Patent Application
20230314450
The invention relates to an in vitro method for detecting in a sample a β-sheet aggregate form of a protein forming β-sheet aggregates (PAU) comprising a step of disaggregating a PAFβ in order to obtain a β-sheet non-aggregate form of the protein forming β-sheet aggregates (PNAFβ) and a step of measuring the PNAFβ content.
The accumulation of often misfolded proteins leads to their aggregation in the cell, causing cellular dysregulations, characteristic of certain neurodegenerative pathologies in particular. β amyloid peptides and more particularly β 1-42 amyloid peptide have been widely studied for the formation of fibrils containing β sheets then of plates in Alzheimer's disease. The aggregation process of β amyloid peptides is well characterized and this process depends on a number of factors in particular their concentration, pH and temperature. Many other proteins, such as RNA and DNA binding proteins, that contain a prion-like domain can also form prion-like aggregates [1]. Proteins containing a prion-like domain are proteins forming β-sheet aggregates (PFβ).
The prion-like domains contain hydrophobic amino acids which promote the formation of fibrils rich in β sheets. The β-sheet aggregate forms of a protein forming β-sheet aggregate (PAFβ) are not soluble in water at neutral pH.
The current diagnosis of these neurodegenerative diseases induced by PAFβ is most often based on imaging, which completes the clinical and neuropsychological examination. It is a cumbersome and expensive procedure that requires several diagnosis steps.
Various diagnosis techniques allowing to demonstrate or quantify the aggregation or oligomerization of PFβ in a biological sample have also been described in the literature.
Structural analysis techniques have allowed to highlight the different levels of aggregation of certain PFβ, in particular β amyloid peptides and TAU or TDP-43 proteins: electron microscopy, atomic force microscopy, Cryo EM, Circular Dichroism, Nuclear Magnetic Resonance, X-ray diffraction. However, these techniques make it difficult to measure the content, even relatively, of aggregates present in a sample. The possible effect of compounds on the level of aggregation (in particular the inhibition of aggregation) of a PFβ cannot therefore really be studied by these techniques.
Techniques capable of separating proteins according to their molecular weight allow to discriminate between high molecular weight PAFβ and low molecular weight PNAFβ. Some of them also allow to discriminate between aggregates of different sizes. Mention may be made, for example, of polyacrylamide gel electrophoresis (PAGE and SDS-PAGE) associated or not with detection by Western Blot. The experimental conditions currently described in these techniques can, however, distort the measurement of the level of aggregation. This is particularly the case for SDS-PAGE in which the combined use of a detergent and a reducing agent (DTT, beta-mercaptoethanol or TCEP) can dissociate all or one portion of the aggregates. Due to their experimental constraints (implementation time, large amount of samples, lack of sensitivity), these techniques are not really suitable for the characterization of compounds capable of modulating the level of PFβ aggregation.
Techniques based on immunodetection have also been described:
The methods described in the prior art therefore aim at directly detecting a PAFβ in a sample, in particular by using one or more ligands of PAFβ. Nevertheless, directly detecting PAFβ can make these techniques difficult to implement, imprecise and/or little suitable for large scale use. In addition, the only PAFβ detection is not enough because, at a given signal, it is not possible to realize the level of aggregation compared to the initial state, unless to compare the measurements to a standard range which necessarily biases the measurements of the tested biological sample.
An easier, faster and more reliable diagnosis of diseases related to the aggregation of PFβ is therefore necessary.
The Applicant has developed a protocol which is simple and easy to implement which allows to disaggregate all or one portion of the PAFβ present in a sample in order to obtain a PNAFβ. This protocol allowed the Applicant to develop a PAFβ detection method carried out through the measurement of the PNAFβ content, which allows to overcome all the constraints related to the detection of PAFβ.
According to a first aspect, the invention relates to an in vitro method for detecting in a sample a β-sheet aggregate form of a protein forming β-sheet aggregates (PAFβ), comprising the following steps:
According to a second aspect, the invention relates to an in vitro method for monitoring the therapeutic efficacy of a treatment for a disease associated with PAFβ, comprising the following steps:
According to a third aspect, the invention relates to an in vitro method for measuring the pharmacological efficacy of a molecule on a disease associated with PAFβ, comprising the following steps:
The expression “protein forming β-sheet type aggregates” or “PFβ” designates a protein capable of forming aggregates rich in β sheets, that is to say a protein capable of forming a multimeric form (or an oligomeric form) rich in β sheets. Generally, a non-aggregate form of PFβ is normal, but an aggregate form thereof is characterized, in particular, by a neurodegenerative disease, such as Alzheimer's disease, Creutzfeldt-Jakob disease, Parkinson's disease or amyotrophic lateral sclerosis (ALS). It can be a human or animal, native or recombinant protein. In the context of the present invention, the non-aggregate form of a PFβ is designated by the expression “β-sheet non-aggregate form of a protein forming β-sheet aggregates” or “PNAFβ”. The aggregate form of a PFβ is referred to as “β-sheet aggregate form of a protein forming β-sheet aggregates” or “PAFβ”.
The PFβ, which can be in aggregate form (PAFβ) or in non-aggregate form (PNAFβ), can be selected from FUS (Fused in sarcoma), TAF15, EWSR1, DAZAP1, TIA-1, TTR (transthyretin), cystatin C, β2-microglobulin, R amyloid peptide (such as β 1-40 amyloid peptide or β 1-42 amyloid peptide), TAU (Tubulin-Associated Unit), α-synuclein, β-synuclein, γ-synuclein, Huntingtin (HTT), SOD1 (superoxide dismutase 1), prion, and TDP-43 (TAR DNA-binding protein 43). For example, when PFβ is TDP-43, “TDP-43 NAFβ” will be considered for the non-aggregate form of TDP-43 and of “TDP-43 AFβ” for the aggregate form of TDP-43.
PFβ listed above are in particular implicated in neurodegenerative diseases. For example, FUS implicated in amyotrophic lateral sclerosis, TAF15 implicated in amyotrophic lateral sclerosis, β amyloid peptides implicated in Alzheimer's disease and hereditary cerebral amyloid angiopathy, prion implicated in Creutzfeldt-Jakob disease and spongiform encephalopathy, α-synuclein implicated in Parkinson's disease, TAU protein implicated in Alzheimer's disease, frontotemporal dementias, transthyretin implicated in senile systemic amyloidosis or familial amyloid polyneuropathy, cystatin C implicated in hereditary cerebral amyloid angiopathy, β2-microglobulin implicated in hemodialysis-related amyloidosis, Huntingtin implicated in Huntington's disease, SOD1 implicated in amyotrophic lateral sclerosis, TDP-43 implicated in amyotrophic lateral sclerosis and frontotemporal dementias. Preferably, the PFβ is selected from β 1-42 amyloid peptide, β 1-40 amyloid peptide, α-synuclein or TDP-43.
The sample in which the method of the invention is implemented can be any sample likely to contain at least one PAFβ. It can be a biological sample of human or animal origin, or a sample of cells or tissue cultured in vitro.
The term “in vitro method” means a method implemented outside the human or animal body, for example on microorganisms, organs, tissues, cells, cellular sub-fractions (for example nuclei, mitochondria) or (native or recombinant) proteins. The term “in vitro” encompasses ex vivo.
