Biochemical markers for neurodegenerative conditions

Abstract

Provided herein is a method of bioassay for the quantification of peptide fragments relevant to neurodegenerative conditions. The method comprises cleaving a Tau protein by a secretase, such as ADAM10, to form a neo-epitope. The method comprises contacting a blood derived sample with an antibody specific for the neo-epitope and determining the level of binding of the antibody to peptide fragments comprising the neo-epitope in the sample. Neo-epitope containing peptide levels are found to be inversely correlated to cognitive function.

Claims

1. A method of detecting fragments of Tau protein formed by secretase cleavage of Tau protein in a human patient, said method comprising: a. obtaining a sample from the human patient; and b. detecting whether said fragments of Tau are present in the sample by contacting the sample with an antibody and detecting binding between the fragments and the antibody wherein said antibody is raised against a synthetic peptide corresponding to a C-terminal or N-terminal neo-epitope amino acid sequence formed by cleavage of Tau protein by a secretase and has specific binding affinity for said C-terminal or N-terminal neo-epitope amino acid sequence, and wherein said antibody has specific binding affinity for any of the following sequences at the N terminal of a peptide: TABLE-US-00005 APVPMP SEQ ID NO 137 APVPMP SEQ ID NO 138 ASLAKQ SEQ ID NO 139 ATLADE SEQ ID NO 140 ATRIPA SEQ ID NO 8 DEAAGH SEQ ID NO 141 DRKDQG SEQ ID NO 142 EAAGHV SEQ ID NO 143 EDHAGT SEQ ID NO 144 EGDTDA SEQ ID NO 145 EVMEDH SEQ ID NO 147 EVMEDH SEQ ID NO 148 EVSASL SEQ ID NO 149 GAAPPG SEQ ID NO 150 GEPPKS SEQ ID NO 151 GSPGTP SEQ ID NO 152 GTPGSR SEQ ID NO 153 HGAEIV SEQ ID NO 154 HKPGGG SEQ ID NO 155 IPAKTP SEQ ID NO 156 KAKTDH SEQ ID NO 157 KSPVVS SEQ ID NO 159 KTPPAP SEQ ID NO 160 LAKQGL SEQ ID NO 46 LATLAD SEQ ID NO 161 LKNVKS SEQ ID NO 162 LPTPPT SEQ ID NO 163 MHQDQE SEQ ID NO 164 MHQDQE SEQ ID NO 165 MVDSPQ SEQ ID NO 166 NATRIP SEQ ID NO 167 NIHHKP SEQ ID NO 168 PMPDLK SEQ ID NO 60 PPAPKT SEQ ID NO 170 PPKSGD SEQ ID NO 171 PPTREP SEQ ID NO 172 PVPMPD SEQ ID NO 173 PVPMPD SEQ ID NO 174 QARMVS SEQ ID NO 67 QLATLA SEQ ID NO 175 QTAPVP SEQ ID NO 176 RENAKA SEQ ID NO 177 RIPAKT SEQ ID NO 178 RKDQGG SEQ ID NO 179 RTPSLP SEQ ID NO 181 SASLAK SEQ ID NO 182 SEKLDF SEQ ID NO 88 SGDTSP SEQ ID NO 183 SGEPPK SEQ ID NO 184 SLAKQG SEQ ID NO 185 SPGSPG SEQ ID NO 186 SPRHLS SEQ ID NO 102 SPSSAK SEQ ID NO 188 SPVVSG SEQ ID NO 189 SRSRTP SEQ ID NO 190 SRTPSL SEQ ID NO 191 STENLK SEQ ID NO 110 TAPVPM SEQ ID NO 192 TAPVPM SEQ ID NO 193 TLADEV SEQ ID NO 194 TPPAPK SEQ ID NO 117 TPPKSP SEQ ID NO 195 TPPSSG SEQ ID NO 196 TPRGAA SEQ ID NO 197 TPSLED SEQ ID NO 198 TRIPAK SEQ ID NO 199 TSPRHL SEQ ID NO 200 VPGGGN SEQ ID NO 201 VSASLA SEQ ID NO 202 where M indicates an oxidised methionine or wherein said antibody has specific binding affinity for any of the following sequences at the C terminal of a peptide: TABLE-US-00006 AAGHVT SEQ ID NO 203 AEIVYK SEQ ID NO 204 AKSRLQ SEQ ID NO 205 APPGQK SEQ ID NO 206 ATRIPA SEQ ID NO 8 DLKNVK SEQ ID NO 207 DQGGYT SEQ ID NO 208 DRSGYS SEQ ID NO 209 EPKKVA SEQ ID NO 210 GAEIVY SEQ ID NO 211 GGGQVE SEQ ID NO 212 GGGSVQ SEQ ID NO 213 GKTKIA SEQ ID NO 214 GNKKIE SEQ ID NO 215 GQANAT SEQ ID NO 216 GQVEVK SEQ ID NO 217 GSRSRT SEQ ID NO 218 GTPGSR SEQ ID NO 153 HAGTYG SEQ ID NO 219 INKKLD SEQ ID NO 220 KAKGAD SEQ ID NO 221 KSPSSA SEQ ID NO 223 LAKQGL SEQ ID NO 46 LGDRKD SEQ ID NO 224 LPTPPT SEQ ID NO 163 MPDLKN SEQ ID NO 225 NKKIET SEQ ID NO 226 PGSRSR SEQ ID NO 227 PKSGDR SEQ ID NO 228 PKTPPS SEQ ID NO 229 PMPDLK SEQ ID NO 60 PMPDLK SEQ ID NO 230 PTREPK SEQ ID NO 232 QDQEGD SEQ ID NO 233 QEGDTD SEQ ID NO 234 QKGQAN SEQ ID NO 235 RSRTPS SEQ ID NO 236 SAKSRL SEQ ID NO 237 SASLAK SEQ ID NO 182 SEKLDF SEQ ID NO 88 SGEPPK SEQ ID NO 184 SNVQSK SEQ ID NO 238 SPRHLS SEQ ID NO 102 SPSSAK SEQ ID NO 188 STENLK SEQ ID NO 110 STGSID SEQ ID NO 239 TPGSRS SEQ ID NO 241 TPPAPK SEQ ID NO 117 VDSPQL SEQ ID NO 242 VPMPDL SEQ ID NO 243 VSKSKD SEQ ID NO 244 YTMHQD SEQ ID NO 245 where M indicates an oxidised methionine.

