises dibenzoylmethane (DBM). DBM is known to efficiently bind and sensitize a range of Ln(lll) ions. Advantageously, ligands comprising DBM and its derivatives enable highly luminescent nanoparticles to be produced. In some embodiments, the ligand comprises or is constituted by structure (I) or (II):

In some embodiments, the complex comprises at least one ligand comprising or constituted by structure (III):

(III) In some embodiments, the complex comprises or is constituted by structure (IV). Although a Eu metal ion is shown, it will be appreciated that alternative metals could be used.

(IV) (EuQSH)

Embodiments of the invention will now be described by way of example and with reference to the accompanying Figures, in which:

Figure 1 is a schematic diagram showing the principle of an embodiment of the assay of the invention;

Figure 2 shows the structures of different luminescent Eu(lll) complexes coated onto gold (A and B) or Si0₂ (C) nanoparticles;

Figure 3 shows a reaction scheme for the synthesis of a ligand for the preparation of a luminescent metal complex;

Figure 4 shows UV-vis spectra showing the change in SPR absorbance upon coating of a gold nanoparticle with: (A) Eu(lll) complexes; (B) kappa light chains; and (C) both Eu(lll) complexes and kappa light chains;

Figure 5 shows a UV-vis spectrum showing the effect of addition of an antibody-FRET acceptor conjugate to nanoparticles coated with Eu(lll) complexes and kappa light chains;

Figure 6 are plots showing the relationship between the lifetime of an excited Eu(lll) complex and the concentration of an antibody-FRET acceptor conjugate when (A) the Eu(lll) complexes are coated onto Au nanoparticles together with kappa light chains; and (B) the kappa light chains are labelled directly with the Eu(lll) complex (no nanoparticle);

Figure 7 are plots showing the change in luminescence upon addition of an antibody-FRET acceptor conjugate to (A) Au nanoparticles functionalized with Eu(lll) complexes and lambda light chains and (B) lambda light chains conjugated directly to the Eu(lll) complex;

Figure 8 shows a reaction scheme for the synthesis of a further ligand for the preparation of a luminescent metal complex; and

Figure 9 shows a UV-vis spectrum showing the change in absorbance upon coating a gold nanoparticle with DBMSS ligands alone or DBMSS ligands and kappa light chains.

Example 1

Methods

Synthesis of a 13 nm gold nanoparticle

Water-soluble citrate-stabilised 13 nm Au nanoparticles were prepared via the reduction of HAuCL» with sodium citrate, in accordance with the methods described by Grablar et al., Anal. Chem., 1995, 67, 735-743, and Osbrorne et al Faraday Discussions, 2015, DOI: 10.1039/C5FD00108K, the contents of which are incorporated herein in their entirety by reference.

Synthesis of H₃QSH

DTPA-bis(anhydride) (4.30 g, 12.0 mmol, 1.0 eq.) was stirred in pyridine (100 mL) to create a suspension into which 6-aminoquinoline (1.70 g, 11.9 mmol, 1.0 eq.) and was added and left to stir under nitrogen for 45 minutes at RT. 4-aminothiolphenol (1.50 g, 12.3 mmol, 1.0 eq.) was added and to the solution and stirring continued under nitrogen at RT for a further 2 h. Water (80 mL) was added to quench the reaction and the solution stirred for 30 minutes under nitrogen. The solvent was removed in vacuo to give a sticky orange solid to which water (~150 mL) was added before the solution was filtered to give a clear yellow solution. Concentrated HCI was added dropwise to the solution under a stream of dinitrogen until pH 3 was reached at which point a yellow precipitate was formed. The clear yellow aqueous layer was decanted to leave a yellow solid that was washed with water (50 mL) and acetonitrile (50 mL) before being triturated in acetonitrile for two days under nitrogen. A bright yellow powder was formed and was collected by suction filtration and washed with acetonitrile (2 x 50 mL) and diethyl ether (2 x 50 mL) to give a yellow powder, which was purified via HPLC. The sample was dissolved in THF / water (1 :1) and an excess of tris(2-carboxyethyl)phosphine hydrochloride was added to reduce any disulphide bonds. The optimum set up for separation was found to be injection of a 1 mL aliquot of 100 mg / mL reduced sample in THF / water (1 :1) onto a preparative HPLC system using a Luna C₁₈ reverse phase column running a water / acetone gradient (0 - 75% acetone over 30 mins) at 8 mL / min. Solvents used contained 0.05 % trifluoroacetic acid. Monitoring the absorbance at 210 nm and 336 nm allowed detection of each species as eluted. After a complete run the column was washed with acetone and water before injection repeated. Three peaks eluted, peak 1 at 11 min, peak 2 at 17 min and peak 3 at 25 min and were identified using mass spectrometry (MALDI-TOF⁺). H₃QSH was contained in peak 2, m/z 626 {M + H}⁺, which was collected and solvent removed in vacuo to afford a yellow solid that was dried further in vacuo to yield H₃QSH (60 mg, 3%).