The sample can for example come from an individual, human or animal, having or being suspected of having a disease associated with a PAFβ, for example a neurodegenerative disease as described above. For example, the sample may be selected from a blood sample, a plasma sample, a serum sample, or a cerebrospinal fluid sample. The sample can also be prepared from tissue or cells from the individual, for example from brain, central nervous system tissue, organs such as spleen and intestine. The sample can therefore be a cell lysate, a cell homogenate, a tissue lysate or a tissue homogenate, such as a brain homogenate. The sample may also comprise cells (for example a cell line), cell sub-fractions (for example nuclei, mitochondria) or (native or recombinant) proteins.
The sample can also come from cells or from a tissue cultured in vitro or ex vivo, preferably from a cell or tissue model of a disease associated with PAFβ. For example, the sample can be selected from cell lysate, cell homogenate, tissue lysate, tissue homogenate, cell culture supernatant, or tissue culture supernatant.
Preferably, the sample is a cell lysate or a cell homogenate.
The term “container” designates a well of a plate, a test tube or any other container suitable for mixing a sample with the reagents necessary for the implementation of the method according to the invention.
Within the meaning of the invention, the term “ligand” denotes a molecule capable of binding to a target molecule. In the context of the invention, the target molecule is PNAFβ. This is then referred to as a “ligand of PNAFβ”. The ligand can be of protein nature (for example a protein or a peptide) or of a nucleotide nature (for example a DNA or an RNA). In the context of the invention, the ligand is advantageously selected from an antibody, an antibody fragment, a peptide or an aptamer, preferably an antibody or an antibody fragment.
Within the meaning of the invention, the term “ligand capable of binding specifically to PNAFβ” or “pair of ligands capable of binding specifically to PNAFβ” designates a ligand or a pair of ligands which binds preferentially to the PNAFβ with respect to the PAFβ, that is to say a ligand or a pair of ligands capable of generating a signal (for example an ELISA signal or a RET signal) with respect to at least one PNAFβ twice higher, for example at least three, at least four, at least five or at least six times higher, than the signal generated with respect to the corresponding PAU. Examples 1-4 and 25 of the present application describe ELISA and FRET methods which allow to easily determine whether a ligand or a pair of ligands is capable of binding specifically to PNAFβ.
Advantageously, the “ligand capable of binding specifically to PNAFβ” or the “pair of ligands capable of binding specifically to PNAFβ” is capable of binding to a PNAFβ with an affinity at least 2 times greater than the affinity for the corresponding PAFβ, for example an affinity at least 2 times higher, at least 3 times higher, at least 4 times higher, at least 5 times higher, at least 6 times higher, at least 7 times higher, at least 8 times higher, at least 9 times higher, at least 10 times higher than the affinity for the corresponding PAFβ. In the case of a pair of ligands capable of binding specifically to PNAFβ, it is not necessary for the two ligands of the pair of ligands to be specific for PNAFβ for the pair of ligands to be specific for PNAFβ. Indeed, it is sufficient for at least one of the two ligands of the pair of ligands to be a ligand specific for PNAFβ for the pair of ligands to be specific for PNAFβ. Nevertheless, both ligands of the pair of ligands may be ligands specific for PNAFβ.
The term “affinity” refers to the strength of all the non-covalent interactions between a ligand and an antigen. Affinity is usually represented by the dissociation constant (Kd). The lower the value Kd, the higher the binding affinity between the ligand and its target. The dissociation constant (Kd) can be measured by well-known methods, for example by FRET, by ELISA or by SPR. The techniques described in the literature therefore make it easy to know whether a ligand or a pair of ligands is specific for PNAFβ.
The term “antibody”, also called “immunoglobulin” refers to a heterotetramer consisting of two heavy chains of approximately 50-70 kDa each (called the H chains for Heavy) and two light chains of approximately 25 kDa each (called the L chains for Light), bound together by intra- and inter-chain disulphide bridges. Each chain is made up, in the N-terminal position, of a variable region or domain, called VL for the light chain, VH for the heavy chain, and in the C-terminal position, of a constant region, made up of a single domain called CL for the light chain and three or four domains called CH1, CH2, CH3, CH4, for the heavy chain. Each variable domain generally comprises 4 “hinge regions” (called FR1, FR2, FR3, FR4) and 3 regions directly responsible for binding with the antigen, called “CDR” (called CDR1, CDR2, CDR3). An “antibody” can be of mammalian (for example human or mouse or rat or camelid), humanized, chimeric, recombinant origin. It is preferably a monoclonal antibody produced recombinantly by genetically modified cells according to techniques widely known to the person skilled in the art. The antibody can be of any isotype, for example IgG, IgM, IgA, IgD or IgE, preferably IgG.
The term “antibody fragment” means any portion of an immunoglobulin obtained by enzymatic digestion or obtained by bio-production comprising at least one disulphide bridge and which is capable of binding to the antigen recognized by the whole antibody, for example Fv, Fab, Fab′, Fab′-SH, F(ab′)2, diabodies, linear antibodies (also called “Single Domain Antibodies” or sdAb, or nanobodies), antibodies with a single chain (for example scFvs). Enzymatic digestion of immunoglobulins with pepsin generates an F(ab′)2 fragment and an Fc fragment split into several peptides. F(ab′)2 is made up of two Fab′ fragments bound by interchain disulphide bridges. The Fab portions are made up of the variable regions and the CH1 and CL domains. The Fab′ fragment is made up of the Fab region and a hinge region. Fab′-SH refers to a Fab′ fragment in which the cysteine residue of the hinge region bears a free thiol group.
The term “tracer” means a chemical or biological agent capable of directly or indirectly emitting a signal which can be detected with an appropriate detection device. It can be a fluorescent, luminescent, radioactive or enzymatic tracer. In a particular embodiment, the tracer is a member of a pair of RET partners.
The term “RET” (from “Resonance Energy Transfer”) designates energy transfer techniques. The RET can be a FRET or a BRET.
The term “FRET” (from “Fluorescence Resonance Energy Transfer”) designates the transfer of energy between two fluorescent molecules. FRET is defined as a non-radiative energy transfer resulting from a dipole-dipole interaction between an energy donor and an energy acceptor. This physical phenomenon requires energy compatibility between these molecules. This means that the emission spectrum of the donor must overlap, at least partially, the absorption spectrum of the acceptor. In accordance with Forster's theory, FRET is a process that depends on the distance separating the two molecules, donor and acceptor: when these molecules are close to each other, a FRET signal will be emitted.
The term “BRET” (from “Bioluminescence Resonance Energy Transfer”) designates the transfer of energy between a bioluminescent molecule and a fluorescent molecule.
The term “pair of RET partners” designates a pair consisting of an energy donor compound (hereinafter “donor compound”) and an energy acceptor compound (hereinafter “acceptor compound”); when in close proximity to each other and when excited at the excitation wavelength of the donor compound, these compounds emit a RET signal. It is known that for two compounds to be RET partners, the emission spectrum of the donor compound must partially overlap the excitation spectrum of the acceptor compound. For example, this is the case of “pairs of FRET partners” when using a fluorescent donor compound and an acceptor compound or of “pair of BRET partners” when using a donor bioluminescent compound and an acceptor compound.
The term “RET signal” designates any measurable signal representative of a RET between a donor compound and an acceptor compound. For example, a FRET signal can therefore be a variation in the intensity or the luminescence lifetime of the donor fluorescent compound or of the acceptor compound when the latter is fluorescent.
«2 Containers» Detection Method
According to a first aspect, the invention relates to an in vitro method for detecting in a sample a β-sheet aggregate form of a protein forming β-sheet aggregates (PAFβ), comprising the following steps:
The general principle of the method according to the invention is illustrated in
Below, the method is described in detail step by step.
