WO2005119265A1

WO2005119265A1 - Protein detection

Info

Publication number: WO2005119265A1
Application number: PCT/GB2005/002153
Authority: WO
Inventors: David Klenerman; Haitao Li
Original assignee: Cambridge University Technical Services Limited
Priority date: 2004-06-03
Filing date: 2005-06-01
Publication date: 2005-12-15
Also published as: GB0412410D0

Abstract

Provided are methods and kits for detecting a protein analyte in a sample, the methods comprising the steps of: (i) providing a sample solution, (ii) providing at least two ligands (e.g. antiboides) capable of binding specifically to the protein analyte, each ligand being labelled with a different fluorophore, which fluorophores are excitable by spectrally separated wavelengths of light, (iii) combining the sample solution and the ligands to form a detection sample such that protein analyte if present is labelled with the at least two ligands, and wherein each ligand is present at a concentration of less than 1 nM,(iv) illuminating the sample solution with at least two coherent light beams having wavelengths capable of exciting the fluorophores, which beams coincide to form a coincident probe volume within the sample solution, (v) detecting the number of coincident fluorescent events within the coincident probe volume over a period of time. The sensitive detection system can be used to detect analytes at very low concentrations, for example in body fluids.

Description

Protein Detection

TECHNICAL FIELD

The present invention relates generally to methods and materials for detecting or quantifying protein analytes in a solution.

BACKGROUND ART

The development of a general method to sensitively detect and quantify protein analytes is important in clinical diagnosis and the field of proteomics since an equivalent of the polymerase chain reaction, widely used for ultra-sensitive DNA detection, does not exist for proteins. Current methods are largely based on protein capture onto a surface, using immobilized antibodies and then detection with a second antibody coupled to an enzyme, which generates an amplified signal by catalysis of a chemical reaction that produces a detectable color. Other more recent methods have used antibodies^1-2 or antibody coated beads³ that are tagged with DNA which can be amplified by PCR to increase sensitivity.

While solution based methods are desirable since the rate of reactions is faster than in the solid phase, one key problem is to be able to distinguish between bound and unbound labels. Ultra-sensitive fluorescence methods have been used previously for this type of analysis where the molecules in the confocal volume of a laser are analysed as they diffuse through the probe volume (see e.g. Bieshke et al . (2000) PNAS: 97: 5468-5473). However these experiments have been performed under conditions where there are, on average, about 10 labels in the probe volume at any one time. Under these conditions it is not possible to distinguish label which is bound to the target. Therefore conditions have been used to ensure there are no unbound molecules and they are all in complexes. Under these conditions it is not possible to make measurements in the more normal and general case when there is an excess of unbound molecules.

Thus it can be seen that novel methods for sensitively detecting and quantify protein analytes would provide a contribution to the art.

DISCLOSURE OF THE INVENTION

The present inventors have used two colour fluorescence coincidence detection to directly count .individual protein analytes (both free in solution, and present as structural components e.g. in viruses). This allowed quantitative measurement of protein analytes at the femtomolar level in complex media such as serum, the relationship being linear over three orders of magnitude .

One embodiment of the invention is illustrated in Fig. 1A. The protein analyte is labelled with red-excited and blue- excited antibodies. Coincidence bursts of fluorescence are detected only in cases where a target molecule labeled with both a red and blue-excited antibody diffuses into the probe volume. In contrast only single color events (red or blue) will be detected for molecules labeled only with antibodies of a single color or unbound antibodies. At the low concentrations used there is a low probability of a red and blue excited antibody entering the probe volume at the same time. This constitutes a statistical background of coincident events below which it is not possible to detect the target. The background from impurities is also significantly reduced, by two orders of magnitude relative to a single color experiment due to the low probability of an impurity fluorescing when red-excited and blue excited simultaneously, owing to their wide spectral separation. As demonstrated below, the method of the invention enables the measurement to be performed in the presence of complex media such as serum. In preferred embodiments the sample preparation methods of the invention are simple, since detection takes place in solution and requires no prior separation. The invention thus provides general methods to detect and quantitate proteins, and to characterize macromolecular complexes, with applications in ultra-sensitive proteomics and clinical diagnostics .