Synthesis of EuQSH

EuCI₃-6H₂0 (23 mg, 0.06 mmol, 1.0 eq.) was added to a stirring solution of H₃QSH (40 mg, 0.06 mmol, 1.0 eq.) in degassed water (5 mL). The pH of the solution was raised to pH 5 by dropwise addition of aqueous tetrabutylammonium hydroxide (40% w/v) under a stream of nitrogen. The volume of the solution was reduced in vacuo to <1 mL to which degassed acetonitrile was added (30 mL) to yield a precipitate. The solid was recovered by suction filtration under nitrogen and washed with degassed acetonitrile (2 x 0 mL) and diethyl ether (2 x 10 mL). The resulting off white powder was dried in vacuo to yield EuQSH (29 mg, 59 %).

Synthesis of EuQuinSAc

The ligand of structure (III) was prepared in accordance with the reaction scheme shown in Figure 2. The preparation of this ligand and its attachment to a gold nanoparticle is further described in Davies et al., Proceedings of the National Academy of Sciences of the United States of America, 109, 1862-1867, the contents of which are incorporated herein in their entirety by reference.

Synthesis of DBMSS

The ligand of structure (II) was prepared according to the synthetic scheme shown in Figure 8. Attaching EuQSH to 13 nm AuNP

To a solution of Au13 (9.4 nM, 2 ml) a solution of EuQSH was added (2.5 mM in MeOH, 40 μΐ.) and the solution stirred at room temperature for 5 min and the SPR was monitored via UV / Vis. The solution was then passed through a sephadex G50 column whereby the red band was collected and the SPR was recorded to confirm the absence of nanoparticte aggregation.

Attaching k FLC to 13 nm AuNP

To a solution of Au13 (9.4 nM, 2 ml) a solution of κ FLC was added (0.7 mg / ml, 80 μΙ_ in PBS) and the solution stirred at room temperature for 5 min and the SPR was monitored via UV / Vis. The solution was then passed through a sephadex G50 column whereby the red band was collected and the SPR was recorded to confirm the absence of nanoparticle aggregation.

Attaching k FLC and Eu(lll) complex to 13 nm AuNP

To a solution of Au13 (9.4 nM, 2 ml) a solution of κ FLC was added (0.7 mg / ml, 8 μί in PBS) and the solution stirred at room temperature for 5 min and the SPR was monitored via UV / Vis. EuQSH (2.5 mM in MeOH, 40 μί) was then added and stirred for a further 5 min and the SPR was monitored via UV / Vis. The solution was then passed through a sephadex G50 column whereby the red band was collected and the SPR was recorded to confirm the absence of nanoparticle aggregation.

Au nanoparticles functionalized with λ FLC and EuQSH were prepared as described above. Synthesis ofAb-AF647

The conjugation of antibody BUCIS04 (specific for kappa light chains) to AF647 was performed using BUCIS04 (1.8 mg / ml, 1 ml) as according to instructions from the Fluoraprobe 647 kit as supplied by Life Technologies. Using the same methodology, an anti-lambda antibody (specific for lambda light chains) was conjugated to AF647. Addition ofAb-AF647 to functionalized nanoparticles

Addition of a solution of anti-K-Ab-AF647 (1.6 mg / ml) in 20 microliter increments to a solution of nanoparticles in concentrations varied from 1-6 nM.

A solution of anti-A-Ab-AF647 was added to nanoparticles (3 nM) to give a final concentration of 110-550 nM anti-A-Ab-AF647.

Fluorescence measurements

Fluorescence data were obtained on an Edinburgh Instruments FLSP920 steady state, and time resolved lifetime spectrometer. Lifetime measurements were carried with the same detection source, using a microsecond flashlamp. F900 software was used to record the data and quartz cuvettes with four transparent polished faces were used to record the data.

