WO2021234261A1 - PROCEDE DE DETECTION D'UNE FORME AGREGEE EN FEUILLETS Bβ D'UNE PROTEINE FORMANT DES AGREGATS DE TYPE FEUILLETS Bβ - Google Patents
PROCEDE DE DETECTION D'UNE FORME AGREGEE EN FEUILLETS Bβ D'UNE PROTEINE FORMANT DES AGREGATS DE TYPE FEUILLETS Bβ Download PDFInfo
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- WO2021234261A1 WO2021234261A1 PCT/FR2021/050854 FR2021050854W WO2021234261A1 WO 2021234261 A1 WO2021234261 A1 WO 2021234261A1 FR 2021050854 W FR2021050854 W FR 2021050854W WO 2021234261 A1 WO2021234261 A1 WO 2021234261A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
Definitions
- the invention relates to an in vitro method of detecting in a sample of an aggregated form in b sheets of a protein forming aggregates of type b sheets (RARb) comprising a step of disaggregation of a RARb in order to obtaining a non-aggregated b-sheet form of the b-sheet aggregate-forming protein (RNARb) and a step of measuring the content of RNARb.
- RARb protein forming aggregates of type b sheets
- amyloid b peptides and more particularly the amyloid b peptide 1-42 have been widely studied for the formation of fibrils containing b sheets and then plaques in Alzheimer's disease.
- the process of aggregation of amyloid b peptides is well characterized and this process depends on a number of factors including their concentration, pH and temperature.
- Many other proteins, such as RNA and DNA binding proteins, which contain a prion-like domain can also form aggregates of the same type [1].
- the proteins containing a prion-like domain are proteins forming b-sheet-like aggregates (RRb).
- the prion-type domains contain hydrophobic amino acids which promote the formation of fibrils rich in b-sheets.
- the aggregated b-sheet forms of a b-sheet aggregate-forming protein (RARb) are not soluble in water at neutral pH.
- the biological sample is filtered through a membrane capable of retaining high molecular weight species and in particular RARb.
- An antibody specific for RARb labeled directly or indirectly with a colorimetric or fluorescent or even luminescent tracer is then applied to the membrane.
- the measured signal is directly proportional to the amount of RARb.
- This method can be adapted to a microplate format to study the aggregation parameters of an amyloid protein or the effect of a compound on its level of aggregation.
- the automation and the throughput of the method are limited due to the filtration and washing steps that this technique requires [2].
- RNARb different ELISA test strategies have been described in order to allow the characterization of the aggregation of RRb.
- the first strategy consists in using the same antibody for the capture of the RARb on the solid phase and as a tracer allowing their detection. This method makes it possible to detect only the RARbs which have several epitopes of the antibody used in capture and detection. Conversely, the capture antibody and the detection antibody will therefore not be able to bind simultaneously to an RNARb because the latter has only one epitope recognized by the antibody used. No signal will therefore be generated in the presence of RNARb [3].
- the second strategy consists in associating in an ELISA test an antibody specifically recognizing RARb with an antibody recognizing all the forms of RRb present in the sample (RARb and RNARb) [4].
- the antibody specific for RARb is generally the capture antibody but a format using this as a detection antibody has also been described [5].
- a third strategy consists in using two different antibodies which both recognize the RARb of interest. This latter strategy seems to improve the specificity of aggregate detection [6].
- kits based on this method have been developed and marketed in order to detect the aggregation of TAU and alpha-synuclein in biological samples (see Cisbio website, commercial references 6FTAUPEG and 6FASYPEG).
- kits make it possible to determine the level of aggregation of the RRb of interest and to determine the effect of a compound on its level of aggregation in miniaturized and high-throughput formats. Nevertheless, these kits require the use of antibodies capable of recognizing RRb epitopes even if these are aggregated, which sometimes proves to be limiting or even blocking in the development of detection tests, given that immunizations are generally performed with RNARb, which can make it difficult to obtain qh ⁇ -RARb antibodies.
- the methods described in the prior art therefore aim to directly detect an RARb in a sample, in particular by using one or more ligands of the RARb.
- directly detecting RARb can make these techniques difficult to implement, imprecise and / or unsuitable for large-scale use.
- detection of RARb alone is not enough because, at a given signal, we cannot realize the level of aggregation compared to the initial state, unless we compare the measurements to a standard range which necessarily bias the measurements of the biological sample tested.
- the Applicant has developed a protocol that is simple and easy to implement which makes it possible to disaggregate all or part of the RARb present in a sample in order to obtain an RNARb.
- This protocol has enabled the Applicant to develop a method for detecting RARb carried out by measuring the content of RNARb, which makes it possible to overcome all the constraints linked to the detection of RARb. Summary of the invention
- the inventive detection in a sample of an aggregated form in b-sheets of a protein forming aggregates of the b-sheet type comprising the following steps: a) In a first container: al) introduce a sample likely to contain an RARb, a2) adjust the pH to a pH ranging from 9.7 to 13.2 to disaggregate all or part of the RARb in order to obtain a non-aggregated b-sheet form of the protein forming b-sheet aggregates (RNARb), a3) adjust the pH to a pH ranging from 6 to 9, b) Measure the RNARb content in the first container with an appropriate immunological method; c) In a second container: c) introduce the same sample as in step a1), c2) adjust the pH to a pH ranging from 6 to 9; d) Measure the RNARb content in the second container with the same method as in step b); e) Compare the contents measured in steps b) and d
- the invention relates to an in vitro method for monitoring the therapeutic efficacy of a treatment of a disease associated with a RARb, comprising the following steps:
- step e) Carry out the method according to the invention on a first sample in which step e) consists in determining the ratio between the content measured in step b) and the content measured in step d) (“Ratio b) / d) of sample 1 ”);
- step B) Carry out the same process as in step A) on a second sample, to determine the “Ratio b) / d) of sample 2”;
- the invention relates to an in vitro method for measuring the pharmacological efficacy of a molecule on a disease associated with a RARb, comprising the following steps:
- step e) Carry out the method according to the invention on a first sample in which step e) consists in determining the ratio between the content measured in step b) and the content measured in step d) (“Ratio b) / d) of sample 1 ”);
- step A) Carry out the same process as in step A) on a second sample, to determine the “Ratio b) / d) of sample 2”; C) Compare the ratios determined in steps A) and B), in which pharmacological efficacy is observed when less than the ratio determined in step A).
- RRb protein forming aggregates of b-sheet type
- RRb protein capable of forming aggregates rich in b-sheets, that is to say a protein capable of forming a multimeric form (or an oligomeric form) rich in layers b.
- a non-aggregated form of RRb is normal, but an aggregated form of it is characterized, in particular, by 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 protein, native or recombinant.
- the non-aggregated form of an RRb is denoted by the expression “non-aggregated form in b-sheets of a protein forming aggregates of type b-sheets” or “RNARb”.
- the aggregate form of an RRb is referred to as "aggregate b-sheet form of a b-sheet aggregate-forming protein" or "RARb”.
- the RRb which can be in aggregated form (RARb) or in non-aggregated form (RNARb), can be chosen from FUS (Fused in sarcoma), TAF15, EWSR1, DAZAP1, TIA- 1, TTR (transthyretin) , cystatin C, b2-microglobulin, amyloid peptide b (such as amyloid peptide b 1-40 or amyloid peptide b 1-42), TAU (Tubulin-Associated Unit), a-synuclein, b-synuclein, g-synuclein, Huntingtin (HTT), SOD1 (superoxide dismutase 1), prion, and TDP-43 (TAR DNA-binding protein 43).
