Patent Application
20160011208
This invention is in the field of assays for aggregatory proteins.
The increased propensity of proteins to misfold and aggregate in the aged organism is thought to underlie many diseases of old age such as Alzheimer's, Parkinson's, motor neurone and prion diseases. These disorders are characterised by aggregates of specific polypeptides and, in the case of Alzheimer's disease (AD), these are extracellular plaques and intracellular tangles. The plaques are of particular interest because overproduction of the constituent peptide, amyloid beta (Aβ), is sufficient to cause rare familial forms of AD.
There is significant interest in understanding the fundamental processes underlying the aggregation that causes these conformational diseases, which should assist with the development of therapeutics to slow or stop the aggregation. Given the central role that protein aggregation has in the pathology of the conformational diseases, there is a need in the art for improved methods for testing the effects of substances on aggregation, in order to identify and develop therapeutics.
The generation of biologically toxic protein aggregates is amenable to study in vitro, where the reaction is seen to occur spontaneously following an initial delay called the lag phase. In a process called seeding, it is also observed that the addition of a few pre-formed aggregates will greatly accelerate the formation of protein aggregates, particularly in those cases where amyloid fibrils are the end product. The presence of such aggregatory seeds has been measured in bulk samples from experimental organisms such as worms and mice and has been observed in human tissue [1-3]. Similar seeding processes are thought to be fundamental to the infectivity that is seen in prion diseases such as bovine spongiform encephalopathy and variant Creutzfeldt-Jacob diseases. Moreover, the spread of pathology within the brain of patients suffering from the common neurodegenerative disorders may occur in a similar way. Indeed, seeding may directly result in the relentless clinical progression that is so distressing to patients and their carers [4].
Current technology allows the measurement of protein aggregation in macroscopic samples using dyes such as Congo red, thioflavin T, thioflavin S and luminescent-conjugated oligothiophenes [5] that generate a fluorescence signal when they are exposed to amyloid-like protein aggregates. However, these bulk phase techniques are insufficiently sensitive for use as a diagnostic test, because background spontaneous seeding will mask the specific effects of seeding and any specific signal is diluted into the bulk of a typical test solution. A highly sensitive and specific test will be required if aggregatory seeds, at low concentrations in peripheral tissues and fluids such as blood and urine, are to be detected [1]. Current diagnostic tests for conformational diseases caused by the aggregation of proteins, such as Alzheimer's disease, typically rely on algorithms based on neuropsychological tests and brain imaging, in part because the protein that aggregates is present in healthy individuals, so the utility of these proteins as biomarkers is limited. There is therefore a need in the art for new and improved diagnostic assays that can accurately detect the presence of clinically relevant species representing a particular conformation of a protein or an aggregate of polypeptides with a particular conformation. Improved assays are also required for screening candidate therapeutics for an ability to modulate clinically relevant processes underlying the aggregation of proteins that cause conformational diseases.
In a first aspect the invention provides a method for detecting the presence of aggregatory seeds of a polypeptide in a sample, comprising:
(i) contacting the sample with monomers of the polypeptide;
(ii) separating the sample into a plurality of microdroplets;
(iii) incubating the microdroplets under conditions suitable for aggregation of the polypeptide; and
(iv) determining the subsequent formation of aggregates of the polypeptide in the microdroplets,
wherein the formation of aggregates of the polypeptide in a microdroplet indicates the presence of one or more aggregatory seeds in the sample.
Aggregatory seeds of a polypeptide are difficult to detect specifically and sensitively when at low concentrations. This is because they are generally detected though aggregation seeding assays whereby samples comprising aggregatory seeds are contacted with monomers of the relevant polypeptide and the formation of polypeptide aggregates seeded by aggregatory seeds is detected. However, when the aggregatory seeds are present at a low concentration, aggregation seeded by aggregatory seeds is swamped by spontaneous aggregation of the polypeptide monomers and it is difficult specifically and sensitively to identify the presence of aggregates seeded by aggregatory seeds above this background spontaneous aggregation. Spontaneous aggregation is relatively slow and spatially homogeneous within the bulk phase solution. In the bulk phase the homogeneous, low level generation of signal cannot be differentiated by the spatially localised, high efficiency generation of signal. This is because in the bulk phase the locally generated signal diffuses into the bulk and so becomes indistinguishable from spontaneously generated signal.
The method of the invention is able to specifically detect seeded aggregation and to distinguish it from background spontaneous aggregation by contacting a sample with monomers of the relevant polypeptide and compartmentalising the sample into a plurality of microfluidic droplets. By separating a sample into a plurality of microdroplets, the local concentration of any aggregatory seeds is increased to the extent that seeded aggregation in microdroplets comprising aggregatory seeds is specifically and sensitively detectable above background spontaneous aggregation.
Therefore, compartmentalising the sample into microdroplets is particularly advantageous for detecting aggregatory seeds using an aggregation assay, because the monomers of the polypeptide that are required for the aggregation assay are liable to spontaneously aggregate, which produces background signal or noise, making the specific detection of aggregates resulting from aggregatory seeds difficult. By analysing a plurality of microdroplets, each of which can be positively determined to have contained or to not have contained aggregatory seeds, the invention allows a sensitive and quantitative analysis of the presence of aggregatory seeds in the initial sample.
Classical biochemical experiments performed in bulk phase are not sensitive or specific enough to usefully report the presence of aggregatory seeds of a protein in a sample. To overcome the insufficiently sensitive detection limits of current methods, the inventors have developed a method that uses compartmentalisation in microfluidic droplets, such as water-in-oil droplets, with μm diameters and picolitre volumes. The fragmentation of a bulk phase into many smaller reactors (for example, about 107/ml) creates higher local concentrations. For example, if only one seed is present in a sample, the effective concentration in one of 107 droplet compartments will be increased 107-fold. The principle underlying the invention is demonstrated schematically in
The method of the invention is particularly useful in the diagnosis and monitoring of disease, because the assay can be used to detect the presence of aggregatory seeds of a polypeptide in patient samples. Therefore, the invention also provides a method for identifying a subject at risk of a conformational disease caused by the aggregation of a polypeptide or for diagnosing, assessing or monitoring a conformational disease caused by the aggregation of a polypeptide in a subject, comprising detecting the presence of aggregatory seeds of the polypeptide in a sample obtained from the subject, wherein the detecting comprises:
(i) contacting the sample with monomers of the polypeptide;
(ii) separating the sample into a plurality of microdroplets;
(iii) incubating the microdroplets under conditions suitable for aggregation of the polypeptide; and
(iv) determining the subsequent formation of aggregates of the polypeptide in the microdroplets,
wherein the formation of aggregates of the polypeptide in a microdroplet indicates the presence of one or more aggregatory seeds in the sample, and the presence of aggregatory seeds in the sample is correlated with the risk, presence, or severity of disease.
The inventors have developed a method for sensitively and specifically detecting the presence of aggregatory seeds at very low concentrations. Therefore, the present invention makes it possible to detect the presence of aggregatory seeds in samples obtained from human subjects and to correlate the presence of aggregatory seeds with the risk, presence, or severity of a conformational disease. As noted above, by analysing a plurality of microdroplets, each of which can be positively determined to have contained or to not have contained aggregatory seeds, the invention allows a sensitive and quantitative analysis of the presence of aggregatory seeds in the initial sample, and this enables a useful diagnosis or prognosis to be made.
The method of the invention is particularly advantageous, because it enables a new class of biomarker to be analysed in the diagnosis of conformational diseases. Conformational diseases are often caused by endogenous proteins that are present in healthy individuals, which limits the utility of simply measuring the concentration of these proteins as biomarkers of disease. This is because it is the conformation of the protein or the self-association of several copies of the polypeptides in particular conformations that generates the toxic species. By sensitively and specifically detecting aggregatory seeds, the present invention allows the detection of a clinically relevant pathological form of the protein underlying the conformational disease. In addition, by detecting aggregatory seeds, the method of the invention allows earlier diagnosis of conformational disease, before large aggregates have been formed. Also, the invention represents a significant advancement because it involves quantifying an activity (seeding of aggregation), rather than merely reporting the presence of a structural epitope in a sample. This seeding activity is of fundamental importance in disease progression, so such measurements have diagnostic and prognostic value in the clinic.
Furthermore, the proteins underlying conformational diseases generally aggregate in the brain, which is inaccessible for simple diagnostic tests. By sensitively and specifically detecting aggregatory seeds, the present invention allows the detection of a clinically relevant pathological form of the protein in peripheral fluids, such as blood and urine, which is practical for diagnostic testing. Aggregatory seeds of proteins underlying conformational diseases are present in peripheral fluids, whilst larger aggregates, which might be assayed more easily, are only present in less accessible tissues, such as the brain.
Significantly, the sensitivity of the method of the invention makes it possible to detect aggregatory seeds at the very low concentrations that they are found in, in peripheral tissues such as blood and urine. As explained above, through contacting a sample with monomers of the relevant polypeptide, compartmentalising the sample into a plurality of microfluidic droplets and performing an aggregation assay to detect the formation of polypeptide aggregates, the method of the invention allows the specific and sensitive detection of aggregatory seeds at low concentrations.