Results

Principle of the FRET-based competitive assay

With reference to Figure 1 , a nanoparticle (NP) is functionalized with kappa or lambda light chains (FLCs) and a luminescent Eu(lll) complex which constitutes a FRET donor. The nanoparticles are mixed with an FLC-specific antibody conjugated to a suitable FRET acceptor. As shown in (A), binding of the antibodies to the NP-immobilised FLCs brings the FRET donor and acceptors into close proximity, allowing FRET to occur. When the sample is added (B), FLCs present in the sample occupy the antibody binding sites, thereby reducing complex formation between the antibodies and the NP-immobilised FLCs. Since the FRET donors and acceptors are not brought into close proximity, FRET is reduced. A difference in the fluorescence signal in the absence and presence of the sample is therefore indicative of the presence of FLCs in the sample.

Eu complex designs

Figure 2 shows the structures of luminescent metal complexes which can be coated onto gold (A and B) or silica (C) nanoparticles for use in the present invention. Common to all of the complexes is a metal ion (Eu, in the embodiments shown, although it will be appreciated that other metals ions may be used) and an organic ligand comprising at least one aromatic sensitizer, at least one linker comprising a surface active group for binding the complex to the metal surface, and at least two donor atoms (O and/or N) which coordinate to the metal ion. Figure 2A shows a luminescent Eu(lll) complex known as EuQSH. The ligand of EuQSH has a backbone which forms a cage-like structure and comprises nine donor oxygen and nitrogen atoms which coordinate to the Eu(lll) ion. The ligand further comprises a quinoline sensitizer and an aromatic linker having a terminal thiol (SH) group for binding to a gold nanoparticle. In an alternative embodiment (Figure 2B), the ligand may comprise two quinoline sentitizer groups and a pair of linkers, each comprising a hydrocarbon chain terminating in a thiol group. This ligand may be prepared in accordance with the synthetic scheme shown in Figure 3.

In a further embodiment (Figure 2C), three ligands are attached to the central metal ion (i.e. coordination number of 6), each ligand comprising a dinzolymethane sensitizer group and a linker terminating in a silane group for bonding to the surface of a silica NP.

Characterization of NPs

UV-vis spectra were used to characterize the formation and functionalization of gold nanoparticles (AuNPs). The UV-vis spectra of the prepared AuNPs showed a surface plasmon resonance (SPR) peak at 519 nm, indicating that the size of the AuNPs prepared was 13 nm. A change in the SPR wavelength was observed as the surfaces of the AuNPs were functionalized with EuQSH (max. wavelength 524 nm) (Figure 4A), kappa FLCs (max. wavelength 524 nm) (Figure 4B) and both EuQSH and kappa FLCs (max. wavelength 528 nm) (Figure 4C).

Characterization of functionalized NPs in the presence of antibody-FRET acceptor conjugate An antibody specific for kappa FLCs was conjugated to AF647 dye (Ab-AF647). Upon addition of Ab-AF647 to the functionalized NPs, a change in the absorption peak was observed, indicating energy transfer between the metal complex donors on the NPs and the AF647 acceptor attached to the antibody (Figure 5).

As the concentration of Ab-AF647 was increased, the lifetime of the Eu(lll) complex decreased, indicating energy transfer between the FRET donors and acceptors. As shown in Figure 6A, a linear, inverse relationship was observed between the Ab-AF647 concentration and the Eu(lll) lifetime. However, this linear relationship was not observed when the Ab-AF647 conjugate was added to FLC labelled directly with EuQSH (i.e. no nanoparticle present) (Figure 6B). Furthermore, changes in the Eu(lll) lifetime were detected at much lower concentrations of Ab- AF647 when using the functionalized nanoparticle (Figure 6A). This demonstrates that FRET from a functionalized NP lowers the concentration of the antibody-FRET acceptor probe required to detect a signal.

In a further experiment, an anti-lambda antibody was conjugated to AF647 dye to give Ab- AF647. Upon addition of Ab-AF647 (550 nM) to nanoparticles (3 nM) functionalized with lambda light chains and EuQSH, a decrease in the Eu luminescence lifetime was observed (Figure 7A). It was found that a decrease in the luminescence lifetime could be detected for an antibody concentration as low as 110 nM. It was further observed that if the Eu complex is conjugated directly to the light chain, instead of to the nanoparticle, micromolar concentrations of the Eu-light chain conjugate is required in order to detect the change in luminescence lifetime (Figure 7B), in contrast to the nanomolar concentration of nanoparticles required.