2. The method of claim 1, wherein the neo-epitope is formed by cleavage of a Tau protein by ADAM10 or BASE-1.

3. The method of claim 1, wherein said antibody has specific binding affinity for the following sequence at the N terminal of a peptide: TPRGAAPPGQ (SEQ ID NO 246).

4. The method of claim 1, wherein said antibody is a monoclonal antibody or a fragment of a monoclonal antibody having specific binding affinity.

5. The method of claim 1, wherein said method is conducted as a competition immunoassay in which said antibody and a competition agent are incubated in the presence of said sample and the competition agent competes with the peptide fragments in the sample to bind to the antibody.

6. The method of claim 5, wherein said competition agent is a synthetic peptide or is a purified native peptide formed by cleavage of the protein from which said epitope comes so as to reveal said neo-epitope.

7. The method of claim 6, wherein said competition agent comprises a peptide comprising the N-terminal sequence TPRGAAPPGQ (SEQ ID NO 246).

8. The method of claim 1, wherein the sample is a sample of urine, serum, blood, plasma, or saliva.

Description

(1) The invention will be further explained and illustrated with reference to the accompanying drawings, in which:

(2) FIG. 1 shows and example of a standard curve with either the selection peptide or the elongated peptide at concentrations of 0, 0.782, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 ng/mL.

(3) FIG. 2 shows competitive ELISA test results measured in extracted brains from either control or Tg4510 mice (a model of Alzheimer's disease).