In addition to methods of diagnosis, the assay of the invention will be useful for preparing reagents or products that are free from aggregatory seeds and for quantifying the amount of aggregatory seeds produced in a test reaction. For example, the assay of the invention will be useful for assessing the ability of a reagent to effect the formation of aggregatory seeds.
In certain embodiments, the microdroplets comprise droplets in an emulsion, such as a water-in-oil emulsion, and the method of the invention comprises forming an emulsion, such as a water-in-oil emulsion. Emulsions can be generated easily using standard equipment and techniques. Microdroplets can be generated in microfluidic devices, are easy to handle with microfluidic devices and they are particularly amenable to the high throughput screening, which is useful for analysing a large number of aliquots, as in the present invention. There are also a number of methods for producing bulk emulsions including vigorous stirring, vortexing, shaking and extrusion, although the resulting droplets are polydisperse.
In certain embodiments, the microdroplet comprises a gel-forming agent and the microdroplet is transformed into a gel bead. Preferably, the microdroplets are transformed into gel beads after incubating the microdroplets under conditions suitable for aggregation of the polypeptide. By transforming the microdroplet into a gel bead, polypeptide aggregates are retained in the bead despite thorough washing. This has the advantage of allowing very sensitive and specific detection of polypeptide aggregates, because un-aggregated monomers can be washed away. Furthermore, the gel beads are stable and resilient and can be transferred between different reaction conditions, allowing further detailed analysis of the polypeptide aggregates, whilst still maintaining the compartmentalisation of the initial subject sample. Therefore, in certain embodiments, the microdroplet is transformed into a gel bead and polypeptide monomers are removed from the gel bead by washing. In certain embodiments, the gel beads are subjected to a plurality of different assays.
The advantages of performing the aggregation assay in a microdroplet comprising a gel-forming agent and transforming the microdroplets into gel beads is applicable to a second aspect of the invention. In this second aspect, the invention provides a method for measuring the effect of a test substance on the aggregation of a polypeptide, comprising:
forming a microdroplet comprising the test substance, monomers of the polypeptide and a gel-forming agent in liquid form;
(ii) incubating the microdroplet under conditions suitable for aggregation of the polypeptide;
(iii) transforming the microdroplet into a gel bead; and
(iv) determining the amount of aggregates of the polypeptide present in the gel bead and comparing the amount to a control bead.
The inventors have developed a method for assaying modulators of polypeptide aggregation that is particularly sensitive and efficient. By performing the assay in microdroplets comprising a gel-forming agent and transforming the microdroplets into gel beads, polypeptide aggregates that are formed are immobilised in the gel beads. The assay of the invention is particularly sensitive because the gel beads can be washed extensively to remove, for example, polypeptide monomers and other reactants, and polypeptide aggregates will be retained in the gel beads. This decreases background signal from, for example, unaggregated material. The method of the invention is particularly amenable to high-throughput screening, because the gel beads are stable, resilient and easy to handle. Therefore, the assay of the invention allows large numbers of gel beads to be analysed. By accommodating the analysis of a large number of gel beads, the methods of the invention provide increased sensitivity, because detecting very rare events is possible. In addition, the ability to test a large number of beads rapidly allows many compounds to be tested. Furthermore, the resilience of the gel beads means that they can be washed and subjected to different conditions without the aggregates being lost and whilst still maintaining the compartmentalisation of the initial subject sample, which allows multiple assays to be performed.
The porous structure of the bead matrix serves to retain aggregated protein, whilst small molecule components and buffer are free to enter or exit the bead. The beads thus become a ‘time capsule’ for aggregated product, which can be subjected to a number of downstream analysis methods. The ability to wash resulting gel beads and remove small oligomers and molecules enhances the sensitivity of, for example, fluorescence-based assays, because the relative signal from trapped protein aggregates increases while background from un-aggregated material is removed. The small droplet volumes (for example, 47.7 pL) provide for economical experiments and multiple redundancy. For example, a total reaction volume of 1 μL can generate up to 105 identical droplets and beads, which constitutes an enormous pool for either averaging techniques or individual analysis.
Therefore, the invention provides a novel assay for the study of protein self-assembly reactions where emerging protein aggregates are confined in microbeads made up of a gel ‘cage’. The beads have unique properties that permit aggregation reactions to proceed at near physiological concentrations. The ‘cage’ is formed by co-compartmentalising liquid gel with the reagent containing solute (e.g. an aqueous solution containing peptide and drug etc.) during the droplet formation step. The droplets can be formed in an oil phase at elevated temperatures in liquid form and stored as an emulsion for the aggregation reactions to proceed. The emulsion can be cooled on ice, leading to solidification of the beads before or after the process to be studied is completed. The oil phase is then removed and the beads are washed. The porous internal structure of the gel matrix (such as an agarose polymer matrix) prevents large aggregates from escaping the beads, whilst soluble peptide, small molecules or solvent are readily removed. The retained aggregates can be readily analysed e.g. by fluorescence or other methods in a highly repeatable and quantitative fashion. The technology affords great flexibility in handling, manipulating and analysing the self-assembly of neurotoxic proteins: reagents can be added or removed, concentrations varied, and ensuing aggregates analysed optically informing on the kinetics of aggregation and the morphology of ensuing amyloid structures. The invention provides the first demonstration of this technology and shows that the aggregation of proteins, such as mutants of Aβ, can be studied in a quantitative manner at near physiological concentrations. The method of the invention can be used to apply several optical techniques to analyse the retained aggregates, including fluorescence lifetime imaging and optical super-resolution imaging, giving direct insight on the morphology of forming aggregates. In addition, the process of aggregate formation can monitored over time by quantification of the fluorescence of the retained aggregate (by measuring intrinsic fluorescence, the fluorescence of the products of seeded co-aggregation with fluorescent monomers or by measurements of fluorescence after staining of the emerging aggregate with amyloid dyes such as e.g. thioflavin T and S, Congo Red), e.g. by flow cytometric (FACS) analysis or imaging techniques. The technology is particularly useful for the rapid screening of agents which may act as modifiers of aggregation.
This aspect of the invention also provides a method for manufacturing a pharmaceutical composition for treating a conformational disease caused by the aggregation of a polypeptide comprising: measuring the effect of a test substance on the aggregation of a polypeptide, in accordance with the method described above; selecting a substance that inhibits aggregation of the polypeptide; optionally modifying the substance; and combining the substance with a pharmaceutically acceptable carrier or excipient. The sensitivity of the assay of the invention, and its amenability to high-throughput screening provides a more efficient method for manufacturing therapeutics that directly affect the polypeptide aggregation that underlies conformational diseases.
The term “aggregatory seed” as used herein refers to a soluble oligomer of a protein or polypeptide. Typically, an oligomer comprises monomers associated through non-covalent interactions. Aggregatory seeds shorten the nucleation-associated lag phase in the kinetics of aggregate formation. Therefore, an aggregatory seed initiates and accelerates the aggregation of similar or identical monomers. For example, aggregatory seeds accelerate the fibrilisation of monomeric protein with sequence homology. Aggregatory seeds may also act via corruptive molecular templating.
Generally, an aggregatory seed comprises a single peptide species and it seeds aggregation of monomers of the same peptide species. Generally, where a polypeptide has a native structure, an aggregatory seed comprises peptides that are at least partially misfolded, but not completely denatured. Therefore, in certain embodiments, an aggregatory seed is a soluble oligomer of misfolded peptides. In certain embodiments, an aggregatory seed comprises 2-20 monomers.
Microdroplets
A “microdroplet” is small droplet of a sample that can be manipulated using standard microfluidic technology and devices. In certain embodiments of the invention, a microdroplet has a volume of less than 1 μl, less than 500 nl, less than 100 nl, less than 10 nl, less than 1 nl, less than 500 μl, less than 400 μl, less than 300 μl, less than 200 μl, less than 100 μl, or less than 50 pl. In certain embodiments of the invention, a microdroplet has a volume of between 0.5 pl and 1 μl, between 0.5 μl and 500 nl, between 1 μl and 200 nl, between 20 μl and 100 nl, between 20 μl and 1 nl, between 20 μl and 500 μl, between 20 μl and 300 μl, between 20 μl and 200 μl, between 20 μl and 100 μl, between 30 μl and 100 μl, or between 30 μl and 80 μl. In certain embodiments, a microdroplet has a diameter of less than 1 mm, less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm, less than 100 μm, less than 80 μm, less than 70 μm, less than 60 μm, or less than 50 μm. In certain embodiments, a microdroplet has a diameter of between 1 μm and 1 mm, between 10 μm and 500 μm, between 10 μm and 250 μm, or between 20 μm and 100 μm. When used in the detection of aggregatory seeds, the size of the microdroplet allows seeded aggregation to proceed faster than spontaneous aggregation. This may also depend on the concentration of the monomers of the protein. The skilled person is able to determine the appropriate size of microdroplet and the skilled person is able to determine an appropriate concentration of the monomers of the protein.