Thus in first aspect of the present invention there is provided a method for detecting a protein analyte in a sample, the method comprising the steps of: (i) providing a sample solution, (ii) providing at least two ligands capable of binding specifically to the protein analyte, each ligand being labelled with a different fluorophore, which fluorophores are excitable by spectrally separated wavelengths of light, (iii) combining the sample solution and the ligands to form a detection sample such that protein analyte if present is labelled with the at least two ligands, and wherein each ligand is present at less than 1 nM, (iv) illuminating the sample solution with at least two coherent light beams having wavelengths capable of exciting the fluorophores, which beams coincide to form a coincident probe volume within the sample solution,

(v) detecting the number of coincident fluorescent events within the coincident probe volume over a period of time, (vi) optionally using the result from step (v) to quantify the protein analyte in the sample solution.

Although the principle of simultaneous detection of the fluorescence of two independently excited fluorophores has previously been used in the fluorescence detection of DNA ^4~9, it has not previously been used for the ultra-sensitive detection of proteins. By "coincident fluorescent event" is meant the presence in the coincident probe volume of at least two distinguishable fluorophores .

Preferably each ligand is present at less than 500, less than 100 or most preferably less than 50 pM. In a system exemplified herein (see also reference 4 - Li, H . ; Ying, L . ; Green, J. J.; Balasubramanian, S.; Klenerman, D. Anal . Chem .2003, 75, 1664-1670 ) the effective focus volume (V_eff) was estimated as 0.34 fl and the overlap volume was measured as 30 % of the total confocal volume excited by the red and blue lasers. At the low concentrations used in the present invention there is a low probability of a red and blue excited antibody entering this probe volume at the same time - thus the simultaneous presence of the ligands in the coincident probe volume is indicative that they are bound to a protein analyte. Therefore the method permits molecule by molecule direct and quantitative counting of ligand-protein complexes in the coincident probe volume, and hence permits the quantification of the protein analyte in the sample solution. Since the number of protein analytes is counted directly, the measurement is quantitative with a large dynamic range.

In one preferred embodiment, step (vi) may comprise comparing the result from step (v) with a calibration curve generated using a known range of concentrations of analyte in the presence of ligands at the concentration given in step (iii) .

Where the value of step (v) does not exceed a background value (i.e. no analyte present), step (vi) may comprise repeating steps (iii) to (v) with a series of lower ligand concentrations and comparing the results from step (v) with respective calibration curves generated using a known range of concentrations of analyte in each of said ligand concentrations . Some particular aspects of the invention will now be discussed in more detail:

Analytes and samples

By protein analyte is meant an analyte it is desired to detect, which contains, consists essentially of, or consists of a protein (including lipoproteins and glycoproteins e.g. an enzyme; hormone; toxin; receptor or structural protein) . The invention may be practised on any such analyte for which specific ligands can be made available.

Thus the protein analyte may be an individual protein, or may be part of a target complex or other structure, for example a virus or bacterium containing one or more proteins (e.g. envelope proteins) . Other targets may be pathological protein aggregates e.g. prions, amyloid, particularly small size nucleation aggregates, and so on. In the latter case the ligands can bind to the same or different proteins or epitopes of the single target - provided only that the single target molecule or complex is able to bind the two or more ligands. Thus unless the target provides more than one of the same epitope or protein, generally the ligands will be specific for different parts of the target so that they do not sterically interfere and compete for binding sites. This can also improve the specificity of the method by reducing the likelihood that a non-target analyte will bind both of the ligands.

The analyte complex may be constituted by two interacting entities e.g. two proteins, each labeled with one of the ligands. This embodiment may be used e.g. in proteome research .

As shown in the examples the present invention is capable of sensitive detection without the need for any separation or amplification steps. Thus in one embodiment the ligands may be added directly to the sample solution to form the detection sample.

In another embodiment it may be preferred to dilute or concentrate the analyte prior to analysis e.g. using conventional techniques such as microfluidic devices.¹⁷

Ligands

The ligands used in the present invention may be labelled directly with the fluorophore, or may optionally be labelled via a secondary ligand - this can increase the intensity of labelling, hence enhance the signal to noise, and also makes the method very general and easily applicable.