Step a)
This phenomenon of disaggregation at pH ranging from 9.7 to 13.2 is quite surprising since the disaggregation methods described in the prior art rather consist in treating the PNAFβ at acid pH, with powerful detergents and/or or by sonication. For example, the work described in [7] shows different methods for obtaining relatively monomeric amyloid proteins from amyloid fibers. In a first approach, a preparation of amyloid fibers is treated by combining an acid, 88% formic acid, with a strong surfactant type detergent, Sodium Dodecyl Sulfate (SDS). An alternative is to combine a chaotropic agent, a saturated solution of Guanidine Thiocyanate (6.8M) with SDS. In both cases, the authors were able to note the disappearance of amyloid fibers in favor of relatively monomeric amyloid proteins (mixture of monomers and dimers). Another study [8] describes different methods of treating biological samples to disaggregate the amyloid proteins (β 1-42 amyloid peptide) they contain before assaying them via an ELISA test. Extracts of mouse brains or cerebrospinal fluids of patients are treated with a solution of fluorinated alcohol (HFIP) or acid (TFA) coupled with sonication for 15 minutes to disaggregate amyloid proteins. The HFIP or TFA are then removed by drying under a constant flow of nitrogen. The dry samples, containing the disaggregated amyloid proteins, are then taken up in a 1% NH4OH solution before being analyzed. The authors suggest that these 2 types of treatment allow to disaggregate the 13142 amyloid peptide aggregates and thereby improve their quantification.
Nevertheless, the applicant has shown that the disaggregation methods described in the prior art do not allow to measure the PNAFβ content with immunological methods, which need to be implemented at a physiological pH (cf. Examples 5 to 17).
The method of the invention does not require treatment at an acid pH to disaggregate the PAFβ. On the contrary, the Applicant has not only shown that disaggregation was possible by treating a PAFβ at a pH ranging from 9.7 to 13.2, but also that this treatment was entirely compatible with the subsequent implementation of an immunological method aiming at measuring the PNAFβ content.
Advantageously, step a2) consists in adjusting the pH to a pH ranging from 10 to 13.2, for example a pH ranging from 11 to 13.2, a pH ranging from 11.5 to 13.2, a pH ranging from 12 to 13.2, a pH ranging from 12.5 to 13.2, a pH ranging from 12.8 to 13.2, a pH ranging from 10 to 13, a pH ranging from 11 to 13, a pH ranging from 12 to 13, for example about 12.8.
The pH is adjusted with a base, for example a strong base such as KOH or NaOH, preferably NaOH.
The duration of step a2) may depend on various parameters such as the base used or the PFβ tested. In particular, step a2) may last at least 30 seconds, for example at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, for example between 30 seconds and 60 min or between 5 minutes and 30 minutes. The person skilled in the art will have no difficulty in adapting the duration of step a2) to manage to disaggregate all or one portion of the PAFβ in order to obtain a PNAFβ.
Step a3) consists in adjusting the pH to a pH ranging from 6 to 9. This step is important since it allows to implement the immunological method of step b).
Advantageously, step a3) consists in adjusting the pH to a pH ranging from 6.4 to 9, for example a pH ranging from 6.4 to 8.4, a pH ranging from 6 to 8.5, a pH ranging from 7 to 8.5.
The pH is adjusted with an acid, for example a strong acid, such as HCl.
Step b)
Step b) consists in measuring the PNAFβ content in the first container with an appropriate immunological method (or appropriate “immunoassay”).
It is necessary that the immunological method of step b) allows to obtain contents which are comparable with each other. However, it is not necessary for the immunological method in step b) to be quantitative, although it can be. The immunological method of step b) must at least allow the measurement of a relative content that can be compared to another relative content measured by the same immunological method.
In a particular embodiment, the immunological method implemented in step b) uses:
The ligand (i) can be selected from an antibody, an antibody fragment, a peptide or an aptamer, preferably an antibody. The pair of ligands (ii) can be selected from a pair of antibodies, a pair of antibody fragments, a pair of peptides or a pair of aptamers, preferably a pair of antibodies.
The person skilled in the art will have no difficulty in obtaining and/or selecting antibodies or antibody fragments having the desired properties, for example by immunizing a mouse with a PFβ, by carrying out a lymphocyte hybridization from the lymphocytes of the spleen of the immunized mouse in order to generate hybridomas and by testing the antibodies of each hybridoma for their capacities to bind specifically to PNAFβ. Examples of a complete protocol for the generation of anti-PNAFβ antibodies then for the selection of specific antibodies are detailed in examples 1-4 and 25. It is therefore very easy to obtain antibodies or pairs of antibodies directed against any PNAFβ for the implementation of the method according to the invention, for example by implementing the methods described in Examples 1-4 and 25.
Obtaining PNAFβ and PAFβ is within the reach of the person skilled in the art. For example, for proteins called “prion-like” proteins, such as TDP-43, FUS, TAF15 and EWSR1, the production of these proteins fused to Gluthatione-S-Transferase (GST) (N- or C-terminal fusion depending on proteins) allows to obtain GST fusion proteins whose turbidimetric analysis shows that they are in non-aggregate form [9][10][13]. When a Tobacco etch Virus protease (TEV) cleavage site is inserted between the PFβ of interest and the GST, a treatment of the GST proteins with TEV followed by a purification step allows to obtain the GST proteins without label. When said proteins are left for 1 h 30 at room temperature with stirring, turbidimetry analysis shows that they are in aggregate form [10][13]. PFβ fused to GST are also commercially available, for example from Abnova: GST-TDP-43 (Ref. H00023435-P02), GST-FUS1 (Ref. H00002521-P01), GST-TAF15 (Ref. H00008148-P02) GST-EWSR1 (Ref. H00002130-Q01).
Amyloid peptides, in particular the β 1-40 and β 1-42 forms, are also available from many suppliers in powder form. The solubilization of these powders in HFIP or NH4OH allows to obtain stock solutions containing more than 90% of non-aggregate forms. The extemporaneous use of these solutions previously diluted in buffers with physiological pH values allows to have samples of non-aggregate forms. Conversely, an incubation of several hours to several days, for example an incubation of more than 24 hours, of these same stock solutions in a physiological buffer at room temperature allows to obtain a sample containing very high proportions of aggregate forms [11].
Samples containing PNAFβ and PAFβ can also be obtained from the company StressMarq (www.stressmarq.com). This is particularly the case for alpha, beta and gamma synucleins, the TAU protein, the Cu/Zn superoxide dismutase 1 (SOD1) protein or Transthyretin (TTR).
Advantageously, the immunological method implemented in steps b) is an ELISA method or a RET method, such as a FRET or a BRET.
Depending on the method used, the measurement of step b) can be done directly in the first container by adding the appropriate reagents or in another container with all or one portion of the contents of the first container. For example, reagents for a RET method can be added directly to the first container. Conversely, the ELISA method will preferably be carried out in another container adapted to the implementation of the ELISA method, in particular in a container at the bottom of which a PNAFβ ligand has previously been immobilized.
Obviously, for the implementation of the RET method, the first ligand and the second ligand must not compete for binding to PNAFβ, for example the first ligand and the second ligand must not bind the same epitope on PNAFβ. It is easy to select an appropriate pair of ligands by carrying out the method described in examples 1-4 and 25.