The number of microdroplets that a sample is compartmentalised into will depend on the volume of the sample and the volume of the microdroplets. It is not necessary that the entirety of a sample of interest, for example a clinical sample, is tested and compartmentalised into microdroplets. The portion of the sample that is tested must be sufficient to provide useful information regarding the whole sample. The skilled person is able to determine the appropriate portion of sample to test. In certain embodiments, a “plurality of microdroplets” is at least 103, 104, 105, 106, 107, 108 or 109 microdroplets.
Microdroplets may be produced by emulsifying the aqueous reaction mixture in a non-aqueous liquid, for example oil, comprising a surfactant. Any suitable surfactant may be used. Techniques for the production of microfluidic aqueous droplets are well known in the art [6-12].
Microdroplets are preferably monodisperse i.e. they preferably have a uniform size.
Classically, assays for protein aggregation are carried out in microwell plates [13], the standard format for pharmaceutical and biochemical experimentation. However, recently assays have been reported that have been scaled down to nano- or even pico-litre volumes [14,15]. This miniaturization is achieved by compartmentalisation of assay components in water-in-oil emulsion droplets generated within microfluidic devices [16-19]. The devices are capable of generating and manipulating droplets at rates of up to 10 kHz. Fully automated modular workflows can be designed [20] to incorporate physical unit operations such as droplet sorting [21-23] and fusion [24,25], and even biochemical processes can be incorporated, such as PCR [26,27], in vitro protein expression [28], enzymatic reactions [25,29] or cell-based assays [30-32]. The minute droplet volume between 0.5 pL and 4 nL also offers advantages for solution-based compounds screening. These attributes, coupled with advanced analytical techniques [33-37], permit droplet assays to be performed in high-throughput fashion. In addition to saving reagents, the compartmentalisation afforded by the droplet boundary enables one to design experiments and access information that is masked in conventional ensemble experiments. Furthermore, effects arising from cell-like confinement or interfacial phenomena can be mimicked inside droplet compartments.
Microdroplets may be prepared and handled using any appropriate means. For example, microfluidic devices constructed using conventional soft lithographic techniques, FF-chips or commercial glass chips may be used.
Polypeptides Prone to Aggregation
The methods of the invention are suitable for use with any polypeptide or protein that is prone to aggregation. In certain embodiments, the protein is Amyloid β peptide, such as Aβ1-40 or Aβ1-42, tau protein, prion protein, α-Synuclein, TDP-43, FUS, huntingtin, a serpin, transthyretin, Notch 3, glial fibrillary acidic protein, seipin, islet amyloid polypeptide (IAPP), pro-IAPP, apolipoprotein AI, AII or AIV, gelsolin, β2 microglobulin, rhodopsin, keratin, keratoepithelin, tubulin, surfactant protein C, odontogenic ameloblast-associated protein, calcitonin, atrial natriuretic factor, serum amyloid A, medin, or lysozyme.
In preferred embodiments, the protein is a protein that forms amyloid aggregates, such as Amyloid β peptide, Aβ1-40, Aβ1-42, IAPP, α-Synuclein, prior protein, or huntingtin. The seeding of aggregation by aggregatory seeds is particularly important in the formation of amyloid aggregates. In preferred embodiments, the protein is a protein that causes a neurodegenerative disorder. The detection of aggregatory seeds that cause neurodegenerative disorders is particularly important, because the aggregatory protein accumulates in the brain, so it is useful to detect aggregatory seeds in peripheral tissues. In preferred embodiments, the protein is a prion protein. In particularly preferred embodiments, the protein is an Amyloid β peptide, such as Aβ1-40 or Aβ1-42. Amyloid beta is a peptide of 36-43 amino acids that is processed from the Amyloid precursor protein by secretases. Aβ1-40 is the predominant form found circulating in plasma, cerebrospinal fluid and brain interstitial fluid. Aβ1-42 is more aggregatory and is associated with the development of disease, and with more severe disease. Both Aβ1-40 and Aβ1-42 are found within plaques.
Detection and Analysis of Aggregates
The use of microdroplets, and optionally gel beads, in the methods of the invention allows rapid, high throughput analysis of aggregates that are formed, which is useful for detecting the aggregatory seeds in a sample and for measuring the effect of a test substance on the aggregation of a protein.
In certain embodiments of either aspect of the invention, the method comprises determining the subsequent formation of aggregates of the polypeptide, which may comprise determining the presence of aggregates of the polypeptide in a microdroplet. In certain embodiments of either aspect of the invention, the protein monomers are labelled and determining the presence of aggregates of the protein comprises detecting the label. In certain embodiments, the protein monomers are fluorescently labelled and determining the presence of aggregates of the protein comprises detecting a fluorescent signal. In preferred embodiments, the fluorescent signal is detected using a flow cytometer or a fluorescence activated cell sorter (FACS). In certain embodiments, the magnitude of the fluorescent signal is correlated with the presence of protein aggregates. In certain embodiments, the profile of the fluorescent signal obtained using FACS indicates the presence of protein aggregates. In certain embodiments, the profile of the fluorescent signal indicates the structure of the protein aggregates formed. For example, Examples 2 and 3 demonstrate that aggregates formed through seeded aggregation initiated by an aggregatory seed result in a profile that can be distinguished from aggregates formed through spontaneous aggregation. Therefore, in certain embodiments, the step of determining the presence of aggregates of the protein comprises correlating the magnitude of the fluorescent signal with the amount of protein aggregate present. In certain embodiments, the magnitude of the fluorescent signal indicates the presence of aggregatory seeds in the test sample, or indicates the effect of the test substance on the aggregation of the protein.
As shown in Examples 2 and 3, use of fluorescently labelled protein monomers and detection of fluorescent signals allows specific detection of synthetic aggregatory seeds and aggregatory seeds derived from animal models of disease. There is a significant and readily detectable difference in the fluorescence signal generated by free protein monomers, spontaneously formed aggregates, and seeded aggregates. By incubating aliquots of seed-containing samples with solutions containing fluorescently labelled monomeric Aβ peptides in picolitre volumes, the fluorescence signal from aggregates generated from the process of seeded aggregation of the fluorescently labelled monomeric Aβ peptides was specifically measured.
As shown in Examples 7 and 8, use of fluorescently labelled protein monomers and detection of fluorescent signals allows highly sensitive measurement and monitoring of the aggregation of proteins and modulation of this aggregation.
Optical signals from fluorescently labelled protein monomers are amenable to investigation by a number of biophysical techniques. For example, in certain embodiments, fluorescence lifetime microscopy is used to detect and/or to inspect the aggregates formed. A decrease in fluorescence lifetime is observed when protein monomers aggregate into amyloid structures [37]. In addition, the fluorescence lifetime of different types of aggregates can be distinguished and a longer fluorescence lifetime is correlated with the presence of more disordered aggregates. In a method of diagnosis, more disordered aggregates and/or the presence of seeds that produce more disordered aggregates may be correlated with increased severity or risk of disease. Also, the effect of a test substance on aggregation of a protein may be detected by an alteration in the degree of order in an aggregate as detected, for example, by fluorescence lifetime microscopy.
In certain embodiments, the method used to measure the fluorescence lifetime from reporter labels may include time domain methods such as time gated detection [38], time correlated single photon counting [39], or frequency domain lifetime imaging methods [40].
In certain embodiments, protein aggregation is monitored using fluorescence anisotropy imaging microscopy (FAIM), which monitors changes in the polarisation state of emitted light which occur upon assembly of labelled monomers into aggregated species [41].
The skilled person is aware of techniques that can be used to detect the presence of aggregates in the microdroplets or gel beads in accordance with the invention, and to determine the structure of the aggregates. For example, in certain embodiments a flow cytometer, fluorescence lifetime microscopy, direct stochastic optical reconstruction microscopy (dSTORM), structured illumination microscopy or FRET microscopy is used to detect aggregates or determine the structure of aggregates. In alternative embodiments, circular dichroism is used [42,43].
The use of fluorescently labelled protein monomers allows highly sensitive detection of aggregatory seeds. While fluorescence intensity reports on the amount of aggregate produced, lifetime measurements inform on the type of aggregate formed, such as the size or degree of order, and thus provides a second way to determine whether a droplet contains seeded or un-seeded aggregates. The profile of the fluorescent signal can be used to determine further details regarding the structure of the aggregates. As shown in Example 4, fluorescence lifetime microscopy can be used to distinguish between diseased mouse brain extracts and control mouse brain extracts (which produce a signal identical to beads incubated with buffer alone), based on the presence of aggregatory seeds.