(4) FIGS. 3A-3B show Western blots comparing brain extracts from wt and Tg4510 mice. FIG. 3A) a western blot conducted with an antibody recognizing intact Tau (MAB3420 Chemicon). FIG. 3B) Western blot conducted with in house antibody (NB191).

(5) FIG. 4 shows correlation of the levels of the ADAM10 generated Tau fragment in CSF and serum samples, showing that it can be monitored in both samples.

(6) FIGS. 5A-5B show correlation of the Tau fragment to age, but not BMI.

(7) FIG. 6 shows the ability of the Tau fragment level to follow progression of severe Alzheimer's in women.

(8) FIGS. 7A-7F show results from Example 2 characterising and providing biological validation of the Tau-A assay. FIG. 7A) Standard curves with either the selection peptide or the elongated peptide at concentrations of 0, 0.59, 1.17, 2.34, 4.69, 9.38, 18.75, 37.5, 75, 150, and 300 ng/ml. FIG. 7B) Measurement of Tau-A fragments in in vitro digests of Tau. FIG. 7C) Western blots of extracted tissues, FIG. 7D) ELISA measurement of brain extracts in the presence or absence of ADAM10. FIG. 7E) Tau-A levels in extracted brains from either control or Tg4510mice (a model of Alzheimer's disease) measured using the ELISA. FIG. 7F) Western blots comparing brain extracts from wild type and Tg4510 mice. Left: a western blot conducted with an antibody recognizing intact Tau (MAB3420 Chemicon). Right: Western blot conducted with in-house antibody (NB191).

(9) FIG. 8 shows pathology related changes in Tau-A levels demonstrating an inverse correlation between Tau-A and the Mattis Dementia Rating Scale (MDRS).

(10) The following examples further explain and illustrate the invention.

EXAMPLE 1

(11) In Vitro Cleavage

(12) Recombinant TAU was cleaved either with activated ADAM10 or BACE1. Protease cleavage was performed by mixing 100 μg and 1 μg of enzyme (ADAM10 or BACE1) in secretase buffer (100 mM NaAcetate, pH 4.0) for 3 days. Finally the cleavage was verified by visualization using the SilverXpress® Silver Staining Kit (cat. no. LC6100, Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's instructions.

(13) Peptide Identification

(14) Peptide fragments in the in vitro cleaved samples were identified using matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) and liquid chromatography coupled to electro spray ionization (ESI) tandem mass spectrometry (LC-MS/MS). MALDI-TOF samples were purified using C18 zip-tips (cat. no. ZTC18SO24, Millipore, Billerica, Mass., USA) according to specifications and 0.1 μg of material was eluted onto a MTP 384 ground steel target plate (Bruker-Daltonics, Bremen, Germany). MALDI tandem mass spectra were recorded on a Bruker ultraflex MALDI-TOF/TOF mass spectrometer (Bruker-Daltonics, Bremen, Germany) in positive ion reflector mode. Mass spectra were externally calibrated in the m/z range of 800-4000 using peptides generated by tryptic digestion of bovine β-lactoglobulin. The m/z software “Flexanalysis” (Bruker-Daltonics, Bremen, Germany) was used to analyze spectra. LCMS samples were ultra-filtrated to remove proteins above 10 kDa, the pH was adjusted to 2.0 using formic acid, and a 4 μL sample was analyzed by LC-MS/MS. LC was performed on a nanoACQUITY UPLC BEH C18 column (Waters, Milford, Mass., USA) using a formic acid/acetonitrile gradient. MS and MS/MS were performed on a Synapt High Definition Mass Spectrometry quadruple time of flight MS (QUAD-TOF; Waters, Milford, Mass., USA), with acquisition range of 350-1600 m/z in MS and 50-2000 m/z, in MS/MS. The software “ProteinLynx Global SERVER (PLGS)” (Waters, Milford, Mass., USA) was used to analyze spectra and generate peak lists. To identify peptides, MS and MS/MS data was searched against Tau (FASTA) protein database using the Mascot 2.2 (Matrix Science, Boston, Mass., USA) software with either the MALDI-TOF/TOF or ESI-QUAD-TOF settings.