The ligands can be labeled directly or indirectly. The direct labeling of the ligand by a member of a pair of RET partners can be carried out by conventional methods known to the person skilled in the art, based on the presence of reactive groups on the ligand. For example, when the ligand is an antibody or an antibody fragment, the following reactive groups can be used: the terminal amino group, the carboxylate groups of aspartic and glutamic acids, the amine groups of lysines, the guanidine groups of arginines, the thiol groups of cysteines, the phenol groups of tyrosines, the indole rings of tryptophans, the thioether groups of methionines, the imidazole groups of histidines.
The reactive groups can form a covalent bond with a reactive group carried by a member of a pair of RET partners. The appropriate reactive groups, carried by the member of a pair of RET partners, are well known to the person skilled in the art, for example a donor compound or an acceptor compound functionalized by a maleimide group will for example be capable of binding covalently with the thiol groups carried by the cysteines carried by a protein or a peptide, for example an antibody or an antibody fragment. Similarly, a donor/acceptor compound carrying an N-hydroxysuccinimide ester will be able to bind covalently to an amine present on a protein or a peptide.
The ligand can also be labeled with a fluorescent or bioluminescent compound indirectly, for example by introducing an antibody or antibody fragment into the measurement medium, itself covalently bound to an acceptor/donor compound, this second antibody or antibody fragment specifically recognizing the ligand.
Another very conventional means of indirect labeling consists in attaching biotin to the ligand to be labeled, then incubating this biotinylated ligand in the presence of streptavidin labeled with an acceptor/donor compound. Suitable biotinylated ligands can be prepared by techniques well known to the person skilled in the art; Cisbio Bioassays, for example, markets streptavidin labeled with a fluorophore, the trade name of which is “d2” (ref. 610SADLA).
Advantageously, one of the members of the pair of RET partners is a fluorescent donor or luminescent donor compound and the other member of the pair of RET partners is a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher).
When the RET is a FRET, the donor fluorescent compound can be a FRET partner selected from: a europium cryptate, a europium chelate, a terbium chelate, a terbium cryptate, a ruthenium chelate, a quantum dot, allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives and nitrobenzoxadiazole. When the RET is a FRET, the acceptor fluorescent compound can be a FRET partner selected from: allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives, nitrobenzoxadiazole, a quantum dot, GFP, GFP variants selected from GFP10, GFP2 and eGFP, YFP, YFP variants selected from eYFP, YFP topaz, YFP citrine, YFP venus and YPet, mOrange, DsRed.
When the RET is a BRET, the donor luminescent compound can be a partner of BRET selected from: Luciferase (luc), Renilla Luciferase (Rluc), variants of Renilla Luciferase (Rluc8) and Firefly Luciferase. When the RET is a BRET, the acceptor fluorescent compound is a BRET partner selected from: allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives, nitrobenzoxadiazole, a quantum dot, GFP, GFP variants selected from GFP10, GFP2 and eGFP, YFP, YFP variants selected from eYFP, YFP topaz, YFP citrine, YFP venus and YPet, mOrange, DsRed.
In a particular embodiment, step b) is carried out by an ELISA method and consists in:
The ELISA method is widely described in the prior art and does not have any difficulty of implementation for the person skilled in the art.
Step c)
Step c) consists in, in a first container, c1) introducing the same sample as in step a1).
Contrary to step a), the pH is not adjusted to a pH ranging from 9.7 to 13.2 during step c). The pH is directly adjusted to a pH ranging from 6 to 9 (step c2)), preferably at the same pH as the pH of step a3).
The pH in step c2) can be adjusted with an acid/base mixture, for example a strong acid/strong base mixture. It may be, for example, an NaOH/HCl mixture. Preferably, the acid used in step c2) is the same as that used in step a3) and the base used in step c2) is the same as the base used in step a2).
Step d)
Step d) consists in measuring the PNAFβ content in the second container with the same method as in step b). Step d) is implemented in the same way as in step b) to be able to compare the measurements and implement step e).
Thus, when step b) is carried out by a RET method, step d) consists in:
In the same way, when step b) is carried out by an ELISA method, step d) consists in:
Step e)
Step e) consists in comparing the contents measured in step b) and in step d), a decrease in the content measured in step d) compared to the content measured in step b) indicating that the sample contains a PAFβ.
Obviously, if the first and the second container are swapped, step e) will consist in comparing the contents measured in step b) and in step d), an increase in the content measured in step d) relative to the content measured in step b) indicating that the sample contains a PAFβ.
The person skilled in the art can easily compare the contents measured in steps b) and d) and define a threshold enabling him to qualify the increase or decrease. For example, a difference between the measured contents greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%. The determination of the threshold may depend on the variability inherent in the immunological method chosen.
The person skilled in the art could, for example, calculate the ratio between the contents measured in steps b) and d). In general, the greater the difference between the measured contents, the greater the ratio between the measured contents and the greater the amount of PAFβ in the sample.
When the immunological method is an ELISA method or a RET method, the RET signals or the ELISA signals measured in steps b) and d) will be compared. Therefore, a decrease in RET signal or a decrease in ELISA signal will indicate that the sample contains PAFβ. Advantageously, a ratio will be calculated between the signal measured in the first container (step b)) and the signal measured in the second container (step d)). The calculation of the ratio can be done manually or automatically.
Preferably, the duration between step a3) and step b) and/or between step c2) and step d) will be adapted to prevent the PNAFβ from re-aggregating into PAFβ. The duration will preferably be less than 24 hours, for example less than 5 hours. Advantageously, steps b) and d) are carried out following steps a3) and c2) respectively, that is to say less than 30 minutes after steps a3) and c2). The person skilled in the art will have no difficulty in adapting the duration of step a3) and/or of step c2) to prevent all or one portion of the PNAFβ from re-aggregating into PAFβ before step c) and/or step d).
The comparison of the contents in the two samples in step e) allows to measure the level of aggregation very precisely, in particular compared to most methods of the prior art.
Method for Monitoring Therapeutic Efficacy
The present invention also relates to an in vitro method for monitoring the therapeutic efficacy of a treatment for a disease associated with PAFβ in a patient, comprising the following steps:
The treatment may be a known treatment for the disease associated with PAFβ or an experimental treatment. The sample is preferably from an individual with PAFβ-associated disease.
To be able to monitor the effectiveness of a treatment, the first sample and the second sample are taken from the patient at different times, for example the first sample is taken at a time T1 and the second sample is taken at a time T2. Advantageously, the first sample is taken before the second sample, for example the first sample is taken at a time T1 before the treatment of the patient or during said treatment, and the second sample is taken at a time T2 after said treatment or during said treatment. The time that elapses between the collection of the first sample and the collection of the second sample will be chosen in order to be able to detect the therapeutic effectiveness of the treatment.
Method for Monitoring the Measurement of the Pharmacological Efficacy
The present invention also relates to an in vitro method for measuring the pharmacological efficacy of a drug molecule or a drug candidate on a disease associated with PAFβ in a test sample, comprising the following steps:
The molecule can be a drug or a drug candidate. The sample preferably comes from cells or tissue cultured in vitro. Consequently, the drug molecule or the drug candidate is preferably tested on an in vitro model of disease associated with a PAFβ. Such models are widely described in the literature.
