Method for detecting indicators for determining diseases

Abstract

The invention concerns a method for detecting indicators for determining diseases (disease indicators), in which aggregates of misfolded proteins play a role, and a method for selective quantitation and/or characterization of these disease indicators.

Claims

1. A method for the qualitative and/or quantitative determination of disease indicators in which a sample is tested for at least two aggregate types of endogenous misfolded proteins, wherein the sample is tested for at least two different aggregate types on the same substrate without further processing and/or treatment and the method comprises: (a) application of the sample to be tested to a substrate, (b) addition of probes labeled for detection with fluorescent dyes, which probes label a respective aggregate by specifically binding to it, and (c) detection of labeled aggregates by surface-based Fluorescence Intensity Distribution Analysis (sFIDA), carried out by a method with a temporally and spatially resolved signal and with high spatial resolution wherein a pixel is determined against a respective background, and wherein (b) can be carried out before (a); the disease is selected from tauopathies, AA (amyloid A) amyloidosis, AL (amyloid light chain) amyloidosis, AApoAI (amyloid apolipoprotein AI) amyloidosis, (amyloid apolipoprotein AII) AApoAII amyloidosis, ATTR (amyloid transthyretin) amyloidosis, schizophrenia and other DISC1 (disrupted in schizophrenia 1) opathies, amyotrophic lateral sclerosis, frontotemporal lobar degeneration and other FUS (fused in sarcoma) proteinopathies, diabetes mellitus type 2, Parkinson's disease and other synucleinopathies, chronic traumatic encephalopathy and other TDP-43 (transactive response DNA binding protein 43 kDa) proteinopathies, Huntington's disease, familial visceral amyloidosis and/or Alzheimer's dementia; and the aggregate type of endogenous misfolded proteins is selected from tau aggregates, serum amyloid A protein aggregates, IgG (immunoglobulin G) light chain aggregates, AapoAI aggregates, AapoAll aggregates, ATTR aggregates, DISC1 aggregates, FUS aggregates, IAPP (islet amyloid polypeptide) aggregates, SOD1 (superoxide dismutase 1) aggregates, α-synuclein aggregates, TDP-43 aggregates, huntingtin aggregates, lysozyme aggregates, Aβ aggregates, and mixed aggregates.

2. The method of claim 1, wherein prior to (a) capture molecules are immobilized on the substrate.

3. The method of claim 1, wherein capture molecules immobilized on the substrate and/or the probes comprise specific antibodies to an epitope of the proteins which form the aggregates.

4. The method of claim 1, wherein the sample is tested for at least three aggregate types of endogenous misfolded proteins.

5. The method of claim 1, wherein the method is a method for a differential diagnosis of a disease selected from tauopathies, AA amyloidosis, AL amyloidosis, AApoAI amyloidosis, AApoAII amyloidosis, ATTR amyloidosis, schizophrenia and other DISC1opathies, amyotrophic lateral sclerosis, frontotemporal lobar degeneration and other FUS proteinopathies, diabetes mellitus type 2, Parkinson's disease and other synucleinopathies, chronic traumatic encephalopathy and other TDP-43 proteinopathies, Huntington's disease, familial visceral amyloidosis and/or Alzheimer's dementia vs. another disease selected from the above-mentioned diseases and comprises: (i) quantitative determination of disease indicators according to claim 1; (ii) comparison of obtained data with standard values; (iii) detection of a significantly different quantity of disease indicators as discrepancy in the comparison; and (iv) attribution of the discrepancy to a disease selected from the above-mentioned diseases.

6. The method of claim 1, wherein the method is a method for a differential diagnosis and wherein after a quantification of a disease indicator for a disease selected from tauopathies, AA amyloidosis, AL amyloidosis, AApoAI amyloidosis, AApoAll amyloidosis, ATTR amyloidosis, schizophrenia and other DISC1opathies, amyotrophic lateral sclerosis, frontotemporal lobar degeneration and other FUS proteinopathies, diabetes mellitus type 2, Parkinson's disease and other synucleinopathies, chronic traumatic encephalopathy and other TDP-43 proteinopathies, Huntington's disease, familial visceral amyloidosis and/or Alzheimer's dementia, obtained data are compared with standard values, a significantly different quantity of disease indicators is detected as discrepancy in the comparison and the discrepancy is attributed to an above-mentioned disease.