(15) The following peptide fragments were identified:

(16) M indicates an oxidized methionine

(17) TABLE-US-00004 Tau + BACE 1 Peptide SEQ ID NO 46 LAKQGL SEQ ID NO 91 SLAKQGL SEQ ID NO 6 ASLAKQGL SEQ ID NO 25 EVSASLAK SEQ ID NO 87 SASLAKQGL SEQ ID NO 135 VSASLAKQGL SEQ ID NO 93 SPGSPGTPGSRS SEQ ID NO 26 EVSASLAKQGL SEQ ID NO 34 HVPGGGNKKIE SEQ ID NO 21 EVMEDHAGTYG SEQ ID NO 78 RKDQGGYTMHQD SEQ ID NO 58 NIHHKPGGGQVEVK SEQ ID NO 116 TLADEVSASLAKQGL SEQ ID NO 7 ATLADEVSASLAKQGL SEQ ID NO 131 TSPRHLSNVSSTGSID SEQ ID NO 47 LATLADEVSASLAKQGL SEQ ID NO 15 EAAGHVTQARMVSKSKD SEQ ID NO 68 QLATLADEVSASLAKQGL SEQ ID NO 22 EVMEDHAGTYGLGDRKD SEQ ID NO 23 EVMEDHAGTYGLGDRKD SEQ ID NO 101 SPQLATLADEVSASLAKQGL SEQ ID NO 79 RKDQGGYTMHQDQEGDTD SEQ ID NO 55 MVDSPQLATLADEVSASLAKQGL SEQ ID NO 132 TSPRHLSNVSSTGSIDMVDSPQL SEQ ID NO 129 TPSLEDEAAGHVTQARMVSKSKD SEQ ID NO 16 EAAGHVTQARMVSKSKDGTGSDDKKAKGAD SEQ ID NO 24 EVMEDHAGTYGLGDRKDQGGYTMHQDQEGD SEQ ID NO 48 LKNVKSKIGSTENLKHQPGGGKVQIINKKLD SEQ ID NO 32 HGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQL SEQ ID NO 133 TSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL SEQ ID NO 49 LKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSK Tau + ADAM 10 Peptide SEQ ID NO 117 TPPAPK SEQ ID NO 8 ATRIPA SEQ ID NO 67 QARMVS SEQ ID NO 110 STENLK SEQ ID NO 102 SPRHLS SEQ ID NO 60 PMPDLK SEQ ID NO 88 SEKLDF SEQ ID NO 50 LPTPPTR SEQ ID NO 13 DEAAGHVT SEQ ID NO 36 HVPGGGSVQ SEQ ID NO 3 APVPMPDL SEQ ID NO 99 SPGTPGSRS SEQ ID NO 17 EDHAGTYG SEQ ID NO 65 PVPMPDLK SEQ ID NO 18 EGDTDAGLK SEQ ID NO 66 PVPMPDLK SEQ ID NO 103 SPSSAKSRL SEQ ID NO 111 TAPVPMPDL SEQ ID NO 4 APVPMPDLK SEQ ID NO 5 APVPMPDLK SEQ ID NO 134 VPGGGNKKIE SEQ ID NO 92 SPGSPGTPGSR SEQ ID NO 121 TPPKSPSSAK SEQ ID NO 37 IPAKTPPAPK SEQ ID NO 80 RTPPKSPSSA SEQ ID NO 1 AAPPGQKGQAN SEQ ID NO 14 DRKDQGGYT SEQ ID NO 89 SGDTSPRHLS SEQ ID NO 104 SPSSAKSRLQ SEQ ID NO 83 RTPSLPTPPT SEQ ID NO 69 QTAPVPMPDL SEQ ID NO 112 TAPVPMPDLK SEQ ID NO 126 TPRGAAPPGQK SEQ ID NO 113 TAPVPMPDLK SEQ ID NO 93 SPGSPGTPGSRS SEQ ID NO 27 GAAPPGQKGQAN SEQ ID NO 26 EVSASLAKQGL SEQ ID NO 63 PPTREPKKVA SEQ ID NO 34 HVPGGGNKKIE SEQ ID NO 33 HKPGGGQVEVK SEQ ID NO 62 