The test sample corresponds to a sample which allows to test a drug or a drug candidate in vitro, it may be for example a sample of cells cultured in vitro or a sample of tissue cultured in vitro. The test sample corresponds to what is commonly called an “in vitro model of disease associated with PAFβ”. Thus, in order to be able to measure the pharmacological efficacy of a drug molecule or a drug candidate, the first sample and the second sample are taken from the test sample at different times, for example the first sample is taken at a time T1 and the second sample is taken at a time T2. Advantageously, the first sample is taken before the second sample, for example the first sample is taken at a time T1 before the treatment of the test sample with a drug molecule or a drug candidate, and the second sample is taken at a time T1 after said treatment. The time that elapses between taking the first sample and taking the second sample will be chosen in order to be able to detect pharmacological efficacy of the drug molecule or the drug candidate.
The present invention will now be illustrated by the following non-limiting examples.
Generating Anti-PNAFβ Antibodies
Mice Immunizations
BALB/c mice are immunized by injection of the PNAFβ protein previously diluted in phosphate buffer prepared under physiological conditions. The absence of the presence of PNAFβ multimers or aggregates is virified in the buffer intended for the injections in order to direct the immune response of the mice to the non-aggregate forms. The first injection is followed by three boosters at monthly intervals.
Fifteen days after each injection, blood punctures are carried out on the mice to verify the presence of an immune response by titration of the antibodies.
Titration of Immune Sera in Anti-PNAFβ Antibodies by ELISA Tests
For this purpose, ELISA-type immunodetection tests are implemented depending on the nature of the PFβ. For amyloid-type PFβs or peptides, PNAFβ is previously labeled on its primary lysines with biotin using a reagent composed of biotin, a carbon linker and an NHS (N-Hydroxysuccinimide) reactive group. For non-amyloid or amyloid PFβ, PNAFβ in GST fusion is directly immobilized on 96-well plates via the GST tag by using ELISA microplates functionalized with a glutahion group. Biotin-labeled proteins are immobilized on 96-well ELISA plates via biotin using microplates functionalized with streptavidin. For this purpose, 100 μl of GST fusion and/or biotinylated PNAFβ solution are added to each well and then incubated for 2 h at room temperature. The wells are then washed three times in PBS buffer supplemented with 0.05% Tween-20. After removing the washing solution, each well is then incubated overnight at 4° C. with 200 μL of a blocking solution composed of PBS, 5% BSA.
The serial dilutions by a factor of 10 to 100 million of the blood samples (immune sera) are then added, in doublets, at a level of 100 μL per well in PBS+0.1% BSA buffer and incubated with stirring for 2 hours. The non-specific antibodies not bound to the immobilized PNAFβ are eliminated by three washing steps of 200 μl in PBS 1× buffer, 0.05% Tween20. The possible presence of specific antibodies is detected using 100 μL per well of mouse anti-Fc secondary antibody coupled to HRP (horseradish peroxidase) (Sigma #A0168 diluted to 1/10000 in PBS, BSA 0.1%). After 1 hour of incubation at room temperature with stirring then three washes under 200 μL in PBS 1× buffer, 0.05% Tween20, the revelation of the HRP is carried out by colorimetric assay at 450 nm following incubation of its TMB substrate (3,3′,5,5′-Tetramethylbenzidine, Sigma #T0440) for 20 min at room temperature and with stirring. This blocking solution is then removed by aspiration and the plates are stored at 4° C. for future use.
In order to ensure that the antibodies detected by the ELISA test are indeed directed against the PNAFβ protein and not against the GST tag or biotin, the same punctures are tested on the ELISA test after pre-incubation with an excess of another orthogonal protein tagged with GST or biotin. Thus, the anti-TAG antibodies bind to the tagged orthogonal protein and therefore not to the PNAFβ protein immobilized at the bottom of the wells; in which case no HRP signal or a decrease in HRP signal is detected.
Fusion & Cloning
The mice having the best anti-PNAFβ antibody titers (signal, that is to say high optical density at 450 nm) and the least drop in signal in the anti-TAG control case are selected for the next step of lymphocyte hybridization, also called fusion. The mouse spleen is extracted to isolate a mixture of lymphocytes and plasma cells. This multi-cell sample is fused in vitro with a myeloma cell line in the presence of a cell fusion catalyst of the polyethylene glycol (PEG) type. A mutant myeloma cell line, deficient for the enzyme HGPRT (Hypoxanthine Guanosin Phosphoribosyl Transferase) is used to allow selection of hybrid cells, called hybridomas. These cells are cultured in a medium containing hypoxanthine, aminopterin (methotrexate) and thyamine (HAT medium), to allow the elimination of unfused myeloma cells and thus select the hybridomas of interest. Unfused spleen cells, on the other hand, die since they are unable to proliferate in vitro. Thus, only hybridomas survive this selection pressure in vitro.
These hybridomas are cultured in culture plates. The supernatants of these hybridomas are tested to assess their ability to produce anti-PNAFβ antibodies. For this purpose, an ELISA test as described above is carried out. The minimum threshold used to select a clone is four times that of the non-specific. The best hybridomas are cloned with a limiting dilution step in order to obtain stable hybridoma clones.
The clones selected are cultured with a view to forming a bank of hybridomas, tested for cell viability and stored in liquid nitrogen. At this step, the antibodies produced by the clones can be easily sequenced by methods well described in the prior art in order to be able to produce the antibodies, for example, in producer cells. Alternatively, antibodies are produced as described below.
Production of Anti-PNAFβ Antibodies
The clones of hybridomas of interest are returned to culture and the cellular inoculum is then injected into BALB/c mice (intraperitoneal injection, IP) in order to allow the production of antibodies in large amounts in the liquid of ascites.
After characterization of the content of ascites fluids by various techniques aiming at quantifying and qualifying the antibody content, said antibodies are then purified after optional precipitation with salts, via affinity chromatography on columns including resins grafted with protein A. After washing the column to remove the constituents apart from the antibodies, the antibody content is eluted by shocking at acid pH in glycine buffer. After pH neutralization and dialysis against a buffer at neutral pH, the anti-PNAFβ antibodies are ready for storage at 4° C. or freezing and subsequent use/characterization (isotyping, assay, functional tests).
Selection of a Pair of Antibodies Capable of Binding Specifically to a PNAFβ (ELISA Method)
In order to select a pair of antibodies preferentially recognizing a PNAFβ vis-à-vis a PAFβ, an ELISA type test is implemented. For this purpose, one of the antibodies of the tested pair is biotinylated using the Lightning-Link Rapid Biotin Type A kit (Expedeon, reference SKU 370-0005) according to the supplier's recommendations. The second antibody of the tested pair, diluted beforehand in PBS buffer at concentrations comprised between 1 and 20 μg/mL, is adsorbed onto 96-well plates of the “high binding” ELISA type. For this purpose, 100 μL of antibodies are added to each well then incubated for 20 hours at 4° C. followed by three washes in PBS 1× buffer, 0.05% Tween20. After removing the washing solution, each well is then incubated overnight at 4° C. with 200 μL of a blocking solution composed of PBS, 5% BSA. This blocking solution is then removed by aspiration and the plates are stored at 4° C. for future use.