7. The method of claim 1, wherein the aggregates comprise small, freely diffusing oligomers.

8. The method of claim 1, wherein a quantitative determination of disease indicators is carried out, which determination comprises a determination of composition, size and/or shape of aggregates.

9. The method of claim 7, wherein a quantitative determination of disease indicators is carried out, which determination comprises a determination of composition, size and/or shape of aggregates.

10. The method of claim 1, wherein the aggregates comprise one or more peptides or monomers of SEQ ID Nos: 1-9 and 12-14.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings graphically represent results of the sFIDA assays described below under the headings “3. Use with aggregates of recombinant protein”, “4. Use on native samples” and “5. Differential diagnostic use of sFIDA”. In particular,

(2) FIGS. 1A to 1F show sFIDA readouts at various concentrations of different protein aggregates;

(3) FIGS. 2A and 2B show sFIDA readouts of protein aggregates in cerebrospinal fluid samples;

(4) FIG. 3 shows the results of a specificity (cross-reactivity) analysis in an ALys-specific sFIDA assay carried out with recombinant ALys aggregates of other protein aggregates; and

(5) FIG. 4 shows Aβ-specific sFIDA readouts at various concentrations in the presence and absence of tau protein.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE PRESENT INVENTION

Examples

(6) 1. Materials and Methods

(7) In the assay, multiwell plates with a 170 μm thick glass bottom were used as sample carriers (SensoPlate Plus 384 Greiner Bio-One, Kremsmünster, Austria). All reagents and solutions used were obtained with the highest degree of purity and sterilized until particle-free prior to use.

(8) In the first step, each sample chamber (well) was filled with 100 μL sodium hydroxide solution (5 M), incubated for 15 minutes at room temperature, rinsed three times with water, mixed with 100 μL hydrochloric acid and again incubated for 15 minutes at room temperature. After washing thee times with water and twice with ethanol, the wells were dried under a nitrogen atmosphere.

(9) In order to produce amino groups on the glass surface, 20 μL of ethanolamine (5.6 M) was placed in each of the wells and incubated overnight at room temperature. The wells were washed three times with DMSO, twice with ethanol, and dried under a nitrogen atmosphere.

(10) Heterobiofunctional polyethylene glycol (NHS-PEG-COOH, MW 3.400 Da) was dissolved to 50 mM in DMSO at 70° C. for 1 min, cooled to room temperature, and adjusted with 2% triethylamine. 15 μL of this solution was added to each of the wells, and incubation was carried out for at least one hour at room temperature. The solution was removed from the wells, and the wells were washed three times with water.

(11) In order to activate the PEG coating, NHS and EDC (carbodiimide) were diluted to 100 mM each in MES buffer (0.1 M, pH 5, MES: 2-N-morpholinoethanesulfonic acid) and mixed in a 1:1 ratio to final concentrations of 50 mM respectively. 30 μL each of this solution was added to the wells and incubated for 30-60 minutes. After removal of the solution, the wells were washed three times with MES buffer (0.1 M, pH 5).

(12) The capture antibodies were diluted to 30 ng/μL in PBS. 15 μL of this solution was added to the wells and incubated for 1-3 hours at room temperature. The solution was then removed, and washing was carried out three times with PBST (PBS with Tween 20) and then three times with PBS.

(13) 3% BSA was first centrifuged at 100,000 g (1 hour at 4° C.). 50 μL each of the supernatants was added to the wells and incubated for one hour at room temperature. The BSA solution was removed, and washing was carried out three times with PBST.

(14) The sample (e.g. aggregates of recombinant protein and natural patient sample) was—if necessary—diluted in PBS, 15 μL each of this solution was added to the wells. The multiwell plate was centrifuged at 1,000 g for one hour at 4° C. in a swing-out centrifuge. The supernatant was removed and the wells were washed three times with PBST and three times with PBS.