- FUS Fused in sarcoma
- TAF15 FUS
- EWSR1 Epoxyribon
- DAZAP1 TIA- 1
- TTR transthyretin
- the RRb listed above are in particular involved in neurodegenerative diseases.
- FUS involved in amyotrophic lateral sclerosis
- TAF15 involved in amyotrophic lateral sclerosis
- amyloid b peptides involved in Alzheimer's disease and hereditary cerebral amyloid angiopathy
- Ga-synuclein involved in Parkinson's disease TAU protein involved in Alzheimer's disease, frontotemporal dementias, transthyretin involved in senile systemic amyloidosis or amyloid familial polyneuropathy
- cystatin C involved in hereditary cerebral amyloid angiopathy
- Huntingtin involved in Huntington's disease SOD1 involved in amyotrophic lateral sclerosis
- TDP-43 involved in amyotrophic lateral
- the sample in which the method of the invention is implemented can be any sample capable of containing at least one RARb. It may be a biological sample of human or animal origin, or a sample of cells or tissue cultured in vitro.
- in vitro method means a method implemented outside the human or animal body, for example on microorganisms, organs, tissues, cells, cellular sub-fractions (eg nuclei, mitochondria) or cells. proteins (native or recombinant).
- in vitro includes ex vivo.
- the sample can for example come from an individual, man or animal, having or being suspected of having a disease associated with a RARb, for example a neurodegenerative disease as described above.
- the sample can 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 can also include cells (eg cell line), cell subfractions (eg nuclei, mitochondria) or proteins (native or recombinant).
- the sample can also come from cells or from a tissue cultivated in vitro or ex vivo, preferably from a cell or tissue model of a disease associated with a RARb.
- the sample can be selected from a cell lysate, a cell homogenate, a tissue lysate, a tissue homogenate, a cell culture supernatant or a tissue culture supernatant.
- the sample is a cell lysate or a cell homogenate.
- container denotes 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.
- the term “ligand” denotes a molecule capable of binding to a target molecule.
- the target molecule is RNARb.
- RNARb ligand This is then referred to as “RNARb ligand”.
- the ligand may be of a protein nature (eg a protein or a peptide) or of a nucleotide nature (eg a DNA or an RNA).
- the ligand is advantageously chosen from an antibody, an antibody fragment, a peptide or an aptamer, preferably an antibody or an antibody fragment.
- RNARb or“ pair of capabPNAF ligands ”designates a ligand or a pair of ligands which binds preferentially to RNARb over RARb, that is to say a ligand or a pair of ligands capable of generating a signal (e.g. an ELISA signal or a RET signal) vis-à-vis an RNARb at least two times higher, for example at least three, at least four, at least five or at least six times higher, than the signal generated vis -to the corresponding RARb.
- Examples 1-4 and 25 of the present application describe ELISA and FRET methods which make it possible to easily determine whether a ligand or a pair of ligands is capable of specifically binding to RNARb.
- the "ligand capable of binding specifically to RNARb” or the “pair of ligands capable of binding specifically to RNARb” is capable of binding to an RNARb with an affinity at least 2 times greater than 'affinity for the corresponding RARb, 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 greater, at least 9 times greater, at least 10 times greater than the affinity for the corresponding RARb.
- the two ligands of the ligand pair can be specific ligands for RNARb.
- affinity refers to the strength of all the non-covalent interactions between a ligand and an antigen. Affinity is generally represented by the dissociation constant (Kd). The lower the Kd value, 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, ELISA or SPR. The techniques described in the literature therefore make it possible to easily know whether a ligand or a pair of ligands is specific for RNARb.
- antibody also called “immunoglobulin” denotes 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 ( say L chains for Light), linked together by intra- and inter-chain disulfide bridges.
- Each chain consists, in the N-terminal position, of a region or variable domain, called VL for the light chain, VH for the heavy chain, and in the C-terminal position, of a constant region, consisting 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 includes 4 "hinge regions” (called FRI, FR2, FR3, FR4) and 3 regions directly responsible for binding with the antigen, called “CDR” (called orps "can be of mammalian origin (eg human or mouse or rat or camelid) ), humanized, chimeric, recombinant. It is preferably a monoclonal antibody produced recombinantly by cells genetically modified according to techniques widely known to those skilled in the art. Any isotype, for example IgG, IgM, IgA, IgD or IgE, preferably IgG.
- antibody fragment means any part of an immunoglobulin obtained by enzymatic digestion or obtained by bio-production comprising at least one disulfide 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 string (eg scFvs).
- Enzymatic digestion of immunoglobulins by pepsin generates an F (ab ') 2 fragment and an Fc fragment split into several peptides.
- F (ab ') 2 is formed from two Fab' fragments linked by inter-chain disulfide bridges.
- the Fab parts consist of the variable regions and the CH1 and CL domains.
- the Fab 'fragment consists of the Fab region and a hinge region.
- Fab'-SH refers to an Fab 'fragment in which the cysteine residue of the hinge region carries a free thiol group.
- tracer is understood to mean 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.
- RET Resonance Energy Transfer
- FRET Fluorescence Resonance Energy Transfer
- BRET (from the English “Bioluminescence Resonance Energy Transfer”) designates the transfer of energy between a bioluminescent molecule and a fluorescent molecule.
- air of RET partners denotes a pair consisting of an energy donor compound (hereinafter mposed energy acceptor (hereinafter “acceptor compound”), when they are close to l 'from each other and when excited at the excitation wavelength of the donor compound, these compounds emit a RET signal.
- RET signal denotes any measurable signal representative of a RET between a donor compound and an acceptor compound.
- a FRET signal can therefore be a variation in the intensity or the luminescence lifetime of the fluorescent donor compound or of the acceptor compound when the latter is fluorescent.
- the invention relates to an in vitro method for detecting in a sample of an aggregated form in b-sheets of a protein forming aggregates of the b-sheet type (RARb), comprising the following steps: a ) In a first container: al) introduce a sample likely to contain an RARb, a2) adjust the pH to a pH ranging from 9.7 to 13.2 to disaggregate all or part of the RARb in order to obtain a non- form aggregated in b-sheets of the protein forming aggregates of the b-sheet type (RNAEb), a3) adjust the pH to a pH ranging from 6 to 9, b) Measure the content of RNARb in the first container with an appropriate immunological method; c) In a second container: c) introduce the same sample as in step a1), c2) adjust the pH to a pH ranging from 6 to 9; d) Measure the RNARb content in the second container with the same method as in step b); e
- Step a) Step a1) consists in, in a first container, introducing a sample capable of containing a RARb.
- the tnt prepared in order to be able to carry out the steps of the process suitably, in particular the step of measuring the RNARb content.
- the sample is a tissue or cells, it can be prepared beforehand by lysing it, grinding it, filtering it and / or putting it in solution in a suitable solvent such as water.
- a suitable solvent such as water.
- Step a2) consists in adjusting the pH to a pH ranging from 9.7 to 13.2 to disaggregate all or part of the RARb in order to obtain a non-aggregated form in b sheets of the protein forming b-sheet aggregates (RNARb).
- a pH ranging from 9.7 to 13.2 makes it possible to break down the RARb. This disaggregation phenomenon makes it possible to obtain an RNARb from the RARb.
- the method of the invention does not require treatment at an acidic pH to disaggregate the RARb.
- the Applicant has not only shown that a Disaggregation was possible by treating a RARb at a pH ranging from 9.7 to 13.2, but also that this treatment was subsequently implemented of an immunological method aimed at measuring the content of RNAEb.
- 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.
- a base for example a strong base such as KOH or NaOH, preferably NaOH.
- step a2) may depend on various parameters such as the base used or the RRb tested.