Serial dilutions by a factor of 1 to 1/100th of samples containing the same initial concentration of PNAFβ or of PAFβ are added at a level of 100 μL/well and incubated for 2 hours at room temperature with stirring. The PNAFβ or PAFβ not bound to the antibody adsorbed on the plates are eliminated by three washing steps in PBS 1× buffer, 0.05% Tween20. The biotinylated antibody, previously diluted in PBS buffer to a concentration comprised between 10 and 200 ng/mL, is added at a level of 100 μL/well and incubated for 2 hours at room temperature with stirring. The biotinylated antibodies not bound to PNAFβ or PAFβ are eliminated by three washing steps in PBS 1× buffer, 0.05% Tween20. The detection of bound antibodies is carried out using a streptavidin-HRP (R&D Systems, Ref. DY998) diluted to 1/10th in PBS, 0.1% BSA. After 30 minutes of incubation at room temperature with stirring, then three washes in PBS 1× buffer, 0.05% Tween20, the revelation of the HRP is carried out by measuring the optical density at 450 nm (O.D. 450 nm) following the incubation of its TMB substrate (3,3′,5,5′-Tetramethylbenzidine, Sigma #T0440) for 20 min at room temperature with stirring.
In a first step, the reference dilution of the samples of PNAFβ or PAFβ is determined by analyzing the O.D. 450 nm measured with the different dilutions of PNAFβ. As indicated in
In a second step, the O.D. 450 nm obtained with the reference dilution of the sample of PNAFβ is compared with the O.D. 450 nm measured with an identical dilution of the sample of PAFβ. As shown in
Selection of a Pair of Antibodies Capable of Binding Specifically to a PNAFβ (FRET Method)
In order to select a pair of antibodies preferentially recognizing a PNAFβ over a PAFβ, a FRET test is set up. This test is based on Cisbio Bioassays HTRF® Technology. The principle of this technique is based on a fluorescence energy transfer between a donor molecule, a Terbium cryptate (Donor), and a fluorescent energy acceptor molecule d2 (Acceptor). These two fluorescent molecules are grafted by covalent coupling to antibodies to implement an immunological assay as illustrated in
In a first step, the reference dilution of the samples of PNAFβ or PAFβ is determined by analyzing the HTRF signal measured with the different dilutions of PNAFβ. As indicated in
In a second step, the HTRF signal obtained with the reference dilution of the sample of PNAFβ is compared with the HTRF signal measured with an identical dilution of the sample of PAFβ. As illustrated in
An alternative method for obtaining samples containing TDP-43 AFβ or TDP-43 NAFβ consists in culturing HeLa cells and treating them or not for at least 6 hours with staurosporine. Analysis of cell lysates by SDS-PAGE and Western-Blot shows that untreated HeLa cell lysates contain TDP-43 NAFβ while HeLa lysates treated with staurosporine essentially contain TDP-43 AFβ [12].
The method is based on the FRET technology (HTRF® technology from Cisbio Bioassays) described in example 1.
Preparation of the Antibodies to be Tested
Five antibodies directed against TDP-43 were labeled respectively with the Donor and with the Acceptor. These labelings were carried out using the labeling kits marketed by Cisbio Bioassays (Commercial references 62EUSUEA and 62D2DPEA). The labeling rates obtained (number of fluorescent molecules per antibody) were in line with expectations. Donor marking rates were comprised between 5.9 and 7.9. Acceptor labeling rates were comprised between 2.3 and 3.3.
The table below gives the characteristics of the antibodies which were tested.
To be tested, all the antibodies were diluted in a buffer to obtain respective concentrations of 3 nM for the antibodies labeled with the Donor and 30 nM for those labeled with the Acceptor.
Preparation of a Sample of TDP-43 NAFβ (Monomers)
HeLa cells were seeded with 5 million cells in a flask (175 cm2) in complete culture medium (MEM alpha medium+2 mM Hepes+10% decomplemented fetal calf serum+1% antibiotics, Penicillin 5000 U/ml and Streptomycin 5000 μg/ml). After 78 hours, the culture medium was aspirated. The cells were then lysed with a cell lysis buffer. The lysate obtained (hereinafter “Monomer sample”), containing the TDP-43 NAFβ, was frozen at −80° C. with a view to its use. This sample was checked by western blot as recommended in the literature [12] in order to ensure that it essentially contains non-aggregated TDP43 (TDP-43 NAFβ).
Preparation of a Sample of TDP-43 AFβ (Aggregates)
HeLa cells were seeded with 5 million cells in a flask (175 cm2) in complete culture medium (MEM alpha medium+2 mM Hepes+10% decomplemented fetal calf serum+1% antibiotics, Penicillin 5000 U/ml and Streptomycin 5000 μg/ml). After 72 hours, the culture medium was aspirated. A staurosporine solution at 1 μM in complete medium was added to the cells in culture for 6 h. Treatment of cells with staurosporine allows TDP-43 to be aggregated to obtain TDP-43 AFβ. The culture medium was then aspirated before adding cell lysis buffer. The lysate obtained (hereinafter “Aggregate sample”), containing the TDP-43 AM, was frozen at −80° C. with a view to its subsequent use. This sample was checked by western blot as recommended in the literature [12] to ensure that it essentially contains aggregated TDP43 (TDP-43 AFβ).
Screening of Pairs of Antibodies on Monomers or Aggregates
On the day of the test, the Monomer and Aggregate samples were thawed before distribution in 384-well microplates (Greiner reference 784075).
The following reagents were added to the microplates in the order below:
The plates were then incubated overnight at room temperature.
The detection of the FRET signal in the various plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module. For all the pairs of tested antibodies, the signals obtained in FRET on the Monomer sample and on the Aggregate sample were compared by calculating a ratio of the FRET signals (Monomers/Aggregates), as shown in
The ratio of FRET signals (Monomers/Aggregates) was calculated for all the pairs of tested antibodies. Table 2 below summarizes the results obtained. All pairs of antibodies with a ratio (Monomer/Aggregates) greater than 2 are considered selective for TDP-43 NAFβ with respect to TDP-43 AFβ.
7 pairs of antibodies allowed to discriminate between TDP-43 NAFβ and TDP-43 AFβ because they have a Monomer/Aggregate ratio greater than 2. They are listed in Table 3.
The method is based on FRET technology (HTRF® technology from Cisbio Bioassays), as detailed in Example 1.
Pair of Tested Antibodies
The antibodies directed against the beta 1-40 amyloid peptide labeled respectively with the Donor and with the Acceptor are those contained in the Amyloid Beta 1-40 HTRF kit marketed by Cisbio Bioassays (reference 62B40PEG). They were diluted in the reference diluent 62RB3FDG as recommended by the Amyloid Beta 1-40 HTRF kit manual.
Preparation of Beta 1-40 Amyloid Peptide NAFβ (Monomers)
The freeze-dried human beta 1-40 amyloid peptide (ERI275BAS, The ERI Amyloid Laboratory, LLC, Oxford) was resuspended according to the supplier's recommendations then diluted in 10 mM Sodium Phosphate pH 7.4 buffer to a concentration of 30 μM. The solution (hereinafter “Monomer sample”), containing the beta 1-40 amyloid peptide NAFβ, was then frozen at −80° C. A Thioflavin T test was performed on this solution. It indicates that it contains more than 90% beta 1-40 amyloid peptide monomers.
Preparation of Beta 1-40 Amyloid Peptide AFβ (Aggregates)
The freeze-dried human beta 1-40 amyloid peptide (ERI275BAS, The ERI Amyloid Laboratory, LLC, Oxford) was resuspended according to the supplier's recommendations then diluted in 10 mM Sodium Phosphate pH 7.4 buffer to a concentration of 30 μM. This solution is then incubated for 495 hours at 25° C., which allows to aggregate the beta 1-40 amyloid peptide to obtain the beta 1-40 amyloid peptide AFβ. The solution (hereinafter “Aggregate sample”), containing the beta 1-40 amyloid peptide AFβ, was then frozen at −80° C. A Thioflavin T test was performed on this solution. It indicates that it contains more than 90% beta 1-40 amyloid peptide aggregates.