DBMSS-coated nanoparticles (Figure 8) were prepared by titrating DBMSS onto Au13 with a pre-coating of Zonyl FSA surfactant. Coated nanoparticles were purified using sephadex 15. Eu3+ was loaded with addition of EuCI₃.6H₂0 in the presence of TEA. Figure 9 shows the UV- vis spectra of gold nanoparticles (13 nm) alone, gold nanoparticles functionalized with DBMSS, and gold nanoparticles functionalized with DBMSS and kappa light chains (KLC). It was found that the functionalized nanoparticles are highly luminescent and sufficiently stable for use in the assay of the invention.

Example 2

The assay of the invention was used to analyse patient samples. Patient test samples were mixed with nanoparticles (13 nm Au) functionalized with kappa light chains and EuQSH, and anti-kappa antibody conjugated to a FRET acceptor. The lifetime of the Eu complex was measured. The results are shown below in Table 1.

The competition of the excess light chain in the patient sample leads to an increase of the luminescence lifetime, indicating a reduction in FRET. This is expected because the excess light chain in the patient sample competes with the nanoparticle-bound light chains for the antibody. By using such measurements it can be determined whether the patient has an excess of light chain. These measurements also enable us to quantify a range of light chain concentrations that can be detected. Table 1

33380 (Kappa = 148 mg / L), (Lambda = 228 mg / L) Renal failure patient

33381 (Kappa = 41 mg / L), (Lambda = 18 mg / L) Healthy levels of both types of light chain

Claims

1. An assay for determining the presence and/or concentration of an antigen in a sample, the assay comprising:

- providing nanoparticles functionalized with:

(i) one of the antigen and an antibody specific for the antigen; and

(ii) a FRET donor constituted by a luminescent metal complex;

2. The assay according to claim 1 , wherein the nanoparticles are functionalized with the antigen, and the FRET acceptor is conjugated to the antibody specific for the antigen.

3. The assay according to claim 1 or claim 2, wherein measuring the fluorescence signal comprises measuring the intensity and/or duration of emission.

4. The assay according to any one of claims 1 to 3, wherein the antigen is kappa or lambda light chains.

5. A method for detecting or monitoring a monoclonal gammopathy, the method comprising carrying out an assay in accordance with claim 4.

6. The method of claim 5, wherein the monoclonal gammopathy is associated with a disease or disorder selected from malignancies of plasma cells or B lymphocytes, disorders involving monoclonal proteins of abnormal structure, light chain deposition disease (LCDD), cryoglobulinaemia), monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic multiple myeloma.

7. A nanoparticle functionalized with: - kappa or lambda light chains or an antibody specific for either kappa or lambda light chains and

- a luminescent metal complex.

8. A kit comprising:

- a first component comprising nanoparticles in accordance with claim 7, wherein the luminescent metal complex constitutes a FRET donor; and

9. An assay according to any one of claims 1 to 4, or a nanoparticle according to claim 7, wherein the nanoparticle is made from a noble metal.

10. An assay according to any one of claims 1 to 4 or 9, or a nanoparticle according to claim 7 or claim 9, wherein the luminescent metal complex is selected from complexes of any transition metal, p-block or rare earth metal.

11. An assay or a nanoparticle according to claim 10, wherein the rare earth metal is selected from europium (Eu), neodymium (Nd), ytterbium (Yb), erbium (Er) and terbium (Tb).

12. An assay according to any one of claims 1 to 4 or 9 to 11 , or a nanoparticle according to any one of claims 7 or 9 to 11 , wherein the luminescent metal complex comprises at least one ligand comprising:

- at least one donor atom for coordinating to the metal ion;

- at least one linker for binding to the nanoparticle; and

- optionally, at least one aromatic sensitizer.

13. An assay or a nanoparticle according to claim 12, wherein the linker comprises a thiol or silyl group.

14. An assay or a nanoparticle according to claim 12 or claim 13, wherein the sensitizer is a quinoline group.

15. An assay or a nanoparticle according to claim 12 or claim 13, wherein the ligand comprises dibenzoylmethane (DBM).

16. An assay substantially as described herein, with reference to Figure 1.

17. A method for detecting or monitoring a monoclonal gammopathy substantially as described herein.

18. A nanoparticle substantially as described herein, with reference to Figures 2-3.