Preferred ligands are antibodies. Each antibody may be monoclonal or polyclonal. It may also be derived from a monoclonal antibody by expressing all or part of the nucleic acid encoding therefore in a suitable host cell such as to produce a polypeptide comprising all or part of the antigen binding site of the original antibody. Such antibodies and derivatives can be raised using any techniques commonly used in the immunology art (see e.g. Roitt et al in '"Immunology 5th edition" - Pub. 1997 by Moseby International Ltd, London) .

The term "antibody" as used herein should be construed as covering any specific binding substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of Chimaeric antibodies are described in EP-A- 0120694 and EP-A-0125023.

For example, it has been shown that fragments of a whole antibody can perform the function of binding antigens . Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VI and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al., Nature 341, 544-546 (1989) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv) , wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P Holliger et al Proc. Natl . Acad. Sci. USA 90 6444-6448, 1993) .

Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804) . Other ligands include aptamers .

The present invention may include the step of providing ligands such as antibodies and optionally labelling them.

In preferred embodiments only 2 ligands are used. However, it will be appreciated that the invention is also applicable to the use of greater numbers of ligands if that is desired e.g. 3 ligands, each distinctively labelled.

Fluorophores

Suitable fluophores, which can be used to label the ligands (e.g. via functional groups), can be selected from those known in the art, and widely available commercially. The fluorophores are excitable by spectrally separated wavelengths of light - thus preferred combinations of fluorophores will preferably include a red or blue excited fluorophores, preferably both.

Example fluorophores used herein include Alexa Fluor® 488 N- hydroxylsuccinimide ester, Alexa Fluor® 647 N-hydroxyl- succinimide ester from Molecular Probes Europe BV (Leiden, The Netherlands) . Other suppliers are well known to those skilled in the art e.g. Amersham Pharmacia.

In the system used in the examples herein the inventors used 488 and 632 nm excitation- 144 nm separations. These sources are readily available and may be used with a dichroic mirror that reflects both wavelengths while transmiting fluorescence .

Detection and quanti ta tion

Detection may occur within a fixed probe volume of the detection sample, the system being based on diffusion of labelled analyte into this probe volume. However in other embodiments the detection limits of the invention may be further improved by relative movement between the detection sample and the illumination device (s) e.g. by use of a flow cell or use of a laser scanner⁶. In these embodiments the encounter rate of the labelled analyte with the probe volume is increased.

Thus in one embodiment, step (v) may be performed wherein the detection sample is passed or flowed through a coincident probe volume defined by the coincidence of the beams .

In another embodiment, step (v) may be performed wherein a coincident probe volume defined by the coincidence of the beams is scanned within the detection sample.

In one embodiment the invention is used in combination with a microfluidic device for sample preparation and flow, using the methods of the invention for in situ detection.

Since the residence time of the analyte in the probe volume in these embodiments will be reduced so will the fluorescence signal. This can be partially compensated for by increasing the laser power, or using highly labelled ligands e.g. via secondary ligands (see above) .

The bin time used will be determined by the length of time it takes for a molecule on average to diffuse through the probe volume. In examples used herein this was of the order of 1 ms, so the signal was generally integrated over a slightly longer time - 5 ms .

The length of time used for counting may be dictated by the concentration of labelled analyte and required accuracy. A threshold setting may be used in order to distinguish true events from background as discussed above, with a high threshold setting generally requiring a longer count time to make an accurate measurement. As will appreciated by the skilled person, calibration experiments performed with samples of known concentration under the same conditions as the detection sample may be used to ensure consistency and permit quantification.

The detecting of coincident fluorescent events can be performed using conventional equipment e.g. using 2 different detector devices appropriate to the respective emissions of the fluorophores .

Rela ted methods

There are many occasions where screening for (detection and/or identification and quantification) of targeted proteinaceous analytes, often present at trace levels in samples, are required. These include clinical applications (samples may include CSF, saliva, sweat, urine, blood, faeces etc.). Another application is the assessment of pathogens or pollutants in soils, water, plants and other matrices.

Importantly we have demonstrated that detection is possible from samples in pure serum opening up the possibility of direct detection in clinical samples .

Thus in particular the invention provides a method as discussed above where the sample is a biosample of an organism, tissue, cell or body fluid. It further provides a method of diagnosis or prognosis, in an individual, of a disease which is associated with a protein analyte, which method comprises taking a biopsy sample from the individual and detecting or quantifying the analyte using a method as described above.