- 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.
- Those skilled in the art will have no difficulty in adapting the duration of step a2) in order to manage to disaggregate all or part of the RAEb in order to obtain an RNAEb.
- Step a3) consists in adjusting the pH to a pH ranging from 6 to 9. This step is important since it makes it possible to implement the immunological method of step b).
- 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.
- an acid for example a strong acid, such as HCl.
- Step b) consists in measuring the RNAEb content in the first container with an appropriate immunological method (or appropriate “immunoassay”).
- step b) It is necessary for the immunological method of step b) to make it possible to obtain contents which are comparable with one another.
- the immunological method of step b) need not be quantitative, although it can be.
- the immunological method of step b) must at least make it possible to measure a relative content which can be compared with another relative content measured by the same immunological method.
- the immunological method implemented in step b) uses:
- a ligand capable of binding specifically to RNAEb said ligand being labeled with a label
- ligand (ii) a pair of ligands capable of binding specifically to RNAEb, at least one ligand of said pair of ligands being labeled with a label.
- the ligand (i) can be chosen from an antibody, an antibody fragment, a peptide or an aptamer, preferably an aD can be chosen from a pair of antibodies, a pair of antibody fragments, a pair of peptides or a pair of aptamers, preferably a pair of antibodies.
- RNARb for example by immunizing a mouse with an RRb, by carrying out lymphocyte hybridization from the lymphocytes from the spleen of the immunized mouse in order to generate hybridomas and by testing the antibodies of each hybridoma for their abilities to bind specifically to RNARb.
- Examples of a complete protocol for generating anti-RNARb antibodies and then selecting 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 type of antibody. which RNARb for the implementation of the method according to the invention, for example by implementing the methods described in Examples 1-4 and 25.
- RNARb and RARb are within the abilities of those skilled in the art.
- “prion-like” proteins such as TDP-43, FUS, TAF15 and EWSR1
- GST Gluthatione-S-Transferase
- RRbs 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 b 1-40 and b 1-42 forms, are also available from many suppliers in powder form.
- the solubilization of these powders in HFIP or NH 4 OH makes it possible to obtain stock solutions containing more than 90% of non-aggregated forms.
- the extemporaneous use of these solutions diluted beforehand in buffers exhibiting physiological pHs makes it possible to obtain samples of non-aggregated forms.
- 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 makes it possible to obtain a sample containing very high proportions of aggregated forms. [11].
- Samples containing RNARb and RARb can also be obtained from the company StressMarq (www.stressmarq.com). This is particularly the case for alpha, beta and gamma synuclein, TAU protein, Cu / Zn superoxide dismutase 1 (SOD1) protein or Transthyreti
- the immunological method implemented in steps b) is an ELISA method or a RET method, such as a FRET or a BRET.
- the measurement of step b) can be done directly in the first container by adding the appropriate reagents thereto or in another container with all or part of the contents of the first container.
- the reagents for a RET method can be added directly to the first container.
- the ELISA method will preferably be carried out in another container suitable for carrying out the ELISA method, in particular in a container at the bottom of which has previously been immobilized a ligand of RNARb.
- step b) is carried out by a RET method and consists of:
- the first ligand and the second ligand must not compete for binding to RNARb, for example the first ligand and the second ligand must not bind the same epitope on RNARb. It is easy to select an appropriate pair of ligands by performing 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 those skilled in the art, based on the presence of reactive groups on the ligand.
- 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, thiol groups of cysteines, phenol groups of tyrosines, indole rings of tryptophanes, thioether groups of methionines, 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 those skilled in the art, for example a donor compound or an acceptor compound functionalized by a maleimide group will for example be able to bind to covalently with the thiol groups carried by the cysteines carried by a protein or a peptide, for example an antibody or an antibody fragment.
- a donor / acceptor compound carrying an ester dole covalently attaches to an amine present on a protein or peptide.
- the ligand can also be labeled with a fluorescent or bioluminescent compound indirectly, for example by introducing into the measurement medium an antibody or an antibody fragment, itself covalently linked to an acceptor / donor compound. , this second antibody or antibody fragment specifically recognizing the ligand.
- Another very conventional indirect labeling means 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 those skilled in the art; the company Cisbio Bioassays markets, for example, streptavidin labeled with a fluorophore whose trade name is “d2” (ref. 610SADLA).
- 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).
- the fluorescent donor compound can be a FRET partner chosen 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.
- FRET partner chosen 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
- the fluorescent acceptor compound can be a FRET partner chosen from: allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives , nitrobenzoxadiazole, quantum dot, GFP, GFP variants chosen from GFP10, GFP2 and eGFP, YFP, YFP variants chosen from eYFP, YFP topaz, YFP citrine, YFP venus and YPet, mOrange, DsRed.
- FRET partner chosen from: allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives , nitrobenzoxadiazole, quantum dot, GFP, GFP
- the luminescent donor compound can be a BRET partner chosen from: Luciferase (read), Renilla Luciferase (Rluc), variants of Renilla Luciferase (Rluc8) and Firefly Luciferase.
- the fluorescent acceptor compound is a partner of BRET chosen from: allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives, nitrobenzoxadiazole, a quantum dot, GFP, GFP variants chosen from GFP10, GFP2 and eGFP, YFP, YFP variants chosen from eYFP, YFP topaz, YFP citrine, YFP venus and YPet, mOrange, DsRed.
- step b) is carried out by a method
- the ELISA method is widely described in the prior art and does not present any difficulty of implementation for a person skilled in the art.
- Step c) consists in, in a first container, c) introducing the same sample as in step a1).
- 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 c1)), preferably to the same pH as the pH of step a3).
- the pH in step c) can be adjusted with an acid / base mixture, for example a strong acid / strong base mixture. It may for example be an NaOH / HCl mixture.
- an acid / base mixture for example a strong acid / strong base mixture. It may for example be an NaOH / HCl mixture.
- the acid used in step c2) is the same as that used in step a3) and the base used in step c) is the same as the base used in step a2).
- Step d) consists in measuring the RNAEb content in the second container with the same method as in step b). Step d) is implemented in the same way as step b) in order to be able to compare the measurements and implement step e).
- step d) consists of:
- step d) consists of:
- Step e) consists in comparing the contents measured in step b) and in step d), a decrease in the content measured at Eiesurea in step b) indicating that the sample contains a RAEb.
- step e) will consist of comparing the contents measured in step b) and in step d), an increase in the content measured in step d) with respect to the content measured in step b) indicating that the sample contains an RAEb.
- a person skilled in the art can easily compare the contents measured in steps b) and d) and define a threshold allowing him to qualify the increase or the 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.
- a 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 quantity of RARb in the sample will be.
- the immunological method is an ELISA method or a RET method
- we will compare the RET signals or the ELISA signals measured in steps b) and d). Therefore, a decrease in the RET signal or a decrease in the ELISA signal will indicate that the sample contains a RAEb.
- 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 ratio can be calculated manually or automatically.
- the duration between step a3) and step b) and / or between step c2) and step d) will be adapted to prevent the RNAEb from re-aggregating into RAEb.
- the duration will preferably be less than 24 hours, for example less than 5 hours.
- steps b) and d) are carried out immediately after steps a3) and c2) respectively, that is to say less than 30 minutes after steps a3) and c2).
- steps b) and d) are carried out immediately after steps a3) and c2) respectively, that is to say less than 30 minutes after steps a3) and c2).
- steps b) and d) are carried out immediately after steps a3) and c2) respectively, that is to say less than 30 minutes after steps a3) and c2).