(15) Fluorescence-labeled antibodies were used as detection probes. These were diluted to 1-2 ng/μL in PBS and mixed with 1.5% BSA. The batches were centrifuged at 100,000 g for one hour at 4° C. 15 μL each of the supernatant was placed in the wells and incubated for 1-2 hours at room temperature. The solution was then removed and washing was carried out five times with PBST and five times with PBS.

(16) The surface fluorescence was visualized by total internal reflection fluorescence microscopy (TIRFM). Alternatively, the protein particles can be visualized by confocal laser scanning at the level of the glass surface. Up to 50 individual images per well (1000×1000 pixels, 114 nm/pixel) were taken per fluorescence channel with a high-sensitivity CCD camera.

(17) Background signals in the image data were removed by applying an intensity threshold. The mean number of pixels with grayscale values above the threshold was determined (sFIDA readout). If several detection probes were used, only the events colocalized in all fluorescence channels were evaluated.

(18) 2. sFIDA Protocol

(19) 2.1. Pretreatment 15 min NaOH (5M) 100 μL/well 3×H.sub.2O 15 min HCl (1M) 100 μL/well 3×H.sub.2O rinse 2×EtOH rinse dry with N.sub.2

(20) 2.2. Glass Activation (Amino Groups) 20 μL/well ethanolamine (5.6 M) [x] incubation ON (overnight) room temperature or longer 4° C. 3×DMSO 2×EtOH dry with N.sub.2

(21) 2.3. Spacer NHS-PEG-COOH (briefly) dissolve 17 mg PEG in 100 μL DMSO at 70° C., allow to cool, 2 μL triethylamine (TEA) 15 μL/well [x] incubation: min. 1 h 3×H.sub.2O

(22) 2.4. PEG Activation with NHS/EDC (50 mM) dilute 5.8 mg NHS and 9.6 mg EDC in 500 μL MES (0.1M), directly apply 1:1 mixing 30 μL well [x] incubation: 30 min-max. 1 h quickly 3×MES (0.1 M)

(23) 2.5 Capture dilute capture antibodies in PBS (30 ng/μL) 15 μL well.fwdarw.incubation 1-3 h RT or longer 4° C. 3×PBST, 3×PBS

(24) 2.6. Blocking 3% BSA 1 h at 100,000×g, 4° C. (Rotor TLA-45) 50 μl/well [x] incubation 1 h 3×PBST 3×PBS Day 2

(25) 2.7. Target 15 μL target, diluted in PBS incubation 1 h centr., 1000 g, 25° C., HH: 3×0.2% SDS/PBS; 5×PBST; 5×PBS plasma and recombinant aggregates: 3×PBST; 3×PBS

(26) 2.8. Detection Antibodies 1-2 ng/μL in 1.5% BSA with PBS after 100,000×g, 4° C. (Rotor TLA-45) 15 μL each of supernatant/well [x] incubation>=1-2 h, RT 5×PBST 5×PBS

(27) (100 μL of the corresponding solution each was used for washing steps)

(28) 3. Use with Aggregates of Recombinant Protein

(29) The basic feasibility of the sFIDA assay for detection of different aggregate species was first demonstrated. As examples, aggregates of different proteins (AapoAI, AL, SOD1, ALys, AA, TDP-43) were prepared, and corresponding dilution series were tested in an sFIDA assay. In this case, the same antibodies directed against the specific protein respectively were used as capture and detection antibodies. Each detection probe was labeled with Alexafluor 488. All of the aggregates were detectable in a concentration-dependent manner to the picomolar range by means of sFIDA.