In certain embodiments, the protein monomers are labelled with an Alexa Fluor™ dye, such as Alexa647. Alexa Fluor dyes are synthesized through sulfonation of coumarin, rhodamine, xanthene (such as fluorescein), and cyanine dyes. In certain embodiments, the protein monomers are labelled with a HiLyte Fluor™ dye, such as Hilyte 488. Further suitable fluorescence reporters include fluorescein and fluorescein derivatives such as O-methyl-fluorescein or fluorescein isothiocyanate (FITC), phycoerythrin, Europium, TruRed, Allophycocyanin (APC), PerCP, Lissamine, Rhodamine, B X-Rhodamine, TRITC, BODIPY-FL, FluorX, Red 613, R-Phycoerythrin (PE), NBD, Lucifer yellow, Cascade Blue, Methoxycoumarin, Aminocoumarin, Texas Red, Hydroxycoumarin, Alexa Fluor™ dyes (Molecular Probes) such as Alexa Fluor™ 350, Alexa Fluor™ 488, Alexa Fluor™ 546, Alexa Fluor™ 568, Alexa Fluor™ 633, Alexa Fluor™ 647, Alexa Fluor™ 660 and Alexa Fluor™ 700, sulfonate cyanine dyes (AP Biotech), such as Cy2, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, IRD41 IRD700 (Li-Cor, Inc.), NIR-1 (Dejindom, Japan), La Jolla Blue (Diatron), DyLight™ 405, 488, 549, 633, 649, 680 and 800 Reactive Dyes (Pierce/Thermo Fisher Scientific Inc) or LI-COR™ dyes, such as IRDye™ (LI-COR™ Biosciences). In certain embodiments, the protein is a recombinant protein labelled with a fusion protein such as GFP, YFP, mRFP or TFP. The skilled person is familiar with other fusion proteins that may be used to label the protein.
In certain embodiments, the formation of aggregates is detected using a reporter substance. For example, in certain embodiments, the sample and the monomers of protein are mixed with a reporter substance and the step of detecting the formation of aggregates of the protein comprises detecting the reporter substance. In such embodiments, the reporter substance generates a specific signal when in the presence of protein aggregates, such as amyloid aggregates. In certain embodiments, the reporter substance is Thioflavin T, Congo red, or (trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB).
In certain embodiments of either aspect of the invention, certain beads or droplets are isolated based on the degree or structure of aggregates formed. The isolated beads or droplets can then be analysed further. This is particularly useful when gel beads are used, because the beads retain protein aggregates and enable buffers etc. to be exchanged for further analytical reactions to be performed. For example, in certain embodiments, the method of the invention comprises isolating beads or droplets comprising aggregates formed by seeded aggregation, or aggregates of a certain size.
Furthermore, in certain embodiments of the invention, the structure of the aggregates formed during the assay are analysed to provide a more detailed diagnosis. Therefore, in certain embodiments, the step of determining the presence of aggregates of the protein comprises determining the structure or one or more biophysical characteristics of the aggregates. Alternatively, in certain embodiments, the method comprises an additional step of determining the structure or one or more biophysical characteristics of the aggregates. In such embodiments, the structure and biophysical characteristics of the aggregates are used to classify the aggregatory seeds present in the sample obtained from the subject, which is used to provide a diagnosis. For example, in certain embodiments, the presence of aggregatory seeds that seed more disordered aggregates is correlated with increased severity or risk of disease. In certain embodiments, the presence of aggregatory seeds that seed more rapid aggregation of protein is correlated with increased severity or risk of disease.
Therefore, in certain embodiments of the invention, the step of determining the presence of aggregates of the protein in the microdroplets comprises detecting the presence or absence of aggregates of the protein; measuring or quantifying the amount of aggregates of the protein; and/or analysing the structure (for example, the degree of order) of the aggregates of the protein.
In certain embodiments of the invention, the sample to be tested is diluted prior to use in the assay. For example, the sample, such as a clinical sample obtained from a subject, may be diluted 5, 10, 50, 100 or 1000 times. The sensitivity and specificity of the assay and its ability to detect the presence of aggregatory seeds may be improved through such dilution.
The steps of any method disclosed herein may be performed in any order.
Usually the raw data obtained from an assay require some sort of manipulation prior to their use. For instance, the nature of most detection techniques means that some signal will sometimes be seen even if no aggregatory seed or aggregate is actually present and so this noise may be removed before the results are interpreted. Similarly, there may be a background level of the aggregatory seed in the general population which needs to be compensated for. Background signal may comprise a second peak due to background or a possible background rate in creating the second peak.
Data may need scaling or standardising to facilitate inter-experiments comparisons. These and similar issues, and techniques for dealing with them, are well known in the diagnostic and fluorescence microscopy areas.
Various techniques are available to compensate for background signal in a particular experiment. For example, replicate measurements will usually be performed to determine intra-assay variation, and average values from the replicates can be compared. Signal may be adjusted according to distribution in a single experiment. Data transformations of this type are standard in the art for permitting valid inter-array comparisons despite variation between different experiments.
Methods of Diagnosing, Assessing, Monitoring or Identifying Risk for Disease
As discussed above, the methods of the invention are particularly useful for detecting the presence of aggregatory seeds in a patient sample in order to provide a diagnosis, prognosis or to identify risk for developing a disease.
The method of the invention is useful for diagnosis, prognosis and diagnosis of risk for diseases caused by the aggregation of a particular protein. Such diseases are known as conformational diseases.
As will be clear to the skilled person, the method of the invention is used to diagnose, assesses, monitor or identify risk for the conformational disease that is caused by aggregation of the protein of which aggregatory seeds are detected. Therefore, aggregatory seeds of Aβ1-40 or Aβ1-42 are detected in the diagnosis of Alzheimer's disease, and aggregatory seeds of α-synuclein are detected in the diagnosis of Parkinson's disease, etc. The method of the invention is useful for diagnosing, assessing, monitoring and identifying risk for Alzheimer's disease, Parkinson's disease and other synucleinopathies, tauopathies, prion diseases, cerebral β-amyloid angiopathy, CADASIL syndrome, frontotemporal lobar degeneration, FTLD-FUS, amyotrophic lateral sclerosis, Huntington's disease and other triplet repeat disorders, hereditary cerebral hemorrhage with amyloidosis, Alexander disease, seipinopathies, familial amyloidotic neuropathy, senile systemic amyloidosis, serpinopathies, type II diabetes, aortic medial amyloidosis, ApoAI, AII or AIV amyloidosis, familial amyloidosis of the Finnish type, lysozyme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis, inclusion body myositis/myopathy, retinitis pigmentosa with rhodopsin mutations, medullary thyroid carcinoma, cardiac atrial amyloidosis, corneal dystrophy, cutaneous lichen amyloidosis, pulmonary alveolar proteinosis, odontogenic (Pindborg) tumor, atherosclerosis, and systemic amyloidoses such as hereditary non-neuropathic systemic amyloidosis, AA and AL.
The central role that aggregatory seeds play in the pathogenesis of conformational disease is well established. Seeded (prion-like) protein aggregation is understood to spread out from an initial nidus [4]. Murine models of seeded aggregation have implicated both Aβ and also the intracellular protein tau in the pathology of Alzheimer's disease [2,3,44-46]. For both Aβ and tau it has been shown that inoculation of model rodents with preformed aggregates can accelerate the pathological deposition of plaques and tangles, something that would eventually occur spontaneously in these rodents. Furthermore, there is good evidence that similar inoculation can seed novel tau pathology in rodents that would otherwise remain healthy [2,46]. The seeded aggregation of proteins is particularly well understood in relation to prion proteins and the formation of amyloid. The prion protein can misfold and aggregate, either stochastically or as the result of a mutation, and the resulting seeds are able to spread within, and between, individuals creating a cascade of misfolding and neurotoxicity [47,48]. In vitro studies of aggregation and amyloid formation have shown that a pure solution of monomers is often stable for an extended period of time called the lag phase. During this time a stochastic process of seed formation occurs, resulting in a burst of amyloid formation as fibrils grow and fracture and so generate more sites for the rapid addition of monomeric peptide [49,50]. These fibrils, when doped back into a subsequent aggregation reaction will often abolish the lag phase, accelerating the formation of further amyloid. Prion-like infectivity through aggregatory seeds has also been demonstrated in other conformational diseases, such as Huntington's disease [1], Parkinson's disease and motor neurone disease [51]. Therefore, the detection of aggregatory seeds is likely to be useful and effective in the diagnosis of conformational diseases.
In preferred embodiments, the method of the invention is a method for diagnosing, assessing, monitoring or identifying risk for a neurodegenerative disease. The methods of the invention are particularly useful for neurodegenerative diseases, because while the brain is not accessible, nevertheless aggregatory seeds can be found in peripheral fluids, such as blood.
In preferred embodiments, the method of the invention is a method for diagnosing, assessing, monitoring or identifying risk for an amyloidopathy. The methods of the invention are particularly useful for amyloidopathies, because the seeding of aggregation by aggregatory seeds is particularly important in the formation of amyloid aggregates, so a diagnosis that includes detection of aggregatory seeds is likely to be particularly accurate and useful.