PPKSGDRSGYS SEQ ID NO 107 SRTPSLPTPPT SEQ ID NO 73 RIPAKTPPAPK SEQ ID NO 114 TAPVPMPDLKN SEQ ID NO 70 QTAPVPMPDLK SEQ ID NO 2 AAPPGQKGQANAT SEQ ID NO 71 QTAPVPMPDLK SEQ ID NO 84 RTPSLPTPPTR SEQ ID NO 35 HVPGGGNKKIET SEQ ID NO 94 SPGSPGTPGSRSR SEQ ID NO 28 GAAPPGQKGQANAT SEQ ID NO 57 NIHHKPGGGQVE SEQ ID NO 29 GEPPKSGDRSGYS SEQ ID NO 95 SPGSPGTPGSRSRT SEQ ID NO 9 ATRIPAKTPPAPK SEQ ID NO 122 TPPKSPSSAKSRL SEQ ID NO 115 TAPVPMPDLKNVK SEQ ID NO 124 TPPSSGEPPKSGDR SEQ ID NO 90 SGEPPKSGDRSGYS SEQ ID NO 51 LPTPPTREPKKVA SEQ ID NO 105 SPVVSGDTSPRHLS SEQ ID NO 59 PGSPGTPGSRSRTPS SEQ ID NO 127 TPRGAAPPGQKGQAN SEQ ID NO 40 KAKTDHGAEIVYK SEQ ID NO 123 TPPKSPSSAKSRLQ SEQ ID NO 58 NIHHKPGGGQVEVK SEQ ID NO 81 RTPPKSPSSAKSRL SEQ ID NO 96 SPGSPGTPGSRSRTPS SEQ ID NO 53 MHQDQEGDTDAGLK SEQ ID NO 106 SRSRTPSLPTPPTR SEQ ID NO 74 RIPAKTPPAPKTPPS SEQ ID NO 54 MHQDQEGDTDAGLK SEQ ID NO 42 KSPVVSGDTSPRHLS SEQ ID NO 7 ATLADEVSASLAKQGL SEQ ID NO 85 RTPSLPTPPTREPK SEQ ID NO 118 TPPAPKTPPSSGEPPK SEQ ID NO 64 PSSGEPPKSGDRSGYS SEQ ID NO 128 TPRGAAPPGQKGQANAT SEQ ID NO 52 LPTPPTREPKKVAVV SEQ ID NO 82 RTPPKSPSSAKSRLQ SEQ ID NO 19 ENAKAKTDHGAEIVY SEQ ID NO 108 SRTPSLPTPPTREPK SEQ ID NO 43 KTPPAPKTPPSSGEPPK SEQ ID NO 20 ENAKAKTDHGAEIVYK SEQ ID NO 125 TPPSSGEPPKSGDRSGYS SEQ ID NO 31 GTPGSRSRTPSLPTPPTR SEQ ID NO 86 RTPSLPTPPTREPKKVA SEQ ID NO 72 RENAKAKTDHGAEIVYK SEQ ID NO 30 GSPGTPGSRSRTPSLPTPPT SEQ ID NO 109 SRTPSLPTPPTREPKKVA SEQ ID NO 119 TPPAPKTPPSSGEPPKSGDR SEQ ID NO 44 KTPPAPKTPPSSGEPPKSGDR SEQ ID NO 97 SPGSPGTPGSRSRTPSLPTPPT SEQ ID NO 75 RIPAKTPPAPKTPPSSGEPPK SEQ ID NO 98 SPGSPGTPGSRSRTPSLPTPPTR SEQ ID NO 61 PPAPKTPPSSGEPPKSGDRSGYS SEQ ID NO 41 KSKDGTGSDDKKAKGADGKTKIA SEQ ID NO 10 ATRIPAKTPPAPKTPPSSGEPPK SEQ ID NO 120 TPPAPKTPPSSGEPPKSGDRSGYS SEQ ID NO 38 IPAKTPPAPKTPPSSGEPPKSGDR SEQ ID NO 45 KTPPAPKTPPSSGEPPKSGDRSGYS SEQ ID NO 76 RIPAKTPPAPKTPPSSGEPPKSGDR SEQ ID NO 100 SPGTPGSRSRTPSLPTPPTREPKKVA SEQ ID NO 11 ATRIPAKTPPAPKTPPSSGEPPKSGDR SEQ ID NO 39 IPAKTPPAPKTPPSSGEPPKSGDRSGYS SEQ ID NO 77 RIPAKTPPAPKTPPSSGEPPKSGDRSGYS SEQ ID NO 130 TRIPAKTPPAPKTPPSSGEPPKSGDRSGYS SEQ ID NO 12 ATRIPAKTPPAPKTPPSSGEPPKSGDRSGYS SEQ ID NO 56 NATRIPAKTPPAPKTPPSSGEPPKSGDRSGYS