The invention further provides a method of assessing infection or contamination of an environment, wherein the infection or contamination is associated with a protein analyte, which method comprises taking a sample from the environment and assessing the analyte using a method as described above.

The methods of the invention may also be employed to allow the detection and identification and also the measurement of protein-protein, protein-DNA and protein-RNA interactions.

In particular, as discussed above, the methods of the invention may be employed to study the proteome e.g. measuring the amount of a specific protein from a collection of cells.

Another embodiment permits the study of the interactions of low abundance proteins and proteins that are expressed at low levels, whereby labelled, interacting, proteins can be detected as coincident events . This may useful where there is interest in understanding the interaction between proteins .

Kits

In a further aspect of the present invention there are disclosed kits or other components for use in carrying out the method of the present invention. These may include any one or more of the following:

1) At least two ligands capable of binding specifically to a protein analyte, each ligand being labelled with a different fluorophore, which fluorophores are excitable by spectrally separated wavelengths of light,

2) One or more standard solutions for calibrating the apparatus and components described above, for instance containing the protein analyte. 3) Instructions for use in a method of the present invention. The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by cross-reference .

FIGURES

Figure 1 Two color single molecule coincidence detection

A) The principle of the method. Coincidence events are detected when a protein molecule labeled with a red-excited and blue-excited antibody enters the probe volume . There are also coincident events when unbound red-excited and blue- excited antibodies enter the probe volume at the same time. This constitutes a statistical background below which it is not possible to detect dual-labeled proteins. Only 50% of the dual-labeled proteins, which can be both red and blue excited, are detected in these experiments but similarly the statistical background is only due to a red-excited and blue- excited antibody entering the probe volume at the same time.

B) Typical data obtained with both labeled antibodies and protein G at 50 pM concentration. Coincident events are marked with asterisks. The top trace represents red, the lower trace blue excitation.

Figure 2 Detection of protein G.

A) Off rate measurement on protein G-IgG complex performed at initial protein G-IgG complex concentration of approximately 50 pM. The off rate was estimated to be 3120+ 800 s by fitting the data to a single exponential decay.

B) Dependence of the number of coincident events with the solution concentration of protein G (0.5-5 pM) in 5 pM solution of IgG labeled with Alexa 647 and 5 pM IgG labeled with Alexa 488. The data was fitted by a linear function. The experimental background with the antibodies only is also shown (open circles) .

C) Dependence of the number of coincident events with the solution concentration of protein G. The Alexa 488 and 647 labeled IgG concentration was the same as the protein G. The data was fitted by a linear function. The experimental background with antibody only is also shown (open circles) .

D) Dependence of the number of coincident events with the solution concentration of protein G from samples measured in human serum (diluted 100 fold with PBS buffer) . The Alexa 488 and 647 labeled IgG concentrations were the same as the protein G. The data was fitted by a linear function. The experimental background with the antibodies only is also shown (open circles) .

Figure 3 Detection of Herpes Simplex Virus .

Dependence of the number of coincident events with the concentration of HSV-1. The concentration of LP2-IgG antibody labeled with Alexa 488 or 647 was five times the HSV-1 concentration. The data was fitted by a linear function.

EXAMPLES

Chemicals and Reagents

Sodium hydrogen carbonate (NaHC0₃) , hydroxylamine, protein G, bovine serum albumin (BSA) , and rabbit immunoglobulin G (IgG, 95%) were all purchased from Sigma-Aldrich Company (Dorset, UK) . Alexa Fluor® 488 N-hydroxylsuccinimide ester, Alexa Fluor® 647 N-hydroxyl-succinimide ester was both purchased from Molecular Probes Europe BV (Leiden, The Netherlands) . PBS (10 mM phosphate, 150 M NaCl, 2 mM NaN₃, pH 7.2), NaHC0₃ (0.1 M, pH ~ 8.3), and hydroxylamine-HCl (1.5 M, pH 8.5) buffers were all prepared using ultra-pure MilliQ water (resistance > 18 MΩ.cm).