In particularly preferred embodiments, the method of the invention is a method for identifying a subject at risk of Alzheimer's disease or for diagnosing, assessing or monitoring Alzheimer's disease.
The subject will typically be a human being. In some embodiments, however, the invention is useful in non-human organisms e.g. horse, pig, cow, sheep, deer, chicken, mouse, rat, rabbit, guinea pig, cat, dog, or non-human primate (monkeys or apes, such as macaques or chimpanzees).
In some embodiments, a method of the invention involves an initial step of obtaining the sample from the subject. In other embodiments, however, the sample is obtained separately from and prior to performing a method of the invention. After a sample has been obtained then methods of the invention could be performed in vitro.
In certain diagnostic methods of the invention, the sample tested is a blood sample (such as a serum or plasma sample), a urine sample, a cerebrospinal fluid sample, a saliva sample, a tears sample, a lymphatic fluid sample or a urine sample. As discussed above, the sensitive methods of the invention are suitable for detecting aggregatory seeds at low concentrations in peripheral fluids. In preferred embodiments of the invention, the sample is a serum or plasma sample.
Detection of aggregatory seeds may be performed directly on a sample taken from a subject, or the sample may be treated between being taken from a subject and being analysed. For example, a blood sample may be treated to remove cells, leaving plasma containing aggregatory seeds for analysis, or to remove cells and various clotting factors, leaving serum potentially containing aggregatory seeds for analysis. For some body fluids, such separation treatments are not usually required (e.g. urine, tears or saliva) but other treatments may be used. For example, various types of sample may be subjected to treatments such as dilution, aliquoting, sub-sampling, heating, freezing, irradiation, etc. between being taken from the body and being analysed e.g. serum is usually stored, frozen prior to analysis. Also, addition of processing reagents is typical for various sample types e.g. addition of anticoagulants to blood samples.
In certain embodiments of the invention, the method additionally comprises performing additional tests and analyses. For example, a diagnosis will generally also include consideration of the subject's age, their family history of disease, other biomarkers, and/or any symptoms. In addition, assessing a subjects risk for a disease will generally include numerous variables including the subject's age, their family history of disease, other biomarkers, and lifestyle. An algorithm may be used to arrive at an assessment of disease risk. The skilled person is aware of how to develop a suitable algorithm.
In certain embodiments of the invention, the presence or levels of aggregatory seeds in a patient's clinical sample may be compared to a control sample. The control sample may be derived from a patient or group of patients that do not have disease, or a patient or group of patients that do have disease. The control sample may be a blank sample. In certain embodiments of the invention, the presence or levels of aggregatory seeds in a patient's sample may be compared to a reference value. In certain embodiments, the presence or levels of aggregatory seeds in a patient's clinical sample may be compared to a threshold value, wherein the presence or levels of aggregatory seeds over the threshold value indicates the presence of the disease condition, or indicates increased risk for the disease condition. The reference or threshold value may be derived from analysis of a large number of patient samples. This analysis may comprise comparing samples from healthy patients and diseased patients. This analysis may be a longitudinal study comparing samples from patients before and after developing disease.
The methods of the invention may comprise monitoring a patient over time, and may comprise testing numerous samples collected over a number of months or years, in order to asses changes in the presence of aggregatory seeds, and in order to provide an improved diagnosis or assessment of risk. If the presence of aggregatory seeds in a sample from a subject increases over time, the risk of disease may increase, or the prognosis may become less positive.
In certain embodiments, the method of the invention is a method of monitoring disease progression in patients undergoing treatment for the disease. In such embodiments, the presence or levels of aggregatory seeds may be measured prior to administration of treatment and after administration of treatment, in order to assess the effectiveness of the treatment. If the presence or levels of aggregatory seeds decreases, treatment may continue. If the presence or levels of aggregatory seeds increases, treatment may be stopped or altered. The treatment may be directly targeted at preventing or reducing aggregation of the aggregatory protein, or the treatment may be targeted at a different mechanism involved in the disease process, such as an upstream mechanism, such as the production of the aggregatory protein. The methods of the invention will be useful for measuring the efficacy of treatments for diseases caused by the aggregation of a particular protein, including treatments that directly interact with the aggregatory protein and treatments that interact with other disease targets. When assessing treatments that interact with other targets or mechanisms involved in the disease process, the presence or levels of aggregatory seeds, as measured using the methods of the invention, may provide an indication of an individual's response to the treatment.
In the methods of the invention, the presence of aggregatory seeds in a clinical sample is correlated with the risk, presence, or severity of disease. In certain embodiments, the absolute or relative quantity of aggregatory seeds in the clinical sample is correlated with the risk, presence, or severity of disease. In certain embodiments, properties of aggregatory seeds are correlated with the risk, presence, or severity of disease. For example, certain species of seeds may be particularly aggregatory and seed more rapid aggregation of protein and therefore be associated with a very high risk for disease or with very severe disease. Also, certain species of seed may seed more disordered aggregates of protein and therefore be associated with a higher risk for disease or with more severe disease. As explained above, the properties of aggregatory seeds in a sample may be determined using the magnitude of a signal, such as total fluorescence signal, or the profile of a signal, such as a fluorescence lifetime signal or changes in emission and excitation spectra.
The level of the aggregatory seeds should be significantly different from that seen in a negative control. Advanced statistical tools (e.g. principal component analysis, unsupervised hierarchical clustering and linear modelling) can be used to determine whether two levels are the same or different.
If a method of the invention indicates that a subject has a conformational disease, further steps may then follow. For instance, the subject may undergo confirmatory diagnostic procedures, such as those involving physical inspection of the subject, and/or may be treated with therapeutic agent(s) suitable for treating the conformational disease.
The invention also provides a software product comprising (i) code that accesses data attributed to a sample, the data comprising measurements of protein aggregates in a microdroplet or a population of microdroplets, and (ii) code that executes an algorithm for assessing the data to represent a level of aggregatory seeds in the sample. The software product may also comprise (iii) code that executes an algorithm for assessing the result of step (ii) to provide a diagnostic indicator of whether the subject has the relevant conformational disease. Suitable algorithms for use in part (iii) include support vector machine algorithms, artificial neural networks, tree-based methods, genetic programming, etc. The algorithm can preferably classify the data of part (ii) to distinguish between subjects with the relevant conformational disease and subjects without based on measured aggregatory seed levels in samples taken from such subjects. The invention also provides methods for training such algorithms. The presence or levels of aggregatory seeds can be used in combination with known biomarkers, as discussed above.
The invention also provides a computer which is loaded with and/or is running a software product of the invention.
The invention also extends to methods for communicating the results of a method of the invention. This method may involve communicating assay results and/or diagnostic results. Such communication may be to, for example, technicians, physicians or patients. In some embodiments, detection methods of the invention will be performed in one country and the results will be communicated to a recipient in a different country.
The method of the invention is useful providing any subject with a diagnosis. In certain embodiments, the subject is at least 30 years old (e.g. >35, >40, >45, >50, >55, >60, >65, >70). In preferred embodiments, the subject is at least 50 years old.
The subject may be pre-symptomatic or may already be displaying clinical symptoms. For pre-symptomatic subjects the invention is useful for predicting that symptoms may develop in the future if no preventative action is taken. For subjects already displaying clinical symptoms, the invention may be used to confirm or resolve another diagnosis. The subject may already have begun treatment.
Because the invention can be implemented relative easily and cheaply, it is not restricted to being used in patients who are already suspected of having a conformational disease. Rather, it can be used to screen the general population or a high risk population e.g. subjects at least 30 years old, as listed above.
In alternative embodiments of the invention, the sample is an environmental sample, such as a sample collected from a field suspected of being contaminated with prion-containing animal faeces e.g. scrapie of sheep. Therefore, in certain embodiments, the method of the invention is used with a sample derived from or obtained from buildings or land used to house animals. In certain embodiments, the method of the invention is used with a sample suspected of comprising prion proteins, such as a food sample.
Gel Beads
The use of gel-forming agents and the formation of gel beads in either aspect of the invention improve sensitivity of the assay and increase its functionality, because un-aggregated monomers and other factors can be washed away, whilst aggregates are retained, and because the gel beads can be readily transferred between assays that may be incompatible.
The gel bead ‘cage’ is formed by co-compartmentalising a gel-forming agent, such as liquid agarose, at elevated temperatures (such as 37° C.) and then later solidifying the interior of the droplet by dropping the temperature (such as to 25° C.) to initiate the sol-gel transition of the agarose matrix. Once solidified, these agarose microbeads retain large protein aggregates despite thorough washing and are amenable to a variety of standard laboratory manipulations, such as flow cytometry (using a flow cytometer or a fluorescence activated cell sorter, FACS). The confinement of seeds into agarose beads prevents diffusional loss of the fluorescence signal into the background medium and enables robust observation of the positive bead. In addition to the increase in sensitivity afforded by measuring in compartments rather than bulk phase, the washing procedure ensures that background fluorescence is removed in the fluorescent aggregate that can be detected with a better signal/noise ratio.