Test of the Pair of Antibodies on Monomers or Aggregates
On the day of the test, the Monomer and Aggregate samples were thawed then diluted in 10 mM Sodium Phosphate pH 7.4 buffer at a concentration of 20.3 ng/mL before distribution in a 384-well microplate (Greiner reference 784075).
The following reagents were added to the microplates in the order below:
The plates were then incubated overnight at a temperature comprised between 2° C. and 8° C.
The detection of the FRET signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module. The signals obtained in FRET on the Monomer sample and on the Aggregate sample were compared by calculating the ratio of FRET signals (Monomers/Aggregates) as shown in
The ratio of the FRET (Monomer/Aggregate) signals of the pair of antibodies is greater than 2 (value of 3.1). This indicates that this pair of antibodies discriminates beta 1-40 amyloid peptide NAFβ from beta amyloid peptide 1-40 AFβ, that is to say this pair of antibodies is specific for beta amyloid peptide 1-40 NAFβ.
The method is based on FRET technology (HTRF® technology from Cisbio Bioassays), as detailed in Example 1.
Pair of Tested Antibodies
Two antibodies directed against the beta amyloid peptide 1-42 labeled respectively with the Donor and the Acceptor were diluted to respective concentrations of 3 nM (Donor) and 30 nM (Acceptor).
Preparation of Beta 1-42 Amyloid Peptide NAFβ (Monomers)
The freeze-dried human beta amyloid peptide 1-42 (The ERI Amyloid Laboratory, LLC, Oxford) was resuspended according to the supplier's recommendations then diluted in 10 mM Sodium Phosphate pH 7.4 buffer to a concentration of 30 μM. The solution (hereinafter “Monomer sample”), containing the beta amyloid peptide 1-42 NAFβ, was then frozen at −80° C. A Thioflavin T test was performed on this solution. It indicates that it contains more than 90% beta amyloid peptide 1-42 monomers.
Preparation of Beta Amyloid Peptide 1-42 AFβ (Aggregates)
The freeze-dried human beta amyloid peptide 1-42 (ERI275BAS, The ERI Amyloid Laboratory, LLC, Oxford) was resuspended according to the supplier's recommendations then diluted in 10 mM Sodium Phosphate pH 7.4 buffer to a concentration of 30 μM. This solution is then incubated for 188 hours at 25° C. The solution (hereinafter “Aggregate sample”), containing the beta 1-42 amyloid peptide AFβ, was then frozen at −80° C. A Thioflavin T test was performed on this solution. It indicates that it contains more than 90% beta amyloid peptide 1-42 aggregates.
Test of the Pair of Antibodies on Monomers or Aggregates
On the day of the test, the Monomer and Aggregate samples were thawed then diluted in 10 mM Sodium Phosphate pH 7.4 buffer at a concentration of 2.4 ng/mL before distribution in 384-well microplates (Greiner reference 784075).
The following reagents were added to the microplates in the order below:
The plates were incubated overnight at a temperature comprised between 2° C. and 8° C.
The detection of the FRET signal in the various plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module. The signal obtained in FRET on the Monomer sample and on the Aggregate sample were compared by calculating the ratio of FRET signals (Monomer/Aggregates) as shown in
The ratio of the FRET signals (Monomer/Aggregate) of the pair of antibodies studied is greater than 2 (value of 4.5). This indicates that this pair of antibodies discriminates beta 1-42 amyloid NAFβ from beta amyloid 1-42 AFβ, that is to say this pair of antibodies is specific for the beta amyloid peptide 1-42 NAFβ.
The HFIP was used pure (100%) or diluted in lysis buffer to obtain 20%, 10% and 2% solutions.
The following reagents were distributed in a 384-well plate in the following order:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The HFIP was diluted in lysis buffer to obtain a 2% solution.
The following reagents were distributed in a 384-well plate in the following order:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The Ratio of the signals obtained was calculated as follows from the FRET signals obtained on the samples:
Ratio of the signals=(sample signal treated with 2% HFIP)/(sample signal treated with lysis buffer).
The urea was diluted in lysis buffer to obtain 7M, 3.5M, 1.75M and 0.88M solutions.
The following reagents were distributed in a 384-well plate in the following order:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The urea was diluted in supplemented lysis buffer to obtain 3.5 M, 1.75 M and 0.88 M solutions.
The following reagents were distributed in a 384-well plate in the following order:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The Ratio of the signals obtained was calculated as follows from the FRET signals obtained on the samples:
Signal ratio=(sample signal treated with 3.5M urea)/(sample signal treated with supplemented lysis buffer).
Guanidinium chloride was diluted in supplemented lysis buffer to obtain 6 M, 3 M and 1.5 M solutions.
The following reagents were distributed in a 384-well plate in the following order:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
FA and TFA were diluted in supplemented lysis buffer to obtain 20%, 10% and 2% solutions.
The following reagents were distributed in a 384-well plate in the following order:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
TFA. Regardless of their concentrations, these reagents reduce the detection signal of TDP-43 NAFβ by more than 75%. Their possible effect on the aggregates cannot therefore be evaluated in this format because they prevent the detection of TDP-43 NAFβ.
The FA was diluted in supplemented lysis buffer to obtain 20% solutions.
The NaOH was diluted in a 450 mM HEPES buffer to obtain a 5N solution
The following reagents were distributed in a 96-well plate in the following order:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The TFA was diluted in supplemented lysis buffer to obtain 20% solutions.
The NaOH was diluted in a 450 mM HEPES buffer to obtain a 5N solution.
The following reagents were distributed in a 96-well plate in the following order:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The TFA was diluted in supplemented lysis buffer to obtain 20% solutions.
The NaOH was diluted in a 450 mM HEPES buffer to obtain a 5 N solution.
The following reagents were distributed in a 96-well plate in the following order:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The ratio of the signals obtained was calculated as follows from the FRET signals obtained on the samples:
Signal ratio=(Sample signal treated with 20% TFA then 5N NaOH)/(Sample signal treated with the TFA/NaOH mixture).
The FA was diluted in supplemented lysis buffer to obtain 20% solutions.
The NH4OH was diluted in a 450 mM HEPES buffer to obtain a 5N solution.
The following reagents were distributed in a 96-well plate in the following order:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The FA was diluted in supplemented lysis buffer to obtain 20% solutions.
The NH4OH was diluted in a 450 mM HEPES buffer to obtain a 5N solution
The following reagents were distributed in a 96-well plate in the following order:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The ratio of the signals obtained was calculated as follows from the FRET signals obtained on the samples:
Signal ratio=(Sample signal treated with 20% FA then 5N NH4OH)/(Sample signal treated with the FA/NH4OH mixture).
The TFA was diluted in supplemented lysis buffer to obtain 20% solutions.
The NH4OH was diluted in a 450 mM HEPES buffer to obtain a 5N solution.
The following reagents were distributed in a 96-well plate in the following order:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The TFA was diluted in supplemented lysis buffer to obtain 20% solutions.
The NH4OH was diluted in a 450 mM HEPES buffer to obtain a 5N solution.
The following reagents were distributed in a 96-well plate in the following order:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The ratio of the signals obtained was calculated as follows from the FRET signals obtained on the samples:
Signal ratio=(Sample signal treated with 20% TFA then 5N NH4OH)/(Sample signal treated with the TFA/NH4OH mixture).