Prepara tion of Alexa 488 and Alexa 647 labeled rabbi t IgG

A protocol that follows the procedure outlined in the Amine- Reactive Probes provided by Molecular Probes was employed to label the rabbit IgG. Briefly, rabbit IgG was dissolved in the 0.1 M NaHC0₃ buffer (pH 8.3) to obtain a concentration of 5 mg/mL, then 1 mL of the freshly prepared IgG solution was added to a vial of 1 rug Alexa Fluor® 488 N- hydroxylsuccinimide ester. After the dye was thoroughly dissolved and mixed with the protein, the resulting solution was allowed for gentle magnetic stirring for 1 hr at room temperature. After which the labeling reaction was terminated by addition of 100 μl of the 1.5 M hydroxylamine-HCl. The resulting mixture was then loaded on a purification column using Bio-Rad BioGel P-30 Fine size exclusion purification resin, and the PBS buffer was used as the eluting buffer. The first yellowish fluorescent band was collected which was the labeled rabbit IgG. The degree of labeling, detected by measuring the UV absorbance at 280 and 494 nm and using the equations provided by the Molecular Probes, was 7 fluorophores per IgG molecule. Alexa 647 labeled rabbit IgG was prepared by the same procedure, except the IgG solution was added to a vial of Alexa Fluor® 647 N-hydroxylsuccinimide ester. The average labeling, detected by measuring the UV absorbance at 280 and 650 nm, was 8 fluorophores per IgG molecule . Production of purified HSV virions and LP2 IgG

A gH-negative mutant of Herpes Simplex Virus Type 1 (HSV-1) which lacks the gene encoding glycoprotein H, and which is named HFEMdelUL22Z ¹⁰ was propagated in a helper cell line, CR1, which supplies gH in trans.¹¹ Tissue culture medium from infected cells was clarified by centrifugation at 2000 x g for 10 minutes, and virus particles were then pelleted from the supernatant by centrifugation at 18,000 rp for 2 h in a Beckman type 19 rotor at 4 °C. The pellets were resuspended in a small volume of PBS and sonicated before being layered on 30ml 15-30% Ficoll gradients in PBS. The gradients were centrifuged at 12,500 rpm for 90 minutes in a Beckman SW28 rotor at 4 °C, and the visible band at the centre of the gradient was harvested, diluted with PBS and pelleted by centrifugation at 21,000rpm in an SW28 rotor. The final pellet was resupended in PBS, and aliquots were stored at -70 °C. Virus particle numbers were estimated by comparison with latex particles of known concentration using negatively stained preparations as described by Watson et al.¹² LP2 is a mouse monoclonal antibody, which recognizes the HSV envelope glycoprotein, gD.¹³ IgG was purified from the tissue culture supernatant from hybridoma cells producing this antibody by immunoaffinity chromatography on a Protein A sepharose column and eluting the IgG with 0. IM glycine pH3. IgG concentrations were determined by measuring the optical density at OD 280. This antibody was labeled by Alexa 647 or Alexa 488 by the same labeling procedure as above. The average labeling numbers are 12 and 14 respectively.

Example 1 - single molecule experimental

The apparatus used to achieve dual-color single molecule fluorescence coincidence detection has been described in a recent publication (Reference 4 below) , the content of which is specifically incorporated herein by reference. Briefly two overlapping laser beams (488 nm, Argon ion, model 35LAP321-230, Melles Griot and 633 nm model 25LHP151 He-Ne laser, Melles Griot) were directed through a dichroic mirror and oil immersion objective (Apochromat 60^χ, NA 1.40, Nikon) to be focused 5 μm into a 1 ml sample solution supported in a Lab-TeK chambered coverglass (Scientific Laboratory Suppliers Ltd, UK) .

Fluorescence was collected by the same objective and imaged onto a 50 μm pinhole (Melles Griot) to reject out of focus fluorescence and other background. Green and red fluorescences were then separated using a second dichroic mirror (585DRLP, Omega Optical Filters) . Green fluorescence was filtered by long-pass and band-pass filters (510ALP and 535AF45, Omega Optical Filters) before being focused onto an avalanche photodiode, APD (SPCM AQ-161, EG&G, Canada) . Red fluorescence was also filtered by long-pass and band-pass filters (565ALP and 695AF55, Omega Optical Filters) before being focused onto a second APD (SPCM AQR-141, EG&G, Canada) . Dark count rates for the two APDs were found to be below 100 counts per second. Outputs from the APDs were coupled to two PC implemented multi-channel scalar cards (MCS-Plus, EG&G, Canada) , the synchronous start output of one MCS card being used to trigger the second.