The gel-forming agent is an agent, for example a polymer such as polysaccharide or polypeptide, which can be solidified from a liquid into a gel, for example by alteration of conditions, such as heating, cooling, or altering pH. Alternatively, the gel-forming agent may be solidified into a gel through a cross-linking reaction. The cross-linking reaction may be initiated by delivering a crosslinker into the droplets or by treating the droplets with UV light. When the microdroplets have been transformed into gel beads (for example, by cooling), they are stable and porous.
Suitable gel forming agents include alginate, gelatine and agarose and other gels having a sol phase sufficiently fluid to move through the channels of a microfluidic device. Preferably agarose, which is a linear polymer made up of the repeating units of a disaccharide (D-galactose and 3,6-anhydro-L-galactopyranose), is employed. In alternative embodiments, the gel forming agent is a polyacrylamide. The gel forming agent may be solidified into a bead by any convenient method, for example by changing the conditions. Preferably the hydrogel forming agent is solidified by altering the temperature, for example by cooling.
For example, when the agent is agarose, it may be solidified by reducing the temperature, for example below 25° C., below 20° C., below 15° C., below 10° C. or below 5° C. The precise gelling temperature is dependent on the type of agarose and its concentration and may be easily determined by the skilled person. For example, 0.5% to 2% w/v of ultra-low melting point agarose Type IX-A(Sigma) has a gelling point of about 17° C. The agarose or other gel-forming agent may be solidified by cooling on ice.
Solidification of the agent within the microdroplet moulds the solidified gel into a bead. The population of solidified beads may be monodisperse. Alternatively, the population may be polydisperse.
Following emulsification and production of a gel bead, the aqueous microdroplets may be de-emulsified. In some embodiments, a deemulsification agent, for example a weak surfactant, may be added to the emulsion to separate the phases and the aqueous phase containing the beads removed. Any suitable mixture of oil and surfactant may be used. The deemulsification agent competes with the surfactant at the oil/water interface and causes it to collapse. Suitable weak surfactants include perfluorooctanol (PFO) and other fluorous compounds with a small hydrophilic group, if fluorinated oil is used, or a buffer containing SDS and Triton and other compounds with a carbon chain on one side and a small hydrophilic group on the other, if mineral oil is used. The deemulsification agent may be added to the emulsion and the mixture agitated, for example with a pipette. In other embodiments, the emulsion may be centrifuged to separate the phases and the aqueous phase containing the beads removed. Suitable techniques for the re-emulsification of gel beads are known in the art [52].
Methods for Assaying the Effect of a Test Substance on Aggregation
The use of microdroplets and gel beads in the methods of the invention allows sensitive analysis of the effect of a test substance on the aggregation of a protein. The method of the invention is particularly amenable to high-throughput screening of candidate therapeutics and identification of substances or compounds that are effective at inhibiting aggregation of a protein, because of the small reagent volumes used and the ease with which gel beads can be handled, for example using microfluidic and flow cytometry technology.
Candidate compounds or test substances may be small organic molecules, proteins, peptides, glycoproteins, nucleic acid molecules, antibodies (or fragments thereof) or any other suitable class of molecule.
The candidate compounds for use in the screening methods of the invention may be obtained using combinatorial library methods known in the art. For example, candidate compounds may be derived from biological libraries, peptoid libraries, spatially addressable parallel solid phase or solution phase libraries, or synthetic libraries. Examples of methods for the synthesis of molecular libraries are known to the skilled person [53-59]. Libraries of compounds can be presented in solution [60], or on beads [61], chips [62], bacteria or spores [63], plasmids [64] or on phage [65-68]. If the test substance is encoded by a nucleic acid, the method preferably comprises a step whereby the nucleic acid is expressed inside a microdroplet.
In certain embodiments of the method of the invention, a population of different test substances is compartmentalised into a plurality of microdroplets. In certain embodiments, following incubation of the microdroplets, transformation of the microdroplets into gel beads and detection of protein aggregates, certain beads are isolated. For example, using standard sorting equipment, beads comprising no or relatively few protein aggregates may be isolated. The test substances present in the isolated beads, which inhibit the formation of aggregates, may then be identified and optionally selected for further analysis or development.
In certain embodiments, the method of the invention comprises contacting a population of test substances (for example, derived from a library as described above) with monomers of the aggregatory protein and a gel-forming agent in the bulk phase. A population of monodisperse microdroplets may then be formed through emulsification, for example using a microfluidics device. The solution may be emulsified such that each microdroplet contains none or one test substance, i.e. the test substances are compartmentalised in the emulsion.
In certain embodiments, the association with a test substance and its gel bead is maintained, to assist identification of effective test substances. The test substance may be retained in the gel bead by any convenient method. In certain embodiments, this is achieved by including particles in the microdroplets that comprise antibodies or tags or other moieties. For example, a library of test substances each comprising a first moiety could be assayed. This library could be compartmentalised into microdroplets with monomers of the aggregatory protein, a gel-forming liquid and particles comprising a second moiety with affinity for the first moiety. Following transformation of the microdroplets into gel beads, each gel bead will retain a protein aggregate formed under the influence of a particular test substance. Each gel bead will also retain the relevant particular test substance, which is associated with a particle via the first and second moieties. The particle used should be larger than the pore size of the gel bead. Such methods are particularly useful when small molecules are assayed.
The particle used to retain the test substance may be a nanoparticle or microparticle. The particle may be a magnetic bead. Suitable nano- or micro-particles are well-known in the art. For example, a particle may be coated with an agent, such as an antibody, which binds to the test substance. In some embodiments, the test substance comprises an epitope tag. A particle may be coated with an antibody or other binding member which binds to the epitope tag. Suitable epitope tags are well-known in the art including, for example, HA (Hemagglutinin), MRGS(H)6, DYKDDDDK (FLAG™), T7-, S-(KETAAAKFERQHMDS), poly-Arg (R5-6), poly-His (H2-10), poly-Cys (C4) poly-Phe(F11) poly-Asp(D5-16), Strept-tag, Strept-tag II (WSHPQFEK), c-myc (EQKLISEEDL), Influenza-HA tag [69], Glu-Glu-Phe tag [70], Tag.100 (Qiagen; 12 aa tag derived from mammalian MAP kinase 2), Cruz tag 09™ (MKAEFRRQESDR, Santa Cruz Biotechnology Inc.) and Cruz tag 22™ (MRDALDRLDRLA, Santa Cruz Biotechnology Inc.). Known tag sequences are reviewed in reference 71. Strep-tag may be used in some preferred embodiments. Antibodies or other binding members may be covalently attached to the particle or may be indirectly attached, for example they may be biotinylated and may be attached to a streptavidin-coated particle through a biotin-streptavidin linkage using standard methods.
Suitable retention agents for retention of test substances include particles, for example non-synthetic particles such as viral particles, or synthetic particles, such as nano- or micro-particles, or polymers including cyclodextrins or dextrans, which are sufficiently large to be entrapped by the gel and unable to diffuse out of the bead.
Alternatively, the test substance may be retained through binding to the matrix of the gel bead itself, for example, through the use of a conjugated moiety with affinity for the matrix. The gel matrix may comprise a moiety (such as streptavidin or biotin, or alternative suitable binding members) that binds the test substance. Larger test substances may be retained in the gel bead as a result of their size.
Test substances, such as small molecule compounds, may be added in comparable ways, for example by using nanoparticles with chemically attached small molecule test substances that are encoded by an optical signal (e.g. quantum dot) or by a characteristic piece of DNA that can be PCR amplified and sequenced for identification [72].
In certain embodiments, an individual test substance is assayed in a population of microdroplets.
In certain embodiments, the invention comprises measuring the effect of a test substance on the disaggregation of a protein aggregate. In this embodiment of the invention the method may comprise a step of incubating a gel bead comprising an aggregate with a test substance and then determining the amount of aggregates of the protein present in the gel bead and comparing the amount to a control bead. Substances identified in this manner may be useful for treating and reversing the progression of conformational diseases.
Once a candidate compound has been identified as a compound that is a modulator of protein aggregation, it may be desirable to perform further experiments to confirm the function of the compound in vitro or in vivo, for example using an animal model.
Kits
The invention also provides kits for carrying out methods of the invention. In certain embodiments, the kit comprises labelled monomers of an aggregatory protein and a gel-forming agent. In certain embodiments, the label is a fluorescent label. In certain embodiments, the gel-forming agent is alginate, gelatine or agarose.
The gel-containing aqueous droplets are then incubated to allow aggregation of the fluorescent reporter peptides onto the sample-derived seeds (G); this reaction occurs within the droplet and, when gel is present, within the substance of the gel matrix. Following the incubation step, the gel beads are released from the oil-droplet emulsion and suspended in aqueous washing buffer that permits the removal of unincorporated fluorescent-labelled peptide solution (H). The brightfield images in (C) and (D) show emulsion droplets (as they emerge from (iv)) and the washed beads, respectively.