- step e The comparison of the contents in the two samples in step e) makes it possible to measure the level of aggregation very precisely, in particular compared to most of the methods of the prior art.
- the present invention also relates to an in vitro method for monitoring the therapeutic efficacy of a treatment of a disease associated with a RAEb in a patient, comprising the following steps: A) Carrying out the method according to the invention on a first sample of said patient, in which step e) consists in determining the concentration in step b) and the content measured in step d) (“Ratio b) / d) sample 1 ”);
- step B) Carry out the same process as in step A) on a second sample of said patient, to determine the “Ratio b) / d) of sample 2”;
- the treatment can be a known treatment for the disease associated with RARb or an experimental treatment.
- the sample is preferably from an individual having a disease associated with RARb.
- 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. time T2.
- 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 of said processing.
- the time which elapses between the taking of the first sample and the taking of the second sample will be chosen in order to be able to detect a therapeutic efficacy of the treatment.
- the present invention also relates to an in vitro method for measuring the pharmacological efficacy of a drug molecule or of a drug candidate on a disease associated with a RARb in a test sample, comprising the following steps:
- step e) Carry out the method according to the invention on a first sample of said test sample, in which step e) consists in determining the ratio between the content measured in step b) and the content measured in step d ) (“Ratio b) / d) of sample 1”);
- step B) Carry out the same process as in step A) on a second sample of said test sample, to determine the “Ratio b) / d) of sample 2”;
- the molecule can be a drug or a drug candidate.
- the sample is preferably from cells or tissue cultured in vitro. Therefore, the drug molecule or drug candidate is preferably tested in an in vitro model of disease associated with rARb.
- Such models are widely described in the literature.
- the test sample corresponds to a sample which makes it possible to test in vitro a drug or a candidate medicamin sample of cells cultured in vitro or a sample of tissue cultured in vitro.
- the test sample corresponds to what is commonly referred to as an "in vitro model of disease associated with RARb".
- 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.
- 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 of a drug candidate, and the second sample is taken at a time. time T1 after said treatment.
- the time which elapses between the taking of the first sample and the taking of the second sample will be chosen in order to be able to detect a pharmacological efficacy of the drug molecule or of the drug candidate.
- Example 1 Method for identifying antibodies specific for a PNAFP
- BALB / c mice are immunized by injection of the PNAFP protein previously diluted in phosphate buffer prepared under physiological conditions. The absence of the presence of multimers or aggregates of PNAFP is verified in the buffer intended to carry out the injections in order to direct the immune response of the mice to the non-aggregated forms. The first injection is followed by three boosters at monthly intervals.
- mice Fifteen days after each injection, blood punctures are performed on the mice to verify the presence of an immune response by titration of the antibodies.
- PNAF amyloid-type PFPs or peptides
- PNAF is labeled beforehand on its primary lysines with biotin by the use of a reagent composed of biotin, a carbon linker and an NHS reactive group (N - Hydroxysuccinimide).
- GST fusion PNAFg is directly immobilized on 96-well plates via the GST tag by the use of ELISA microplates functionalized with a glutahion group. Proteins labeled with biotin are immobilized via biotin by the use of microplates functionalized with streptavidin.
- RNARb solution 100 ⁇ L of GST and / or biotinylated molten RNARb 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 ⁇ L of a saturation solution composed of PBS, 5% BSA.
- serial dilutions by a factor of 10 to 100 million of the blood punctures are then added, in doublets, at a level of 100 ⁇ L per well in PBS + 0.1% BSA buffer and incubated with stirring for 2 h.
- the non-specific antibodies not bound to the immobilized RNARb are eliminated by three stages of washing of 200 ⁇ L in PBS IX buffer, 0.05% Tween20.
- the possible presence of specific antibodies is detected using 100 ⁇ L per well of secondary anti-mouse Fc antibody coupled to HRP (horseradish peroxidase) (Sigma # A0168 diluted to 1/10 000 in PBS, BSA 0.1%).
- the same punctures are tested on the ELISA test after pre-incubation with an excess of another orthogonal protein tagged with GST or biotin.
- the anti-TAG antibodies bind to the tagged orthogonal protein and therefore not to the RNARb protein immobilized at the bottom of the wells; in which case no HRP signal or a decrease in HRP signal is detected.
- mice exhibiting the best qh ⁇ -RNARb antibody titers (signal, that is to say high optical density at 450 nm) and the least decrease in signal in the anti-TAG control case are selected for the The next stage of lymphocyte hybridization, also called fusion.
- the spleen of the mice is extracted in order to isolate a mixture of lymphocytes and plasma cells. This multicellular sample is fused in vitro with a myeloma cell line in the presence of a polyethylene glycol (PEG) type cell fusion catalyst.
- PEG polyethylene glycol
- a mutant myeloma cell line deficient in the HGPRT (Hypoxanthine Guanosin Phosphoribosyl Transferase) enzyme is used to allow selection of hybrid cells, called hybridomas. These cells are cultured in a medium containing hypoxanthine, aminopterine (methotrexate) and thyamine (HAT medium), to allow the elimination of the myeloma cells. non-fused and thus select the hybridomas of interest. As for the non-fused spleen cells, they die off in vitro. Thus, only the hybridomas survive this selection pressure in vitro.
- HGPRT Hypoxanthine Guanosin Phosphoribosyl Transferase
- hybridomas are cultured in culture plates. The supernatants of these hybridomas are tested to evaluate their capacity to produce anti-PNAF antibodies. For this, 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 constituting a library of hybridomas, tested for cell viability and stored in liquid nitrogen.
- 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, the antibodies are produced as described below.
- the hybridoma clones of interest are put back into culture and the cell inoculum is then injected into BALB / c mice (intraperitoneal injection, IP) in order to allow the production of antibodies in large quantities in the liquid. ascites.
- the anti-PNAF antibodies are ready for storage at 4 ° C or freezing and subsequent use / characterization (isotyping, assay, functional tests).
- an ELISA type test is set up.
- 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 pair tested diluted beforehand in PBS buffer at concentrations of between 1 and 20 pg / ml, is adsorbed on 96-well plates of the “high binding” ELISA type. For this, 100 ⁇ L of antibodies are added to each well and then incubated for 20 h at 4 ° C followed by three washes in PBS IX buffer, 0.05% Tween20. After removing the washing solution, each well is then incubated overnight at 4 ° C. with 200 ⁇ L of ise PBS, 5% BSA. This saturation solution is then removed by suction and the plates are stored at 4 ° C for future use.
- RNARb or RARb Serial dilutions by a factor of 1 to 1 / 100th of samples containing the same initial concentration of RNARb or RARb are added at a level of 100 ⁇ L / well and incubated for 2 h at room temperature with stirring.
- the RNARb or RARb not fixed to the antibody adsorbed on the plates are removed by washing in three steps in PBS 1X buffer, 0.05% Tween20.
- the biotinylated antibody diluted beforehand in PBS buffer at a concentration of between 10 and 200 ng / mL, is added at a level of 100 ⁇ L / well and incubated for 2 h at room temperature with stirring.
- the biotinylated antibodies not bound to RNARb or RARb are eliminated by three stages of washing in PBS IX buffer, 0.05% Tween20.
- the detection of the fixed antibodies is carried out using streptavidin-HRP (R&D Systems, Ref. DY998) diluted to 1/10 in PBS, BSA 0.1%.
- streptavidin-HRP R&D Systems, Ref. DY998
- the HRP is revealed by measuring the optical density at 450 nm (OD 450 nm) following the 'incubation of its TMB substrate (3,3', 5,5'-Tetramethylbenzidine, Sigma # T0440) for 20 min at room temperature with stirring.