For single molecule coincidence experiments, protein G and Alexa 488 and Alexa 647 labeled IgG antibodies were diluted to 50 pM in PBS for 30 minutes before experiments. Coverslips modified with BSA were used in all experiments to minimize surface absorption and all experiments were carried out at room temperature. The excitation laser powers were adjusted to 100 μW for the red laser and 300 μW for the blue laser to give comparable counts on both channels. We collected data for 180 minutes for fluorescence coincidence experiment with a 5 ms bin time on both MCS cards. The average brightness of a fluorescence burst was 470 with an average background of 10. A threshold of 50 counts was used on each channel to count coincident events . For experiments in serum we made a stock solution of protein G and IgG complex at concentration of 10 nM in Human Serum (from Sigma) which was diluted to the appropriate concentration in PBS buffer for measurements .

For the single virus coincidence experiments, HSV-1 particles were diluted to 5 pM in PBS then 25 pM Alexa 488 labeled LP2- IgG and 25 pM Alexa 647 labeled LP2-IgG were added. This sample was further diluted for low concentration coincidence counting experiment. All experiments were carried on at room temperature. The excitation laser powers were the same as protein G experiment. We collected data for 180 min for fluorescence coincidence experiment with a 10 ms bin time on both MCS cards. The average brightness of a fluorescent burst was 850 counts and the average background was 18 counts in these experiments. A threshold of 100 on each channel was used to count coincident events.

Results and discussion

We studied the well characterized interaction between protein G (MW 20 kDa) and IgG. The IgG was either labeled with Alexa 488 (blue-excited) or Alexa 647 (red-excited) . Protein G has up to three available binding sites for IgG with a sub- nanomolar dissociation constant.¹⁴

Typical data obtained with both labeled antibodies and protein G at 50 pM concentration is shown in Fig. IB, where coincident events are marked with asterisks. Not all the events are coincident due to two reasons. Firstly single labeled protein G and free antibodies are also present. Secondly the overlap between the red and blue excited probe volumes is imperfect, 30% in these experiments, so some molecules diffuse through a region that is just excited by the red or blue laser only. ⁴ Note the good signal to noise, typically 50:1, on both channels for individual protein- antibody complexes .

We made measurements to evaluate the dissociation of the protein G-IgG complex to determine whether we were working under equilibrium conditions or kinetic control by measuring how the number of coincident events changed during the experiment (Fig. 2A) . We found that the dissociation rate for the complex was 3120 ± 800 s^"1. Thus for a typical 3 hour measurement, the complex has dissociated to reach its equilibrium value during the measurement time and we are therefore working close to equilibrium conditions . We then measured the number of coincident events as a function of protein G concentration, in the presence of 10 pM total IgG (Fig. 2B) ) . There is a clear linear dependence of number of coincident events with protein G concentration. At 1 pM or lower protein G concentration it is not possible to detect coincident events above the statistical background due to red and blue excited IgG randomly being present in the probe volume at the same time.

To obtain higher sensitivity it is necessary to decrease the IgG concentration to reduce the statistical background. The results of doing this are shown in Fig. 2C. In this case the concentration of IgG used was the same as protein G. The number of coincident events is directly proportional to protein G concentration over three orders of magnitude. The number of coincident events counted is significantly higher than the statistical background so that the sensitivity is only limited by the encounter rate of the protein G-IgG complex with the probe volume. In this case, relying only on diffusion, the limit is about 50 fM.

We then repeated the experiment using samples made in human serum (Fig. 2D) . These were diluted a hundred-fold into PBS buffer and the number of coincident events counted. Again this was proportional to the protein G concentration over three orders of magnitude. In this case the background coincident events were significantly higher so that the limit of detection sensitivity was about 50 fM.