Device Fabrication
The microfluidic devices used in this study were constructed using conventional soft lithographic techniques (
Formation of Gel Beads
A microfluidic droplet generator with a flow focusing geometry [73] was used to generate water-in-oil droplets. The contents of the droplets were determined by the content of the supply stream: peptide buffer solution was mixed with agarose gel solution and trapped by HFE7500 oil containing a surfactant, e.g. 0.5% (w/w) fluorous tri-block copolymers [74,75]. Alternatively, a number of alternative oil/surfactant combinations can be used, e.g. mineral oil containing about 3% Abil EM 90, or mineral oil (95% w/w) containing about 4.5% (w/w) Span 80 and about 0.5% (w/w) Tween 80. The emulsion droplets were produced at around a 4.4 kHz frequency. Afterwards, the emulsion droplets were collected and ‘immortalised’ by cooling (e.g. on ice or in a water bath): here an emulsion droplet was created containing liquid agarose and cooled to trigger the transformation of agarose-containing droplets into gel beads. Subsequently, the emulsion was broken by removing the surfactant layer coating of the beads by adding 1H,1H,2H,2H-perfluorooctanol (97%) oil. In the case of mineral oil/non-fluorous surfactant combinations the emulsion can, for example, be broken by freezing the emulsion in a microtube and centrifuging at maximum speed for 5 minutes. The aqueous phase will be below the oil layer.
Eventually, the bare gel beads were washed three times with Napi (Sodium phosphate) buffer (pH 7.4, 50 μM sodium phosphate) and stored in the buffer until an optical assay was carried out. In this final step the non-aggregated peptides were extracted from the beads. In contrast, the large aggregates are trapped inside the beads as they are too bulky to leave the agarose gel matrix. The agarose beads can retain aggregates, but also allow for adding and extraction of components through its gel matrix, which provides a platform for studying aggregates at a variety of settings. A schematic illustrating the formation of the beads is provided in
Therefore, monodisperse droplets are produced in a microfluidic device and loaded with all reagents. After offline incubation for a defined aggregation time interval, the droplets can be retrieved and incubated on ice. The temperature drop prevents further aggregation reactions from taking place and effectively inhibits aggregation. Furthermore, the lowered incubation temperature is below the gelling point of agarose and triggers the transformation of the droplets into porous and stable beads, which is completed within 2 minutes or less. The bead structure persists even upon subsequent temperature elevation to ambient conditions and after removing the droplet oil boundary through addition of 1H,1H,2H,2H-perfluorooctanol (97%) oil or, the case of mineral oil/no-fluorous surfactant combinations by phase separation by centrifugation. The porous structure of the agarose bead matrix serves to retain aggregated amyloid, whilst small molecule components and buffer are free to enter or exit the bead. The beads thus become a ‘time capsule’ for aggregated product, which can be subjected to a number of downstream analysis methods. The ability to wash resulting agarose beads and remove small oligomers and molecules enhances the sensitivity of fluorescence-based assays because the relative signal from trapped amyloids increases while background from unaggregated material is removed. The small droplet volumes (˜50 pL) provide for economical experiments and multiple redundancy: For example, a total reaction volume of 1 μL can generate up to 105 identical droplets and beads, which constitutes an enormous pool for either averaging techniques or individual analysis.
This example demonstrates the successful detection of synthetic aggregatory seeds of AP using microfluidic droplet gel beads. Synthetic seeds were generated from unlabelled Aβ1-42 that was incubated for 10 days (50 Napi buffer, pH 7.4). These seeds were then incubated with fluorescently labelled Aβ1-42 monomers in microfluidic droplets. The total fluorescence of the droplets was measured using FACS analysis and was compared to the fluorescence of droplets incubated without synthetic seeds (i.e. droplets that comprised fluorescently labelled monomer only). As shown in
This example demonstrates the successful detection of aggregatory seeds of Aβ in samples obtained from brain extracts of a Drosophila model of Alzheimer's disease [76,77]. As in Example 2, the total population of beads was considered and the distribution of fluorescence intensities was measured.
This experiment used transgenic flies expressing the Aβ1-42 peptide. Wild type flies were used as a control. Brain extracts were prepared using the protocol outlined below.
1. Male flies were collected in a 1.5 ml snap-top tube at 5, 10 and 25 days old. Flies were snap-frozen for 30 sec in liquid nitrogen and then vortexed for 30 sec.
2. Step 1 was repeated a further 2 times.
3. The contents of the snap-top tube were placed on a weighing boat on a bed of dry ice and the heads were separated from the rest of the bodies.
4. The heads were collected in fresh 1.5 ml snap-top tubes (30-50 heads for control and 30-50 heads for AO flies).
5. Fly heads were homogenized using a disposable plastic pestle in 15 μl of PBS+0.05% (w/v) SDS.
6. The samples were then sonicated in an ice cold water bath at maximum intensity for 8 min.
7. The samples were then centrifuged at 3000 g for 1 min.
8. The protein concentration of the extracts was determined using the BioRad protein assay (or nanodrop) and adjusted to 3 mg/ml.
9. The supernatant was collected and stored at −80° C.
The fly brain samples were incubated with fluorescently labelled Aβ1-42 monomers in microfluidic droplets. The total fluorescence of the droplets was measured using FACS analysis. As shown in Example 2, seeded aggregation of the fluorescently labelled peptide should be accelerated when compared to non-seeded aggregation, so it was expected that beads containing an aggregatory seed (from the non-labelled sample derived from Aβ1-42 flies) should accumulate more aggregate within a particular time and so have a higher fluorescence signal.
We have observed that the signal to-noise ratio for the assay is higher as the extracts are diluted and this is a powerful characteristic of the digital signal that can be generated by microfluidic techniques.
A second way that the production of seeded aggregates can be measured is using fluorescence lifetime imaging. This technique relies on measurement of how the fluorescence signal declines with time after a very brief excitation. Normally a monomeric peptide in solution with the HiLyte fluorescent dye would have a lifetime of approximately 4000 picoseconds. When the peptide aggregates the kinetics of fluorescence decay are accelerated such that the half-life may decrease to about 2500 ps.
Between these extremes we can assess the structural content of the seeded aggregates. Interestingly, we find that spontaneous aggregation (spiked with PBS only) results in a low half-life that is indistinguishable from aggregates resulting from incubation with control mouse brain, while aggregates seeded from diseased mouse brain have a significantly longer half-life. This means that the diseased-seeded aggregates are less structured than the spontaneous aggregates, and this demonstrates a further way in which samples comprising aggregatory seeds can be distinguished from samples that do not comprises aggregatory seeds.
Brain extracts from two strains of mice were made in PBS using the protocol below.
The first strain was a normal laboratory mouse and the second littermate control mouse was derived from the same strain but was transgenic for a disease-linked variant of human amyloid precursor protein (APP). As a consequence the transgenic mice accumulate AP aggregates, show AD-related phenotypes and are used as a model of AD. We compared the biophysical characteristics of the aggregates that were generated when brain extracts were mixed with fluorescence-labelled Aβ1-42 peptides within the agarose-containing microdroplets. Fluorescence lifetime microscopy was used to inspect the structure of the aggregates that were retained within the microbeads following the washing step. As shown in
To follow the spatial distribution of amyloid nucleation and growth within the bead structure we used highly inclined illumination [79], and epi-fluorescence microscopy, as well as direct stochastic Optical Reconstruction Microscopy (dSTORM) [42,43] to look at distribution of the aggregates. The latter is an optical super resolution imaging technique that permits features to be resolved at a resolution down to 20 nm [80].
As the resolution of widefield HiLo is not sufficient to resolve individual aggregates, we performed high resolution dSTORM experiments on Alexa647-labelled Aβ1-40 under similar conditions. It has been reported that conventional fluorophores can be used as efficient photoswitchable fluorescent probes [86]. By irradiation with light of different wavelengths without an activator fluorophore, there can be a reversible cycle for these probes between a fluorescent and a dark state. This is the principle of stochastic optical reconstruction microscopy (STORM) with nanometer accuracy and subdiffraction-resolution images. Significant improvement of the Aβ1-42 structure observation has been proven by dSTORM imaging [87]. For detecting the detailed structure of the aggregates formed inside agarose beads, super-resolution fluorescence imaging was performed by total internal reflection fluorescence (TIRF) configuration equipped with an oil-immersion objective [88]. In order to visualise the clear structure of aggregates inside the agarose bead, a smaller area was selected and magnified (
For the preparation of the dSTORM images the photo switching buffer was prepared and injected into beads reservoir. Oxygen contamination to the switching buffer was prevented by sealing the reservoirs before the imaging process was initiated. 5000 images of a certain area of an agarose bead were recorded. Based on a custom made Matlab code, the images were reconstructed. Clear structure and different length of aggregates can be observed down to a size of ˜100 nm.
This example demonstrates the use of fluorescence lifetime imaging to assess and monitor the aggregation state of amyloid in a bead, which is useful for screening.