- the reference dilution of the RNARb or RARb samples is determined by analyzing the O.D. 450 nm measured with the different dilutions of RNARb. As shown in Figure 2, the reference dilution is that for which 80% of the maximum 450nm O.D. is obtained.
- the O.D. 450 nm obtained with the reference dilution of the sample of RNARb is compared with the O.D. 450 nm measured with an identical dilution of the sample of RARb.
- a pair of antibodies capable of specifically binding to RNARb will give a 50% lower O.D. 450nm on the RARb sample compared to the O.D. 450nm measured on the RNARb sample.
- a FRET test is set up. This test is based on Cisbio Bioassays HTRF® Technology. The principle of this technique is based on a transfer of fluorescence energy between a donor molecule, a Terbium cryptate (Donor), and a fluorescent energy-accepting molecule d2 (Acceptor). These two fluorescent molecules are grafted by covalent coupling to antibodies to implement an immunological assay as illustrated in Figure 4. To do this, one of the antibodies of the tested pair is labeled with the donor Lumi4 Terbium using the kit.
- Terbium Cryptate labeling kit (Cisbio Bioassays, reference 62TBSPEA) according to the manufacturer's recommendations.
- the antibody labeled with the donor is diluted in a Hepes 20mentration buffer of 0.5 nM.
- the second antibody of the pair tested is labeled with the d2 acceptor using the d2 labeling kit (Cisbio Bioassays reference 62D2DPEA) according to the supplier's recommendations.
- RNARb or RARb Serial dilutions by a factor of 1 to 1 / 100th of samples containing the same initial concentration of RNARb or RARb are distributed in a 384 microplate at a level of 16 ⁇ L / well. 2 ⁇ l of the 0.5 nM solution of antibody labeled with the donor and 2 ⁇ l of the 5 nM solution of antibody labeled with the acceptor are then added to each well. The microplate is incubated for 20 h at room temperature. The detection of the FRET signal in the different plates was carried out on a PHERAstar FS Lamp device (BMG Labtech) using an HTRF detection module.
- the reference dilution of the RNARb or RARb samples is determined by analyzing the HTRF signal measured with the different dilutions of RNARb. As indicated in FIG. 5, the reference dilution is that for which 80% of the maximum HTRF signal is obtained, before the saturation plateau of the immunoassay.
- the HTRF signal obtained with the reference dilution of the RNARb sample is compared with the HTRF signal measured with an identical dilution of the RARb sample.
- a pair of antibodies capable of specifically binding to RNARb will give a 50% lower HTRF signal on the RARb sample compared to the HTRF signal measured on the RNARb sample.
- Example 2 Method making it possible to identify pairs of antibodies capable of binding specifically to TDP-43 NAFP (FRET method)
- An alternative method of obtaining samples containing TDP-43 ARb or TDP-43 NARb consists in cultivating HeLa cells and in treating them or not for at least 6 hours with staurosporine.
- the analysis of the cell lysates carried out by SDS-PAGE and Western-Blot shows that the lysates of untreated HeLa cells contain TDP-43 NARb while the lysates of HeLa treated with staurosporine mainly contain TDP-43 ARb [12] .
- the method is based on the FRET technology (HTRF® technology from Cisbio Bioassays) described in Example 1.
- TDP-43 Five antibodies directed against TDP-43 were labeled respectively with the Donor and with the Acceptor. These markings were carried out using the marking kits marketed by Cisbio Bioassays (Commercial references 62EUSUEA and 62D2DPEA). The levels of labeling obtained (number of fluorescent molecules per antibody) were as expected. The donor tagging rates were between 5.9 and 7.9. The omission rates between 2.3 and 3.3.
- HeLa cells were seeded to 5 million cells in a flask (175 cm 2 ) in complete culture medium (MEM alpha medium + 2 mM Hepes + 10% decomplemented fetal calf serum + 1% antibiotics, penicillin 5000 U / ml and Streptomycin 5000 pg / ml). After 78 hours, the culture medium was aspirated. The cells were then lysed with cell lysis buffer. The lysate obtained (hereinafter “Monomer sample”), containing the TDP-43 NAEb, was frozen at -80 ° C. with a view to its use. A control of this sample was carried out by western blot as recommended in the literature [12] in order to ensure that it essentially contains unaggregated TDP43 (TDP-43 NAEb).
- Monomer sample The lysate obtained (hereinafter “Monomer sample”), containing the TDP-43 NAEb, was frozen at -80 ° C. with a view to its use.
- HeLa cells were seeded to 5 million cells in a flask (175 cm 2 ) in complete culture medium (MEM alpha medium + 2 mM Hepes + 10% decomplemented fetal calf serum + 1% antibiotics, penicillin 5000 U / ml and Streptomycin 5000 pg / ml). After 72 hours, the culture medium was aspirated. A 1 ⁇ M staurosporine solution in complete medium was added to the cells in culture for 6 h. Treatment of cells with staurosporine allows TDP-43 to aggregate to obtain TDP-43 AEb. The culture medium was then aspirated before adding a cell lysis buffer.
- MEM alpha medium + 2 mM Hepes + 10% decomplemented fetal calf serum + 1% antibiotics, penicillin 5000 U / ml and Streptomycin 5000 pg / ml After 72 hours, the culture medium was aspirated. A 1 ⁇ M staurosporine solution in complete medium was added to the cells in
- Aggregate sample The lysate obtained (hereinafter “Aggregate sample”), containing TDP-43 AEb, was frozen at -80 ° C. with a view to its subsequent use. A control of this sample was carried out by western blot as recommended in the literature [12] in order to ensure that it essentially contains aggregated TDP43 (TDP-43 AEb). [0136] Screening of the pairs of antibodies on the Monomers or the Aggregates
- the detection of the FRET signal in the different plates was carried out on a PHERAstar FS Lamp device (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 a ratio of the FRET signals (Monomers / Aggregates), as shown in Figure 7 with the Acl-Donor / Ac4-Acceptor pair.
- the ratio of FRET signals (Monomers / Aggregates) was calculated for all pairs of antibodies tested. Table 2 below summarizes the results obtained. All pairs of antibodies exhibiting a (Monomer / Aggregate) ratio greater than 2 are considered selective for TDP-43 NAF vis-à-vis TDP-43 AF.
- the method is based on FRET technology (HTRF® technology from Cisbio Bioassays), as detailed in Example 1.
- the antibodies directed against the beta amyloid peptide 1-40 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 diluent reference 62RB3FDG as recommended by the manual for the Amyloid Beta 1-40 HTRF kit.
- the lyophilized human amyloid beta 1-40 peptide (ERI275BAS, The ERI Amyloid Laboratory, LLC, Oxford) was re-suspended according to the supplier's recommendations then diluted in 10 mM Sodium Phosphate buffer pH 7.4 to a concentration of 30 mM.
- the solution (hereinafter “Monomer sample”), containing the amyloid peptide beta 1-40 NAF, was then frozen at -80 ° C. A Thioflavin T test was performed on this solution. It indicates that this contains more than 90% of beta amyloid peptide 1-40 monomers.
- the lyophilized human amyloid beta 1-40 peptide (ERI275BAS, The ERI Amyloid Laboratory, LLC, Oxford) was re-suspended according to the supplier's recommendations then diluted in 10 mM Sodium Phosphate buffer pH 7.4 to a concentration of 30mM. This solution is then incubated for 495 hours at 25 ° C., which makes it possible to aggregate the beta amyloid peptide 1-40 to obtain the beta amyloid peptide 1-40 AF.
- the solution hereinafter “Aggregate sample”
- the solution was then frozen at -80 ° C.