(18) Selection of Peptide for Immunizations

(19) The first six amino acids of each free end of the sequences identified by MS were regarded as neo-epitopes generated by the protease in question. All obtained protease-generated sequences were analyzed for homology and distance to other cleavage sites and then blasted for homology using the NPS@: network protein sequence analysis.

(20) Reagents and Peptides

(21) All reagents were standard high-quality chemicals from companies such as Merck and Sigma Aldrich. The synthetic peptides used for monoclonal antibody production and validation were: (a) immunogenic peptide: TPRGAAPPGQ-GGC-KLH (SEQ ID NO 248-KLH) (Keyhole-Limpet-Hemocyanin), (b) screening peptide TPRGAAPPGQ (SEQ ID NO 246), (c) de-selection peptide ATPRGAAPPGQ (SEQ ID NO 247) which has been elongated with one amino acid in the N-terminus were purchased from Chinese Peptide Company, Beijing, China. Peptide conjugation reagents were produced by Pierce (Thermofisher, Denmark).

(22) Buffers Used for the ELISAs

(23) Buffer used for dissolving the coating peptide was composed of the following: (150 mM Trizma, 1% BSA, 0.05% Tween-20, 0.36% Bronidox L5, pH 8.0 (Tris-BTB), and reaction stopping buffer composed of 0.1% H.sub.2SO.sub.4.

(24) ELISA-plates used for the assay development were Streptavidin-coated from Roche cat.: 11940279. All ELISA plates were analysed with the ELISA reader from Molecular Devices, SpectraMax M, (CA, USA).

(25) Development of an ELISA

(26) Methods for monoclonal antibody development are previously described ([6]. Briefly, 4-6-week-old Balb/C mice were immunized subcutaneously with 200 μl emulsified antigen and 50 μg TPRGAAPPGQ-GGC-KLH (SEQ ID NO 248-KLH). Consecutive immunizations were performed at 2-week intervals in Freund's incomplete adjuvant, until stable sera titre levels were reached, and the mice were bled from the 2nd immunization on. At each bleeding, the serum titre was detected and the mouse with highest antiserum titre was selected for fusion. The selected mouse was rested for 1 month followed by intravenous boosting with 50 ng TPRGAAPPGQ-GGC-KLH (SEQ ID NO 248-KLH) in 100 μl 0.9% Sodium Chloride solution 3 days before isolation of the spleen for cell fusion.

(27) Fusion

(28) The fusion procedure previously described [16] was followed with SP2/0 as myeloma cells. The fusion cells were cloned in 35-mm cell culture dishes by the semi-solid medium method and the dishes were incubated in a CO.sub.2-incubator. Next, clones were plated into sixteen 96-well microtiter plates and left for three days, followed by screening of culture supernatants.

(29) Antibody Screening

(30) Supernatants were screened in a competitive ELISA setting. Peptide TPRGAAPPGQ (SEQ ID NO 246) was used as the selection peptide and the ATPRGAAPPGQ (SEQ ID NO 247) as the elongated peptide. Cell lines specific to selection peptide and without cross-reactivity to the elongated peptide were selected and the antibodies were purified.