Lifetime shifting of Hilyte488-labelled Aβ1-40 was monitored on a TCSPC (Time-Correlated Single Photon Counting) confocal microscope setup. Agarose beads were imaged by a 60×0.25 oil objective (Leica). The laser beam was generated by a super continuum source (20 mHz) and excitation was set to 488 nm in order to excite the tagged fluorophore Hilyte488. The emission was filtered by a 515 nm longpass filter. In order to probe the lowest possible concentration of Aβ1-40 that could still lead to detectable aggregation, three different concentrations of Aβ1-40 were prepared in Napi buffer solution (5 μM, 2.5 μM and 1 μM) and were mixed with 1.5% agarose solution. The 0.75% agarose emulsion samples were incubated at 37° C. for 2, 4, 6, 8, 24 and 48 hours, respectively. Afterwards, the emulsion droplets were immobilised, broken and washed, as described above, and used for TCSPC analysis.
The fluorescence lifetime of bead-caged amyloid samples was analysed by TCSPC using a custom designed microscopy set-up [89]. We have previously shown that the fluorescence lifetime of fluorescently tagged amyloid aggregates is a sensitive readout of aggregation state and decreasing lifetimes are indicative of increasing degrees of aggregation [37]. A major motivation for the current work was to establish lifetime imaging in conjunction with microdroplet technology as a new platform to investigate molecular mechanisms of aggregation and for the screening of potential aggregation inhibiting small molecule agents.
To investigate this more quantitatively, the aggregation time course was measured for different concentrations of peptides in beads and compared to bulk measurements (
This example describes the use of gel beads to measure spontaneous (non-seeded) aggregation. The number of protein aggregates in beads arising from the HiLyte488 labelled Aβ1-40 peptide was analysed by flow cytometry, and quantified via measurement of ensuing fluorescence from the aggregates retained within bead cages. At a droplet generation rate of 4.4 KHz a population of approximately 5.3×106 droplets was obtained over 20 min, from which aliquots of approximately 2×105 droplets were withdrawn. The droplets were contained throughout the aggregation time course (up to 96 hours) and analysed at periodic intervals.
FACS Analysis
Since the processed gel beads were stored in aqueous buffer solution, they could be tested via flow cytometry or FACS, a technique requiring aqueous carrier fluid, in order to assess the aggregation process and compare different intensity of aggregates inside agarose beads. Incubated and washed agarose beads with Hilyte488-labelled Aβ1-40 aggregates were sent to FACS and the beads with fluorescent aggregates were counted. Empty beads with only agarose and no Aβ1-40 were also prepared as a control group for FACS testing. The percentage of beads containing aggregates was calculated using the program Summit. Conditions: 37° C., Napi pH7.4.
This Example Demonstrates the Use of the Assays of the Invention to Successfully Identify a known inhibitor of Aβ aggregation. Aggregatory Aβ1-42 monomers were incubated in gel beads with scyllo-inositol, which is a known inhibitor of Aβ aggregation. As shown in
Therefore, the assays of the invention are capable of identifying a known inhibitor, and will be useful for identifying further inhibitors of aggregation.
The assays and methods of the invention encompass simplified systems wherein droplet generation and fluorescence detection are integrated. For example, the chip device for droplet and bead formation may be built in harder plastic or glass (instead of PDMS). In addition, a fibre optics-based system can be used to detect fluorescent aggregates in beads as they pass through a capillary to be detected with a portable laser-induced fluorescence (PLIF) reader.
The assays and methods of the invention can be used for monitoring disease progression. The Drosophila model of Alzheimer's disease used in the previous examples provides an important link between link between experimentally observed and computationally predicted aggregation behaviour and neurotoxicity [92], based on the ability to set up a disease cycle from presymptomatic through moderate disease and to death within 30 days. Several neurological assays are available (e.g. locomotor performance, reduced longevity and histological indicators [93,94]) and measurements of aggregatory seeds obtained with the droplet bead assay can be correlated with these known indicators.
Transgenic Drosophila expressing various isoforms of the Aβ peptide (1-40, 1-42 and the arctic variant of 1-42) are generated and aged up to 40 days. At the required age aliquots of 50 flies are anaesthetized briefly in CO2 and then frozen in liquid nitrogen in a snap top tube. Following decapitation by vortexing while frozen, the heads are collected and homogenized in 50 μl ice-cold PBS using a disposable pestle. The homogenate is centrifuged at 10,000 g for 5 min and the pellet is discarded. The protein concentration of the supernatant is estimated using a nanodrop (OD280 nm) and PBS added to make standard extracts of 3 mg/ml. The control samples include: similar flies not expressing a transgene or flies expressing another unrelated neurotoxic aggregation-prone polypeptide such as polyQ [95]. In other controls the Aβ-expressing fly extracts are immunodepleted using two monoclonal antibodies (4G8 and 6E10) to control for non-Aβ effects. Non-transgenic fly extracts are also mock-immunodepleted. The measurements using the microfluidic assays of the invention are correlated with disease phenotypes in the fly, including biochemical markers, such as brain Aβ levels and markers of oxidative stress, and phenotype measurements, such as locomotor assays and mortality, in particular the gradient of the mortality trajectory plot (log of the chance of dying vs. time).
Measurements of aggregatory seeds obtained with the droplet bead assay can also be correlated with disease progression in a murine model of neurodegenerative disease. While for flies brain extracts are the only accessible sample, a mouse model gives access to a variety of different samples that can be analysed in droplet bead assays. For example, a CRND8 mouse model of Alzheimer's disease is available. Multiple tissue extracts (brain (see, for example,
The measurements of aggregatory seeds obtained with the droplet bead assay can also be correlated with progression of human disease. In the same way as for the flies and mice, extracts from frozen post-mortem brain tissue (hippocampus) from 10 AD patients (medium-severe) and 10 age- and sex-matched non-demented controls are collected. CSF samples from the same patients are prepared by the same method used for the mouse urine samples. The assays of the invention are used to detect aggregatory seeds in the brains of subjects with Alzheimer's disease, and the amount of aggregatory seeds is correlated with disease progression, as in the animal models above.
This example demonstrates the successful detection of aggregatory seeds of Aβ in samples obtained from brain extracts of CRND8 [78] mice that are a model of Alzheimer's disease and littermate control mice. As in Example 2, the total population of beads was considered and the distribution of fluorescence intensities was measured.
This experiment used transgenic CRND8 mice that generate human Aβ1-42 peptide in their brains. Wild type, littermate control mice were used as controls. Brain extracts were prepared using the protocol outlined below.
1. 200 mg mouse brain cortex was homogenized using a disposable plastic pestle in 50 μl of PBS+0.05% (w/v) SDS.
2. The samples were then sonicated in an ice-cold water bath at maximum intensity for 8 min.
3. The samples were then centrifuged at 3000 g for 1 min.
4. The supernatant was retained and the protein concentration of the extracts was determined using the BioRad protein assay (or nanodrop) and adjusted to 3 mg/ml by addition of PBS.
5. The samples were stored at −80° C.
The mouse brain samples were incubated with fluorescently labelled Aβ1-42 monomers in microfluidic droplets. The total fluorescence of the droplets was measured using FACS analysis. As shown in Example 2, seeded aggregation of the fluorescently labelled peptide should be accelerated when compared to non-seeded aggregation, so it was expected that beads containing an aggregatory seed (from the non-labelled sample derived from CRND8 mice) should accumulate more fluorescent-labelled aggregate within a particular time and so have a higher fluorescence signal.
This example demonstrates the successful detection of aggregatory seeds of Aβ in samples obtained from the serum of CRND8 [78] mice that are a model of Alzheimer's disease and littermate control mice. As in Example 2, the total population of beads was considered and the distribution of fluorescence intensities was measured.
This experiment used transgenic CRND8 mice that generate human Aβ1-42 peptide in their brains. Wild type, littermate control mice were used as controls. Brain extracts were prepared using the protocol outlined below.
1. 300 μl of murine blood was allowed to clot in a 1.5 ml snap-top tube
2. The samples were then centrifuged at 3000 g for 1 min.
3. The serum supernatant was retained and the protein concentration was determined using the BioRad protein assay (or nanodrop) and adjusted to 3 mg/ml by addition of PBS.
4. The samples were stored at −80° C.
The mouse serum samples at 100 μg/ml were incubated with fluorescently labelled Aβ1-42 monomers in microfluidic droplets. The total fluorescence of the droplets was measured using FACS analysis. As shown in Example 2, seeded aggregation of the fluorescently labelled peptide should be accelerated when compared to non-seeded aggregation, so it was expected that beads containing an aggregatory seed (from the non-labelled sample derived from CRND8 mice) should accumulate more fluorescent-labelled aggregate within a particular time and so have a higher fluorescence signal.
The fluorescence intensity of the population of CRND8-derived beads was higher than for beads from control mouse serum. This indicates that aggregatory Aβ seeds can be detected in a peripheral tissue such as blood or serum at concentrations that are comparable to those in clinical specimens.
It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1303507.6 | Feb 2013 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2014/050573 | 2/26/2014 | WO | 00 |