- a Thioflavin T test was performed on this solution. It indicates that this contains more than 90% aggregates of beta amyloid peptide 1-40.
- the detection of the FRET signal in the different plates was carried out on a PHERAstar FS Lamp device (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 the FRET signals (Monomers / Aggregates) as shown in Figure 8.
- the ratio of the signals of FRET (Monomer / Aggregate) of the pair of antibodies is greater than 2 (value of 3.1) ⁇ This indicates that this pair of antibodies discriminates the beta amyloid peptide 1-40 NAF of the beta amyloid peptide 1-40 AF, that is, this pair of antibodies is specific for beta amyloid peptide 1-40 NARb.
- Example 4 Method making it possible to identify pairs of antibodies capable of binding specifically to the amyloid peptide beta 1-42 NAFP (FRET method)
- the method is based on FRET technology (HTRF® technology from Cisbio Bioassays), as detailed in Example 1.
- the lyophilized human amyloid beta 1-42 peptide (The ERI Amyloid Laboratory, LLC, Oxford) was resuspended according to the supplier's recommendations then diluted in 10 mM sodium phosphate buffer pH 7.4 to a concentration of 30 mM .
- the solution (hereinafter “Monomer sample”), containing the beta amyloid peptide 1-42 NAF, was then frozen at -80 ° C. A Thioflavin T test was performed on this solution. It indicates that this contains more than 90% of beta amyloid peptide 1-42 monomers.
- the lyophilized human amyloid beta 1-42 peptide (ERI275BAS, The ERI Amyloid Laboratory, LLC, Oxford) was resuspended according to the supplier's recommendations then diluted in 10 mM Sodium Phosphate buffer pH 7.4 to a concentration of 30 pM. This solution is then incubated 188 hours at 25 ° C. The solution (hereinafter “Aggregate sample”), containing the beta amyloid peptide 1-42 AF, was then frozen at -80 ° C. A Thioflavin T test was performed on this solution. It indicates that this contains more than 90% aggregates of beta amyloid peptide 1-42.
- the plates were incubated overnight at a temperature between 2 ° C and 8 ° C.
- the detection of the FRET signal in the different plates was carried out on a PHERAstar FS Lamp device (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 the FRET signals (Monomer / Aggregates) as shown in Figure 9.
- the ratio of the signals of FRET (Monomer / Aggregate) of the pair of antibodies studied is greater than 2 (value of 4.5). This indicates that this pair of antibodies discriminates amyloid beta 1-42 NAF from amyloid beta 1-42 AF, i.e. this pair of antibodies is specific for amyloid peptide beta 1 -42 NARb.
- Example 5 Test of the effect of Hexafluoroisopropanol (HFIP) on the detection capacity of a method using a pair of antibodies capable of binding specifically to TDP-43 NAFP
- HFIP was used pure (100%) or diluted in lysis buffer to obtain 20%, 10% and 2% solutions.
- 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 device (BMG Labtech) using an HTRF detection module.
- FIG. 10 gives the signals obtained with the different concentrations of HFIP tested. For concentrations greater than 2%, HFIP decreases the detection signal of TDP-43 NARb by more than 75%. Its possible effect on the aggregates will therefore be evaluated in this format with a concentration of 2%.
- Example 6 Test of HFIP as a disaggregating agent for TDP-43 AFP
- G01791 HFIP has been diluted in 2% tampoi.
- 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 device (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:
- FIG. 11 gives the results obtained.
- the signal ratio is less than or equal to 1 (0.89) which indicates that the treatment with 2% HFIP does not increase the detection of PNAF in the sample containing TDP-43 ARb. It can be concluded from this that the treatment with 2% HFIP does not make it possible to disaggregate, even partially, TDP-43 ARb.
- the urea was diluted in lysis buffer to obtain 7M, 3.5M, 1.75M and 0.88M solutions.
- FIG. 12 gives the signals obtained with the different concentrations of Urea tested. For concentrations greater than 75% the detection signal of TDP-43 NARb. Its possible effect on the aggregates will therefore be evaluated in this format with concentrations less than or equal to 3.5M.
- Example 8 Test for Urea as a TDP-43 AFP Disaggregating Agent
- the urea was diluted in supplemented lysis buffer to obtain 3.5 M, 1.75 M and 0.88 M solutions.
- 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 device (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:
- Figure 13 gives the results obtained.
- the signal ratio is less than or equal to 1 for all Urea concentrations tested, indicating that these treatments do not increase the detection of TDP-43 PNAF in the aggregate sample. It can be concluded from this that a treatment with urea concentrations less than or equal to 3.5M does not make it possible to disaggregate, even partially, TDP-43 ARb.
- Example 9 Test of the effect of Guanidinium chloride on the detection capacity of a method using a pair of antibodies capable of binding specifically to TDP-43 NAFP
- Guanidinium chloride was diluted in supplemented lysis buffer to obtain 6 M, 3 M and 1.5 M solutions.
- 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 device (BMG Labtech) using an HTRF detection module.
- FIG. 14 gives the signals obtained with the different concentrations of Guanidinium chloride tested. Whatever its concentration, Guanidinium Chloride decreases the detection signal of TDP-43 NARb by more than 75%. Its possible effect on the aggregates cannot therefore be evaluated in this format because this compound prevents the detection of TDP-43 NARb.
- AF and TFA were diluted in supplemented lysis buffer to obtain 20%, 10% and 2% solutions.
- 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 device (BMG Labtech) using an HTRF detection module.
- FIG. 15 gives the signals obtained with the different concentrations of AF or TFA tested. Whatever their concentrations, these reagents reduce the detection signal of TDP-43 NAFp 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 NAFp.
- Example 11 Test of the effect of formic acid (FA) followed by neutralization with NaOH on the detection capacity of a method using a pair of antibodies capable of binding specifically to TDP-43 NAFP [0208] The AF was diluted in supplemented lysis buffer to obtain solutions of
- the NaOH was diluted in a 450 mM HEPES buffer to obtain a 5N solution.
- 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 device (BMG Labtech) using an HTRF detection module.
- FIG. 16 compares the RNARb detection signal obtained with the AF treatment then NaOH with that obtained in the absence of treatment.
- the treatment causes an almost total disappearance of the TDP-43 NARb detection signal. Their possible effect on the disaggregation of TDP-43 ARb cannot therefore be evaluated.
- Example 12 Test of the effect of Trifluoroacetic Acid (TFA) followed by neutralization with NaOH on the detection capacity of a method using a pair of antibodies capable of binding specifically to TDP-43 NAFP
- 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 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 device (BMG Labtech) using an HTRF detection module.
- FIG. 17 compares the RNARb detection signal obtained with the TFA treatment then NaOH with that obtained in the absence of treatment. Even if this treatment significantly affects the TDP-43 NAF detection signal (-62%), the remaining signal makes it possible to test this on the disaggregation of TDP-43 ARb.
- Example 13 Test of the effect of Trifluoroacetic Acid (TFA) followed by neutralization with NaOH as a disintegrating agent for TDP-43 AFP
- 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 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 device (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:
- Example 14 Test of the effect of formic acid (FA) followed by neutralization with NH 4 OH on the detection capacity of a method using a pair of antibodies capable of binding specifically to TDP -43 NAFP
- the AF was diluted in supplemented lysis buffer to obtain 20% solutions.
- the NH 4 OH was diluted in a 450 mM HEPES buffer to obtain a 5N solution.
- 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 device (BMG Labtech) using an HTRF detection module.
