DETERMINATION OF DISEASE-SPECIFIC PROTEIN AGGREGATES IN STOOL SAMPLES

Abstract

The invention relates to methods for selective quantification of A-beta or alpha-synuclein aggregates comprising the immobiliZation of anti-A-beta or alpha-synuclein antibodies on a substrate, application of the stool sample to be tested to the substrate, addition of probes labelled for detection which by specific binding to A-beta or alpha-synuclein aggregates mark these and detection of the marked aggregates.

Claims

1.-16. (canceled)

17. A method for the selective quantification and/or characterization of A-beta or alpha-synuclein aggregates in a stool sample, wherein the method comprises: (a) providing a substrate on which capture molecules are immobilized, (b) applying a stool sample to be tested to the substrate, (c) adding probes labelled for detection, which by specific binding to A-beta or alpha-synuclein aggregates mark these, and (d) detection of the marked aggregates by high spatial resolution in which each event is determined in front of the respective background.

18. The method of claim 17, wherein a pre-treatment of the stool sample is carried out.

19. The method of claim 17, wherein the stool sample is pre-treated by homogenization and separation of solid components from liquid components of the stool sample with addition of water or one or more aqueous buffer solutions.

20. The method of claim 17, wherein a substrate of glass is used.

21. The method of claim 20, wherein the substrate of glass has a hydrophilic coating of or based on dextran.

22. The method of claim 17, wherein the capture molecules are covalently bonded to the substrate or to the coating.

23. The method of claim 22, wherein the capture molecules are labelled with one or more fluorescent dyes.

24. The method of claim 17, wherein the capture molecules are anti-A-beta or alpha-synuclein antibodies.

25. The method of claim 24, wherein the capture molecules specifically bind an epitope of an A-beta or alpha-synuclein aggregate.

26. The method of claim 17, wherein A-beta or alpha-synuclein peptide-specific probes are used.

27. The method of claim 17, wherein the probes are fluorescent dye-labelled anti-A-beta or alpha-synuclein antibodies.

28. The method of claim 17, wherein two or more different probes are used.

29. The method of claim 17, wherein two or more probes labelled with different fluorescent dyes are used.

30. The method of claim 17, wherein at least one probe is an anti-A-beta or alpha-synuclein antibody which specifically binds to the N-terminal epitope of the A-beta peptide or to alpha-synuclein.

31. The method of claim 17, wherein the detection is carried out by spatially resolving fluorescence microscopy.

32. The method of claim 31, wherein detection is carried out by one of confocal fluorescence microscopy, fluorescence correlation spectroscopy (FCS), optionally in combination with cross correlation and single particle immunosolvent laser scanning assay, laser scanning microscopy (LSM) or TIRF microscopy, as well as a corresponding super-resolution variant STED, SIM, STORM, dSTORM.

33. The method of claim 17, wherein an internal or external standard is used for the quantification of A-beta or alpha-synuclein aggregates.

34. The method of claim 33, wherein the standard for quantification of A-beta or alpha-synuclein aggregates is a polymer composed of polypeptide sequences which with respect to their sequence are identical to that subregion of the endogenous proteins which forms an epitope or have a homology of at least 50% over the corresponding subregion with those endogenous proteins which cause a protein aggregation disease or an amyloid degeneration or protein misfolding disease, and wherein the polymers do not aggregate and the polypeptide sequence has a biological activity of the epitope.

35. A method for determining the efficacy of active ingredients and/or curative methods for the treatment of Alzheimer's disease or Parkinson's disease, wherein the method comprises carrying out the method of claim 17 and comparing the active ingredients and/or curative methods with respect to their effect on A-beta or alpha-synuclein aggregate formation, those active ingredients and/or curative methods which show lower A-beta or alpha-synuclein aggregate formation compared to a control being selected.

36. A method for making a decision regarding the inclusion of an individual in a clinical trial or test, wherein the method comprises carrying out a quantification and/or characterization of A-beta aggregates according to the method of claim 17 and comparing a measured value with a threshold value.

Description

DESCRIPTION OF THE FIGURES

[0178] FIG. 1 shows diagrammatically the results of the measurements on or examinations of stool samples from three Alzheimer patients on the one hand and five control patients on the other hand as described in Example 9a).

[0179] FIG. 2 shows diagrammatically the results of the measurements on or examinations of stool samples from four Parkinson patients on the one hand and five control patients on the other hand as described in Example 9b).

EXAMPLES

[0180] 1. Preparation of Substrate for Covalent Bonding

[0181] The amination of the glass surface was carried out with APTES and toluene. For this, 250 μl APTES were dissolved in 5 ml toluene in a crystallizing dish and placed exactly in the middle of the desiccator. A glass plate was then removed from the packaging and placed upside down in the desiccator. After drawing vacuum for approx. 10 minutes, the desiccator was flooded with argon via a balloon. After an incubation period of 1 hour, the crystallizing dish was removed and the plate was dried with the vacuum pump attached for 2 hours. The preparation after amination was not paused because the amination is not stable.

[0182] For the covalent coating with dextran, the glass surface was aminated in advance as described above. One mg/ml CMD was activated with 200 mM EDC and 50 mM NHS in 0.1 M MES pH 3.5 and added to the glass plate and incubated for 30 minutes at room temperature. Subsequent washing with water five times removed unbound dextran.

[0183] For the covalent coating with PEG, the glass surface was aminated in advance as described above. Bifunctional PEG (MW 3400 Da, NHS/COOH) was used as spacer. A 4 mM PEG solution was prepared for this purpose. First, 20 μl of PBS was added to each well and then 20 μl of the prepared PEG solution was added accordingly. After an incubation period of 1 hour at room temperature, the wells were washed five times with water. For quenching, a 10 mM ethanolamine solution was prepared and 20 μl was pipetted into each well. After a 15 minute incubation period, the wells were washed five times with water. Activation of the carboxylic acid group was performed using 20 μl of a solution consisting of 200 mM EDC and 50 mM NHS in 0.1 M MES. Following the 30 minute incubation, the wells were washed five times with water.