(30) FIG. 1A-F: sFIDA of different protein aggregates. The protein aggregates were diluted in the indicated concentrations in PBS buffer and analyzed by sFIDA assay. PBS buffer was used as a negative control. A) ApoAI, capture and detection probe (AF488-labeled) EPR 1368Y; B) AL, capture and detection probe (AF488-labeled) EPR 5367; C) SOD1, capture and detection probe (AF488-labeled) EPR 1726; D) ALys, capture and detection probe (AF488-labeled) EPR 2994 (2); E) AA, capture and detection probe (AF488-labeled) EPR 4134; F) TDP-43, capture and detection probe (AF488-labeled) EPR 5810.

(31) 4. Use on Native Samples

(32) In order to demonstrate in an illustrative manner that protein aggregates can be detected in blood and cerebrospinal fluid samples (CSF samples) or urine, saliva, mucosa, or biopsy material, protein aggregates (α-synuclein and DISC1) were diluted in CSF and analyzed by sFIDA. For the detection of α-synuclein, anti-αSyn-Aβ 2B2A11 was used as a capture antibody, and fluorescence-labeled anti-αSyn-ABs 3H2897-AF633 or 211-AF488 were used as detection probes. For detection of DISC1 aggregates, anti-DISC1-AK 14F2 was used as a capture antibody and 14F2-AF633 as a detection probe. Both synuclein and DISC1 aggregates can be detected in a concentration-dependent manner to the picomolar range by means of sFIDA (see FIGS. 2A and B).

(33) In order to investigate the molecular basis of schizophrenia, a rat model was developed at the laboratory of Carsten Korth that strongly expresses the protein DISC1. In order to test whether this protein is natively present in aggregates, CSF samples from a transgenic animal were analyzed by sFIDA. Compared to two controls, the transgenic sample showed a clearly elevated titer of DISC1 aggregates (FIG. 2A, samples WT1, WT2, Tg).

(34) FIG. 2: sFIDA detection of protein aggregates in CSF. Aggregates of recombinant protein were diluted in human CSF and tested in an sFIDA assay. A) DISC1 detection. mAβ 14F2 was used as a capture antibody and detection probe (AF633-labeled). In addition to recombinant DISC1 aggregates (10 pg-10 ng) CSF samples from rats (WT1, WT2, Tg) were subjected to sFIDA analysis. B) Synuclein detection by means of 2D sFIDA. Anti-αSyn-Aβ 2B2A11 was used as a capture antibody and fluorescence-labeled anti-αSyn-ABs 3H2897-AF633 or 211-AF488 were used as detection probes. Only the signals colocalized in both fluorescence channels above a threshold values were taken into consideration.

(35) 5. Differential Diagnostic Use of sFIDA

(36) A sample was distributed to different reaction chambers (wells) coated with different capture antibodies. Alternatively, a mixture of different capture antibodies can be immobilized in a single well, or different capture antibodies can be applied using a “spotting” method to different positions within a single surface, e.g. the bottom of a microtiter plate well.

(37) By using different probes, each carrying a different fluorophore, different aggregate species were therefore analyzed almost simultaneously in a single sample aliquot.

(38) It was shown in this example that an ALys-specific sFIDA assay (anti-lysozyme antibody as probe and capture antibody) shows no cross-reactivity with other protein aggregates over a broad concentration range (FIG. 3). Anti-lysozyme antibodies used as probes and capture antibodies interact only with aggregates of the type ALys.

(39) In a further example, it was shown that the presence of other proteins does not adversely effect the detectability of amyloid beta aggregates. For this purpose, a mixture of amyloid-beta aggregates and tau aggregates was subjected to sFIDA analysis.

(40) FIG. 3. Specificity analysis. In an ALys-specific sFIDA assay with capture antibodies, rabbit monoclonal EPR2994(2) and detection probe EPR2994(2)-AF488, as well as a dilution of recombinant ALys aggregates of other protein aggregates (tau, TDP-43, SOD1, AA, AL, synuclein, ApoAI) were tested for cross-reactivity.

(41) FIG. 4: Aβ-specific sFIDA in the presence of tau protein. Protein aggregates of Aβ were diluted in the indicated concentrations and analyzed with and without the presence of 1 μM tau protein aggregate by Aβ-specific sFIDA assay (detection probe 6E10/Atto488, capture antibody Nab228).