- FIG. 19 compares the TDP-43 NARb detection signal obtained with the AF treatment then NH 4 OH with that obtained in the absence of treatment. Even if this treatment significantly affects the detection signal of TDP-43 NARb (-68%), the remaining signal makes it possible to test this on the disaggregation of TDP-43 ARb.
- Example 15 Test of the effect of formic acid (AF) followed by neutralization with NH 4 OH as a disintegrating agent for TDP-43 AFP
- the AF was diluted in supplemented lysis buffer to obtain 20% solutions.
- the NH 4 OH 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:
- 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 device (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:
- Figure 20 gives the results obtained.
- the signal ratio is less than or equal to 1 (0.78) indicating that the treatment does not increase the detection of TDP-43 NAFP in the aggregate sample. It can be concluded from this that the treatment with 20% AF followed by neutralization with NH 4 OH does not make it possible to break down, even partially, TDP-43 AFp.
- Example 16 Test of the effect of trifluoroacetic acid (TFA) followed by neutralization with NH 4 OH on the detection capacity of a method using a pair of antibodies capable of binding specifically to TDP -43 NAFP
- the TFA was diluted in supplemented lysis buffer to obtain 20% solutions.
- the NH 4 OH was diluted in a 450 mM HEPES buffer to obtain a 5N solution.
- 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 device (BMG Labtech) using an HTRF detection module.
- FIG. 21 compares the RNARb detection signal obtained with the TFA then NH 4 C ) H treatment with that obtained in the absence of treatment. Even if this treatment significantly affects the TDP-43 NAF detection signal (-72%), the remaining signal makes it possible to test this on the disaggregation of TDP-43 ARb.
- Example 17 Test of the effect of trifluoroacetic acid (TFA) followed by neutralization with NH 4 OH as a disintegrating agent for TDP-43 AFP
- the TFA was diluted in supplemented lysis buffer to obtain 20% solutions.
- the NH 4 OH was diluted in a 450 mM HEPES buffer to obtain a 5N solution.
- the following reagents were dispensed into a 96-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 device (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:
- Example 18 effect of NaOH solutions giving pH between 8.2 and 13.3 followed by neutralization with HCl on the method using the TDP-43 N AFP detection reagents
- 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 device (BMG Labtech) using an HTRF detection module.
- Figure 23 shows the results obtained with the different processing modes. Treatment with an NaOH solution resulting in a pH of 13.3 followed by neutralization with HCl does not make it possible to detect TDP-43 NARb. The disintegration power of all other solutions could be tested on TDP-43 ARb.
- Example 19 determination of the minimum and maximum pH to disaggregate TDP-43 AFP with NaOH [0272] NaOH solutions tested
- 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 device (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 (Signal sample treated with NaOH then HCl) / (Signal sample treated with NaOH / HCl mixture).
- FIG. 24 shows the results obtained as a function of the pH induced by the various NaOH solutions tested. Detection of TDP-43 N AFP is possible when the pH ranges from 8.5 to 13.2 during the disaggregation step. However, the pH making it possible to have an optimal amplitude of detection of TDP-43 NAFP is approximately pH 12.8.
- Example 20 determination of the time necessary to disintegrate TDP-43 AFP at a pH of 12.8
- the following reagents were distributed in a 96-well plate in the following order: 1) 60 ⁇ L of a sample of TDP-43 AF prepared according to the protocol described in Example 2 2) 10 pL of a 1.2N NaOH solution (pH of the sample brought to 12.8) or of an NaOH / HCl mixture (obtained by melancholy of 1.2N NaOH and a solution of HCl to IN). The mixture was incubated between 30 seconds and 1 hour at room temperature depending on the wells.
- 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 device (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:
- FIG. 25 gives the results obtained as a function of the contact time with 1.2N NaOH.
- the results show that the treatment with 1.2N NaOH allowed TDP-43 AFP to be at least partially disaggregated independently of the contact time (signal ratio always greater than 1. However, the best disaggregation was obtained for contact times. ranging from 5 minutes to 30 minutes.
- Example 21 determination of the minimum and maximum pH during the measurement of the detection of TDP-43 NAFP
- Signal ratio (Signal sample treated with 1.2N NaOH then HCl) / (Signal sample treated with NaOH / HCl mixture).
- Example 22 Measurement of the Aggregation Level of a Beta Amyloid Peptide 1-42 Using the Housing and the HCl as Neutralizing Agent
- the method described below is based on HTRF technology (see Example 1 for principle).
- samples 1-42 NAF and 1-42 AF were thawed and then diluted in 10 mM sodium phosphate buffer pH 7.4 at a concentration of 2.4 ng / mL before distribution into the microplate.
- the detection of the HTRF signal in the different plates was carried out on a PHERAstar FS Lamp device (BMG Labtech) using an HTRF detection module.
- the Aggregation Ratio is calculated as follows from the HTRF signals obtained on the samples:
- Signal ratio (Signal sample treated with 1.2N NaOH then IN HCl) / (Signal sample treated with the NaOH / HCl mixture).
- Figure 27 shows the signal ratios obtained with samples 1-42 NARb and 1-42 ARb.
- the signal ratio obtained on sample 1-42 ARb is much higher than that obtained on sample 1-42 NAFp. This result tells us that the method is able to measure the level of aggregation of
- Example 23 Effect of treatment with NaOH, potassium hydroxide (KOH) or NH 4 OH followed by neutralization with HCl on the detection capacity of a method using a pair of 'antibody capable of binding specifically to TDP-43 NAFP
- 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 device (BMG Labtech) using an HTRF detection module.
- FIG. 28 shows the TDP-NAFP detection signals obtained with the different treatments. Detection of TDP-43 NAFP is possible regardless of the bases used when a neutralization step with HCl making it possible to have a pH between 7 and 8 is carried out. The disaggregating effect of these bases will therefore be tested on TDP-43 AFp.
- Example 24 Effect of treatment with NaOH, with potassium hydroxide (KOH) or with NH 4 OH followed by neutralization with HCl on the disaggregation of TDP-43 AFP
- 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 device (BMG Labtech) using an HTRF detection module.
- FIG. 29 shows the ratio of the signals obtained with the different treatments.
- the two treatment conditions with NaOH (1.2N and 0.8N), giving respectively pHs of 12.8 and 9.6, make it possible to detect TDP43-NAF confirming their disaggregating effect on TDP-43 ARb.
- the 2 NH 4 C ) H treatments (ION and 1.2N) which give pH similar to the NaOH treatments do not allow TOR43-NARb to be detected. This result shows that NH 4 OH has no disaggregating effect of TDP-43 ARb.
- Example 25 Method for identifying pairs of antibodies capable of binding specifically to Alpha-Synuclein NAFP (Alpha-Syn NAFp)
- the method is based on FRET technology (HTRF® technology from Cisbio Bioassays), as detailed in Example 1.
- a pair of antibodies directed against Alpha-Synuclein labeled respectively to the Donor and to the Acceptor is that contained in the Total-Alpha-Synuclein HTRF kit marketed by Cisbio Bioassays (ref. 6FNSYPEG). Each antibody was diluted in the kit diluent as recommended by the user manual.
- Alpha-Syn NARb and Alpha-Syn ARb 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 15.6ng / mL in the lysis buffer of the HTRF Total-A Synuclein kit.
- the ratio of the signals of FRET (Monomer / Aggregate) 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 NARb compared to Alpha-Syn ARb.
- Example 26 effect of a 1.2N NaOH solution followed by neutralization with IN HCl on the detection capacity of a method using a pair of antibodies capable of binding specifically to Alpha-Syn NAFP
- the Alpha-Syn NARb 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 15.6ng / mL.
- the HTRF Total-A Synuclein kit donor and acceptor antibodies were diluted as recommended by the supplier in the kit manual.