Example 2 - detection of protein analytes in a virus

To show the generality of the method and demonstrate the potential for virus detection we then performed experiments on Herpes Simplex Virus (HSV) . The virus particle is approximately 120 nm in diameter, comprising an icosahedral nucleocapsid surrounded by a lipid bilayer embedded with multiple virus-specific membrane glycoproteins, including glycoprotein D (gD).¹⁵ We used fluorophore labeled antibodies to gD for these experiments. We found in this case that the binding was irreversible over the three hour measurement time. Experiments with antibodies only gave no detected coincidence events and hence a zero statistical background. This is because multiple antibodies bind to the virus, so a higher fluorescence threshold was used for coincidence detection, rejecting two differently labeled antibodies in the probe volume at the same time. Based on the mean fluorescence intensity for a labeled virus compared to an antibody alone there were 10 antibodies in total per virus under the conditions of the experiments. This is in agreement with the ratio of antibody to virus used in these experiments (10:1) and the observation of a very slow antibody off-rate that ensures that virus-bound antibodies do not dissociate during the experiment. The number of coincident events was proportional to the number of viruses over three orders of magnitude, down to a concentration of 50 fM where the encounter rate with the probe volume was again limiting (Fig.3) . Example 3 - quantification of analytes in samples of unknown concentration

The examples above demonstrate that direct counting of individual target molecules or viruses gives a linear dependence of signal with analyte concentration over three orders of magnitude allowing quantitative measurements . The detection limit for both protein G and HSV, in PBS buffer, is not due to signal to noise, or background coincidence, but rather the rate the analyte molecules encounter the probe volume (and thus the acquisition time for an experiment) . The fact that we can quantitatively detect both viruses of 120 nm diameter and protein G molecules of diameter 3.5 nm using the same apparatus with a similar detection limit supports that the method is general for the detection of most biological particles and complexes. The only requirement is for a ligand (preferably an antibody) labeled with two different fluorophores .

Typically, for a sample of unknown concentration, firstly calibration experiments are performed on analyte samples of known concentrations (e.g. as in protein G in Example 1) which can be prepared from stock solutions made by serial dilution and using fixed fluorophore labeled antibody concentrations (e.g. 100 pM) . The analyte solutions are diluted, ensuring that sufficient time is permitted for equilibrium had been reached before measurement for these experiments. Different dilutions are assessed until the coincident events detected have reached the background level.

The calibration curve is then be repeated with a lower antibody concentration e.g. 10 pM, 1 pM , 100 fM to give a series of calibration curves at different fixed antibody concentrations . The unknown sample is then run firstly at the highest fixed antibody concentration to determine if the number of coincident events is above background, in which case this number is determined and the amount of target read off the calibration curve. If the number was no higher than background then the experiment is repeated at the next lower antibody concentration until the coincident events are above background and this concentration can be determined.

In an another embodiment the number of coincident events may be directly converted to a bound-analyte concentration by reference to the overlap probe volume and detection efficiency of coincident events which may be measured in separate calibration experiments. The true concentration may then be determined from the dissociation constant or off-rate for the ligand using conventional thermodynamic equations.

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Claims

1 A method for detecting a protein analyte in a sample, the method comprising the steps of: (i) providing a sample solution,

(ii) providing at least two ligands capable of binding specifically to the protein analyte, each ligand being labelled with a different fluorophore, which fluorophores are excitable by spectrally separated wavelengths of light, (ϋi) combining the sample solution and the ligands to form a detection sample such that protein analyte if present is labelled with the at least two ligands, and wherein each ligand is present at a concentration of less than 1 nM, (iv) illuminating the sample solution with at least two coherent light beams having wavelengths capable of exciting the fluorophores, which beams coincide to form a coincident probe volume within the sample solution,

(v) detecting the number of coincident fluorescent events within the coincident probe volume over a period of time,

(vi) optionally using the result from step (v) to quantify the protein analyte in the sample solution.

2 A method as claimed in claim 1 wherein each ligand is present in the detection sample at a concentration of less than 500pM in step (iii) .

3 A method as claimed in claim 2 wherein each ligand is present in the detection sample at a concentration of less than lOOpM in step (iii) .

4 A method as claimed in claim 3 wherein each ligand is present in the detection sample at a concentration of less than 50pM in step (iii) .

A method as claimed in any one of the preceding claims wherein each ligand is present in the detection sample at a concentration in excess of the concentration of analyte in step (iii) . 6 A method as claimed in any one of the preceding claims wherein the detection sample in step (iii) is prepared such as to contain a concentration of analyte, if present, of greater than 50 fM.