[0184] 1a. No Pretreatment is Necessary for Non-Covalent Coating of the Glass Surface with Capture Molecule.

[0185] 2. Immobilization of Antibodies as Capture Molecules on the Coated Substrate (Covalent Binding).

[0186] The solution of antibodies was added to the wells and incubated for 1 hour at room temperature. By the activation of the carboxylic acid groups of PEG and dextran as described above, the amine groups of the capture molecules were linked to PEG or dextran and thus covalently bound to the glass surface. To quench the unoccupied activated carboxylic acid groups, a 10 mM ethanolamine solution in water was used and incubated in the wells for 15 minutes.

[0187] For non-covalent binding, the capture antibody was dissolved in a suitable buffer, in this case PBS or carbonate. The concentration of 2.5 μg/ml of capture antibody used depended on the biomarker and was determined in advance by various titration experiments. Incubation was overnight at 4° C.

[0188] After the capture antibody incubation, unbound antibodies were removed by washing five times with TBS and 0.05% Tween® 20 (non-ionic surfactant; a polyethylene glycol sorbitan fatty acid ester) and washing five times with TBS.

[0189] 3. Blocking

[0190] For both covalent as well as non-covalent binding, a blocking step occurred after incubation with the capture antibody. For this, BSA, casein or milk powder were dissolved in TBS at a concentration between 0.5-5.0% and incubated for 90 minutes at room temperature in the wells.

[0191] Alternatively, antibacterial and antifungal substances such as ProClin 300 or sodium azide can be added to the blocking solution. Subsequently optionally another washing step (washing five times with TBS and 0.05% Tween 20 and washing five times with TBS) can be done.

[0192] 4. Immobilization of A-Beta or Alpha-Synuclein Aggregates on the Pre-Treated Substrate

[0193] The samples to be measured were incubated on the substrate for 1 hour at room temperature (20° C.) or 37° C. and then washed five times with TBS.

[0194] 5. Linking the Probes with Fluorescent Dye to Label them

[0195] The detection probes, here Nab228 (anti-A-beta 1-11) or 211 (anti-alpha-synuclein 121-125) or EP1536Y (anti-phospho-alpha-synuclein (S129)) were each labelled with CF dyes, here CF633 succinimidyl ester and CF488A succinimidyl ester. The purification was carried out by size exclusion chromatography as known to the person skilled in the art.

[0196] 6. Marking of the Aggregates with the Probes

[0197] The amount of antibody used depends on the desired degree of marking and a high signal-to-noise ratio, which was titrated in advance within the assay development. The probes were added and incubated for 1 hour at room temperature in the dark. Unbound probes were removed by washing five times with TBS.

[0198] 7. Detection of the Aggregates and Measurement of the Samples.

[0199] The measurement was performed here with a Celldiscoverer 7 (Carl Zeiss, Jena, Germany) equipped with a confocal laser scanning microscope or a TIRF microscope (Leica, Wetzlar, Germany). The fluorescence intensity of an area of 1000×1000 pixels (TIRF) or 2752×2208 or 512×512 pixels (Celldiscoverer 7) was determined. Since different probes were used, a colocalization analysis was performed (test where measured values of both probes occurred at the same location (in this case a radius of approx. 10 nm); e.g. a red emitting fluorophore and a green emitting fluorophore can be observed). In order to obtain representative values, several areas of the glass surface (4 wells) were measured. The measurement was performed using ZEN software from Carl Zeiss or LAS AF software from Leica.

[0200] 8. Preparation of Stool Samples

[0201] The stool samples can undergo different preparation steps known to the person skilled in the art.

[0202] Here, the sample was homogenized in a first buffer of Tris-buffered saline (TBS) with ProClin 300 and BSA and then centrifuged, and the supernatant was subsequently diluted in a second buffer of TBS or PBS with BSA, detergent and sodium azide.

[0203] 9. Analysis of Stool Samples

[0204] Two studies were carried out; one in relation to Alzheimer's patients and one in relation to Parkinson's patients. [0205] a) Eight stool samples from different patients were analyzed with the method according to the invention. The stool samples were each from three Alzheimer's patients and five control patients (healthy with regard to protein aggregation diseases, different ages). The results are summarized in Table 1. Also in FIG. 1.

TABLE-US-00001 TABLE 1 sFIDA measurements sample average of measurements standard deviation P1 136082.9 88274.6 P2 11263.6 6799.7 P3 39574.7 20329.0 K1 995.1 289.0 K2 578.3 404.5 K3 655.1 171.0 K4 1221.8 686.4 K5 527.8 253.5 [0206] b) Nine stool samples from different patients were analyzed with the method according to the invention. The stool samples were each from four Parkinson's patients and five control patients (healthy with respect to protein aggregation diseases, different ages). The results are summarized in Table 2. Also in FIG. 2.

TABLE-US-00002 TABLE 2 sFIDA measurements sample average of measurements standard deviation P1 26470.6 13028.8 P2 742.6 891.7 P3 24470.5 4188.5 P4 1245.8 400.4 K1 44.9 21.8 K2 118.1 114.0 K3 20.1 4.3 K4 16.2 1.5 K5 43.1 8.9

[0207] The results prove that a clear distinction between the groups is possible. The average of alpha-synuclein oligomers in the Alzheimer's or Parkinson's group each was significantly higher than in the respective control groups. It was therefore possible to draw conclusions about the health status of the patients from the test results of the stool samples.