- 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 device (BMG Labtech) using an HTRF detection module.
- Figure 31 gives the results obtained. They show that the treatment with 1.2N NaOH followed by neutralization with IN HCl has little effect on the detection of Alpha-Syn NARb by the antibodies. The effect of this treatment to disaggregate Alpha-Syn ARb can therefore be evaluated.
- Example 27 Measurement of the Level of Aggregation of Alpha-Synuclein Using NaOH as Disaggregating Agent and HCl as Neutralizing Agent
- Alpha-Syn NAF and Alpha-Syn AF 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.
- the detection of the FRET signal in the different plates was carried out on a PHERAstar FS Lamp device (BMG Labtech) using an HTRF detection module.
- Figure 32 shows the signal ratios obtained with the samples of Alpha-Syn NAF and Alpha-Syn ARb.
- the signal ratio obtained on the Alpha-Syn AF sample is much higher than that obtained on the Alpha-Syn NARb sample.
- This result indicates to us that the method according to the invention makes it possible to measure the level of aggregation of alpha-synuclein.
- rpiq._ll Figure 1 illustrates the principle qe
- the comparison of the RNARb (Signal Ratio) contents therefore makes it possible to know whether the sample tested includes a RARb.
- the signal ratio for a sample not including RARb will be equal to 1.
- the signal ratio for a sample including RARb will be greater than 1.
- FIG. 2 represents the ELISA signal (D.O.) obtained for different dilutions of RNARb.
- O.D. ELISA signal obtained for different dilutions of RNARb.
- the value of O.D. corresponding to 80% of the maximum signal makes it possible to determine the reference dilution to be used in the remainder of the experiment.
- FIG. 3 represents the results obtained in an ELISA test aimed at determining whether a pair of antibodies selectively recognizes a RNARb or not.
- FIG. 4 shows the principle of an HTRF immunoassay.
- This type of immunoassay makes it possible to know whether two antibodies, respectively labeled with the Donor and with the Acceptor, are capable of creating a pair of antibodies capable of simultaneously recognizing an antigen of interest.
- A) the 2 antibodies tested are not capable of creating a pair of antibodies capable of recognizing the antigen of interest; there is no energy transfer between the Donor and the Acceptor and therefore no fluorescence signal emitted by the Acceptor.
- B) The two antibodies tested are capable of simultaneously recognizing the antigen of interest. An energy transfer then takes place between the Donor and the Acceptor. This results in a specific fluorescence emission at 665 nm which is emitted by the acceptor.
- FIG. 5 represents the HTRF signal obtained for different dilutions of RNARb.
- the HTRF signal value corresponding to 80% of the maximum signal makes it possible to determine the reference dilution to be used in the remainder of the experiment.
- FIG. 6 represents the results obtained in an HTRF test aimed at determining whether a pair of antibodies selectively recognizes an RNARb or not.
- Figure 7 shows the calculation of the signal ratio between a TDP-43 ARb aggregate sample and a TDP-43 NARb monomer sample obtained with a pair of antibodies directed against the TDP-43 protein. A signal ratio greater than 2 indicates that the tested pair is selective for TDP-43 NARb.
- FIG. 8 represents the calculation of the signal ratio between an aggregate sample of amyloid peptide beta 1-40 and a monomer sample of this same peptide obtained with a pair of antibodies directed against the amyloid peptide beta 1-40. The value of the signal ratio greater than 2 indicates that the tested pair is selective for the amyloid peptide 1-40 NARb (monomeric form).
- Figure 9 shows the calculation of the signal ratio between an aggregate sample of amyloid peptide beta 1-4e same peptide obtained with a pair of antibodies directed against the amyloid peptide beta 1-42. The value of the signal ratio greater than 2 indicates that the tested pair is selective for the amyloid peptide 1-42 NAF (monomeric form).
- Figure 10 shows the effect of Hexafluoroisopropanol (HFIP) on the detection capacity of an HTRF method using a pair of antibodies capable of specifically binding TDP-43 NARb.
- HFIP Hexafluoroisopropanol
- Figure 11 shows the lack of disaggregating effect of Hexafluoroisopropanol (HFIP) on a sample of TDP-43 ARb.
- HFIP Hexafluoroisopropanol
- Figure 12 shows the effect of Urea on the detection capacity of an HTRF method using a pair of antibodies capable of specifically binding TDP-43 NARb.
- Figure 13 shows the absence of urea disaggregating effect on a sample of TDP-43 ARb.
- Figure 14 shows the effect of Guanidinium Chloride on the detection capacity of an HTRF method using a pair of antibodies capable of specifically binding TDP-43 NARb.
- Figure 15 shows the effect of Formic Acid (A) or TFA (B) on the detection capacity of an HTRF method using a pair of antibodies capable of specifically binding TDP-43 NARb.
- Figure 16 shows the effect of Formic Acid followed by NaOH neutralization on the detection capacity of an HTRF method using a pair of antibodies capable of specifically binding TDP-43 NARb.
- FIG. 17 shows the effect of TFA followed by neutralization with NaOH on the detection capacity of an HTRF method using a pair of antibodies capable of specifically binding TDP-43 NARb.
- FIG. 18 shows the absence of a disaggregating effect of TFA followed by neutralization with NaOH on a sample of TDP-43 ARb.
- Figure 19 shows the effect of Formic Acid followed by NH 4 OH neutralization on the detection capacity of an HTRF method using a pair of antibodies capable of specifically binding TDP-43 NARb.
- FIG. 20 represents the absence of a disaggregating effect of Formic Acid followed by neutralization with NH 4 OH on a sample of TDP-43 ARb.
- Figure 21 shows the effect of TFA followed by NH 4 OH neutralization on the detection capacity of a ps method capable of specifically binding TDP-43 NARb.
- FIG. 22 represents the absence of a disaggregating effect of TFA followed by neutralization with NH 4 OH on a sample of TDP-43 ARb.
- Figure 23 represents the effect of NaOH solutions giving pH between 8.2 and 13.3 followed by neutralization with HCl on the detection capacity of an HTRF method using a pair of antibodies capable of binding specifically to the. TDP- 43 NARb.
- Figure 24 shows the determination of the minimum and maximum pH for disaggregating a sample of TDP-43 ARb with NaOH solutions.
- Figure 26 shows the determination of the minimum and maximum pH when measuring the detection of TDP-43 NARb.
- Figure 27 shows the measurement of the level of aggregation of a beta amyloid peptide 1-42 using NaOH as a disintegrating agent followed by neutralization at mci.
- Figure 28 shows the effect of solutions of NaOH, KOH and NH 4 OH followed by neutralization with HCl on the detection capacity of an HTRF method using a pair of antibodies capable of specifically binding to the HCl. TDP-43 NARb.
- Figure 29 shows the effect of solutions of NaOH, KOH and NH 4 OH followed by neutralization with HCl on the disaggregation of TDP-43 NARb.
- Figure 30 represents the calculation of the signal ratio between an aggregate sample of Alpha-Synuclein (Alpha-Syn ARb) and a monomeric sample of this same protein (Alpha-Syn NARb) obtained with a pair of antibodies directed against Alpha-Synuclein. The value of the signal ratio greater than 2 indicates that the tested pair is selective for Alpha-Syn NARb.
- Figure 31 shows the effect of an NaOH solution followed by neutralization with HCl on the detection capacity of an HTRF method using a pair of antibodies capable of specifically binding to Alpha- Syn NARb.
- Figure 32 shows the measurement of the level of alpha-synuclein aggregation using NaOH as a disintegrating agent followed by neutralization with HCl.
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