7 A method as claimed in any one of the preceding claims wherein step (vi) comprises comparing the result from step (v) with a calibration curve generated using a known range of concentrations of analyte in the presence of ligands at the concentration given in step (iii) .

8 A method as claimed in any one of the preceding claims wherein, if the number given in step (v) does not exceed a background value in absence of analyte, step (vi) comprises repeating steps (iii) to (v) with a series of lower ligand concentrations and comparing the results from step (v) with respective calibration curves generated using a known range of concentrations of analyte in each of said ligand concentrations .

9 A method as claimed in any one of the preceding claims which is preceded by the step of providing the ligands and labelling them.

10 A method as claimed in any one of the preceding claims wherein the ligands are conjugated directly with the fluorophore .

11 A method as claimed in any one of claims 1 to 9 wherein the ligands are labelled with the fluorophore via a secondary ligand. 12 A method as claimed in any one of the preceding claims wherein the ligands are antibodies.

13 A method as claimed in any one of the preceding claims wherein two ligands are provided.

14 A method as claimed in any one of the preceding claims wherein the different fluorophores are red and blue excited fluorophores .

15 A method as claimed in any one of the preceding claims wherein coincident fluorescent events are detected within a probe volume of fixed location within the detection sample.

16 A method as claimed in any one of claims 1 to 14 wherein relative movement between the detection sample and the illumination device is introduced such that coincident fluorescent events are detected within a probe volume at a plurality of locations within the detection sample.

17 A method as claimed in claim 16 wherein the detection sample is passed or flowed through a coincident probe volume defined by the coincidence of the beams.

18 A method as claimed in claim 16 wherein a coincident probe volume defined by the coincidence of the beams is scanned through the detection sample.

19 A method as claimed in any one of the preceding claims wherein the detection sample is provided by and\or illuminated within a microfluidic device.

20 A method as claimed in any one of the preceding claims wherein the protein analyte is selected from: an enzyme; a hormone; a toxin; a receptor or a structural protein. 21 A method as claimed in any one of the preceding claims wherein the protein analyte is an individual protein.

22 A method as claimed in any one of claims 1 to 20 wherein the protein analyte is part of a target complex.

23 A method as claimed in claim 22 wherein the target complex is selected from a virus; a bacterium; a pathological protein aggregate.

24 A method as claimed in any one of claims 1 to 20 wherein the protein analyte is a complex or aggregate comprising at least one protein entity.

25 A method as claimed in claim 24 wherein the protein analyte is a complex or aggregate consisting of two protein entities .

26 A method as claimed in claim 25 wherein the protein entities are labelled in step (iii) by different ligands.

27 A method as claimed in claim 25 or claim 26 wherein the protein entities are low abundance proteins of the proteome.

28 A method as claimed in any one of the preceding claims for quantitation of protein complexes in a sample, which comprises performing step (vi) .

29 A method as claimed in any one of the preceding claims wherein the sample is a biosample of an organism, tissue, cell or body fluid.

30 A method as claimed in claim 29 wherein sample is selected from: CSF, saliva, sweat, urine, blood, faeces.

31 A method as claimed in any one of claims 1 to 28 wherein the sample is an environmental sample.

32 A method as claimed in claim 31 wherein sample is selected from: soil; water; plant material.

33 A method of diagnosis or prognosis, in an individual, of a disease which is associated with a protein analyte, which method comprises taking a biopsy sample from the individual and detecting or quantifying the analyte using a method as claimed in claim 29 or claim 30.

34 A method of assessing infection or contamination of an environment, wherein the infection or contamination is associated with a protein analyte, which method comprises taking a sample from the environment and assessing the analyte using a method as claimed in claim 31 or claim 32.

35 A method for detecting or identifying the interaction of two binding entities to form a protein analyte target complex, which method comprises performing a method as claimed in any one of claims 25 to 27.

36 A kit for use in carrying out the method of any one of the preceding claims, which kit includes any two or more of the following: i) at least two ligands capable of binding specifically to a protein analyte, each ligand being labelled with a different fluorophore, which fluorophores are excitable by spectrally separated wavelengths of light, ii) one or more standard solutions for calibrating the apparatus or components of the method, iii) instructions for use in a method of any one of the preceding claims.