NaOH Tested Solutions
HCl Tested Solutions
NaOH/HCl Tested Mixtures (Obtained by Mixing an Identical Volume of an NaOH Solution and an HCl Solution)
The following reagents were distributed in a 96-well plate:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
NaOH Tested Solutions
HCl Tested Solutions
NaOH/HCl Tested Mixtures (Obtained by Mixing an Identical Volume of an NaOH Solution and an HCl Solution)
The following reagents were distributed in a 96-well plate:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The ratio of the signals obtained was calculated as follows from the FRET signals obtained on the samples:
Ratio of the signals=(Sample signal treated with NaOH then HCl)/(Sample signal treated with NaOH/HCl mixture).
The following reagents were distributed in a 96-well plate in the following order:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The ratio of the signals obtained was calculated as follows from the FRET signals obtained on the samples:
Ratio of the signals=(Sample signal treated with 1.2N NaOH then 1N HCl)/(Sample signal treated with NaOH/HCl mixture).
NaOH Tested Solutions
1.2N NaOH solution (allows to bring the pH of the sample to 12.8)
HCl Tested Solutions
NaOH/HCl Tested Mixtures (Obtained by Mixing an Identical Volume of an NaOH Solution and an HCl Solution).
The following reagents were distributed in a 96-well plate:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The Ratio of the signals obtained was calculated as follows from the FRET signals obtained on the samples:
Signal ratio=(Sample signal treated with 1.2N NaOH then HCl)/(Sample signal treated with NaOH/HCl mixture).
The method described below is based on HTRF technology (see Example 1 for principle).
The 1.2N NaOH and 1N HCl solutions were prepared as described in the previous examples.
Preparation of samples containing monomeric (1-42 NAFβ) or aggregated (1-42 AFβ) beta 1-42 amyloid peptide: freeze-dried human beta 1-42 amyloid peptide (ERI275BAS, The ERI Amyloid Laboratory, LLC, Oxford) was re-suspended according to the supplier's recommendations then diluted in 10 mM Sodium Phosphate pH 7.4 buffer to a concentration of 30 μM (1-42 NAFβ). An identical solution of beta 1-42 amyloid peptide is incubated for 188 hours at 25° C. to obtain 1-42 AFβ. The 2 samples were frozen at −80° C. before future use. Before use, the level of aggregation of the 2 samples was checked by the Thioflavin T method.
On the day of the test, the samples 1-42 NAFβ and 1-42 AFβ were thawed then diluted in 10 mM sodium phosphate buffer pH 7.4 at a concentration of 2.4 ng/mL before distribution in the microplate.
The following reagents were distributed in a 384-well plate in the following order:
The plates were incubated overnight at a temperature comprised between 2° C. and 8° C.
The detection of the HTRF signal in the various plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The Aggregation Ratio is calculated as follows from the HTRF signals obtained on the samples:
Signal ratio=(Sample signal treated with 1.2N NaOH then 1N HCl)/(Sample signal treated with the NaOH/HCl mixture).
Preparation of NaOH, KOH, NH4OH and HCl Solutions to Treat TDP43-AFβ Lysates
HCl Tested Solutions
Base/HCl Tested Mixtures (Obtained by Mixing an Identical Volume of an NaOH Solution and an HCl Solution).
The following reagents were distributed in a 96-well plate:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
Preparation of NaOH, KOH, NH4OH and HCl Solutions to Treat TDP43-AFβ Lysates
HCl Tested Solutions
Base/HCl Tested Mixtures (Obtained by Mixing an Identical Volume of an NaOH Solution and an HCl Solution)
The following reagents were distributed in a 96-well plate:
A portion of the volume contained in the wells of the 96-well plate was transferred to a 384-well plate before adding the FRET detection reagents with:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The Ratio of the signals obtained was calculated as follows from the FRET signals obtained on the samples:
Ratio of the signals=(Sample signal treated with Base then HCl)/(Sample signal treated with Base/HCl mixture).
The method is based on FRET technology (HTRF® technology from Cisbio Bioassays), as detailed in Example 1.
Pair of Tested Antibodies
A pair of antibodies directed against Alpha-Synuclein labeled respectively with the Donor and with the Acceptor is that contained in the HTRF Total-Alpha-Synuclein kit marketed by Cisbio Bioassays (ref. 6FNSYPEG). Each antibody was diluted in the kit diluent as recommended by the user manual.
Preparation of Alpha-Syn NAFβ and Alpha-Syn NAFβ Samples
Alpha-Syn NAFβ and Alpha-Syn AFβ were obtained from StressMarq (StressMarq Active Human recombinant A53T Mutant Alpha Synuclein Protein Monomer, ref SPR-325 and Active Human recombinant A53T Mutant Alpha Synuclein Protein Preformed Fibrils Type 1 ref. SPR-326). They were diluted to 15.6 ng/mL in the lysis buffer of the HTRF Total-A Synuclein kit.
Test of the Pair of Antibodies on Alpha-Syn NAFβ and Alpha-Syn
The following reagents were distributed in a 384-well plate in the following order:
The detection of the FRET signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The signals obtained in FRET on the Alpha-Syn NAFβ sample (Monomers) and on the Alpha-Syn AFβ sample (Aggregates) were compared by calculating the ratio of the FRET signals (Monomers/Aggregates) as shown in
The ratio of the FRET (Monomer/Aggregate) signals of the pair of antibodies is greater than 2 (value of 6.64). This indicates that this pair of antibodies is able to specifically bind Alpha-Syn NAFβ over Alpha-Syn AFβ.
The Alpha-Syn NAFβ (StressMarq, Active Human recombinant A53T Mutant Alpha Synuclein Protein Monomer, ref. SPR-326) was diluted in the lysis buffer of the HTRF Total-Alpha Synuclein kit (Cisbio Bioassays, ref. 6FNSYPEG) at 15.6 ng/mL. Donor and acceptor antibodies in the HTRF Total-A Synuclein Kit were diluted as recommended by the supplier in the kit manual.
The following reagents were distributed in a 384-well plate in the following order:
The plates were incubated overnight at room temperature. The detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
The method is based on FRET technology (HTRF® technology from Cisbio Bioassays), as detailed in Example 1.
Pair of Tested Antibodies
The antibodies directed against Alpha-Synuclein labeled respectively with the Donor and with the Acceptor are those contained in the HTRF Total-Alpha-Synuclein kit marketed by Cisbio Bioassays (ref. 6FNSYPEG). They were diluted in the kit diluent as recommended by the user manual.
Preparation of Alpha-Syn NAFβ and Alpha-Syn NAFβ Samples
The Alpha-Syn NAFβ and the Alpha-Syn AFβ were obtained from the company StressMarq (StressMarq Active Human recombinant A53T Mutant Alpha Synuclein Protein Monomer, ref SPR-325 and Active Human recombinant A53T Mutant Alpha Synuclein Protein Preformed Fibrils Type 1 ref SPR-326). They were diluted to 15.6 ng/mL in the lysis buffer of the HTRF Total-Alpha Synuclein kit.
Test of the Pair of Antibodies on Alpha-Syn NAFβ and Alpha-Syn
The following reagents were distributed in a 384-well plate in the following order:
The detection of the FRET signal in the various plates was carried out on a PHERAstar FS Lamp apparatus (BMG Labtech) using an HTRF detection module.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR2004948 | May 2020 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2021/050854 | 5/17/2021 | WO |