(31) Tau-A ELISA Methodology

(32) In preliminary experiments, we optimized the reagents, their concentrations and the incubation periods by performing several checkerboard analyses. The Tau-A ELISA was developed as follows: A 96-well ELISA plate pre-coated with streptavidin was further coated with 6 ng/ml of the synthetic peptide TPRGAAPPGQ-Biotin (SEQ ID NO 246-Biotin) dissolved in Tris-BTB buffer at 20° C. for 30 min by constant shaking at 300 rpm. The plate was washed five times in washing buffer and 20 μl of sample was added, followed by 100 μl of peroxidase conjugated anti-human mAb-Tau-A solution (50 ng/ml). The plate was incubated for 1 h at 20° C. in 100 mM Tris-BTB buffer during which time it was shaken at 300 rpm.

(33) The plate was again washed five times followed by addition of 100 μl tetramethylbenzinidine (TMB) (Kem-En-Tec cat. 438OH). The plate was incubated for 15 min in darkness and shaken at 300 rpm. In order to cease the reaction, 100 μl of stopping solution (95-97% H2SO4, Merck Cat. No.: 1.00731) was added and the plate was analysed in the ELISA reader at 450 nm with 650 nm as the reference.

(34) Standards

(35) A standard curve was performed by serial dilution of TPRGAAPPGQ-biotin (SEQ ID NO 246-biotin). Standard concentrations were 0, 0.782, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 ng/ml.

(36) Samples for Testing Native Reactivity of the Antibodies

(37) During assay development and validation serum from healthy adult subjects of different age and gender were used. Serum samples are obtained from young healthy volunteers 23-45 years of age. We also tested serum samples from different species including mouse and rat to determine the level of interspecies cross reactivity. Finally, we also tested cerebrospinal Fluid (CSF) and plasma samples.

(38) Animal Samples

(39) Flash frozen brains from 5 wildtype and 5 Tg4510 Tauopathy mice aged 5 months [9] were extracted according to the following protocol:

(40) The tissue was pulverized using a Bessman pulverizer and weighed. The tissue was extracted using 250 mg tissue/mL extraction buffer (50 mM Tris-HCl, 50 mM HEPES, 15% glycerol, 1 mM EDTA, 0.5% sodium deoxycholate, Roche protease inhibitor (cat#05 056 489001), final pH8.3). The lysate was cleared by sonication, and the supernatants were collected after centrifugation at 4° C./5 min/10000 rpm. Protein concentration was determined using the DC Protein Assay from BioRad.

(41) Western Blotting

(42) 100 μg of each extract was loaded onto an SDS-PAGE gel. The gel was run and the samples were transferred to nitrocellulose membranes as described by [23]. The levels of Tau-A fragments and total Tau protein were detected by incubation with the primary antibodies diluted to 100 ng/mL in TBS-T containing skim milk powder [22]. A secondary antibody recognizing mouse IgG conjugated to horse-radish peroxidase was then used, and finally the blot was visualized using enhanced chemiluminescence as previously described [21].

(43) Human Samples

(44) Two sets of human samples were used. One set (51 samples) was a collection of serum samples from an osteoarthritis study, this study contained participants with ages from 18-75, as well as Body Mass Index data [7].

(45) The second set of samples was paired serum and CSF samples from patients with severe Alzheimer's disease. These samples contained a subset of samples collected within the same individuals at baseline and follow-up (18 months later).

(46) Statistical Analysis

(47) For assay validation, optical density was fitted against analyte concentration applying a four-parameter logistic regression to the calibration curve. Average, standard deviations, percentage coefficient of variation (% CV), and differences from theoretical values were calculated for all standards and samples. Quantitative data were analysed using GraphPad Prism 5 (GraphPad Software, San Diego, Calif., USA). Significant differences between means were determined using the Student's two-tailed unpaired t-test, not assuming Gaussian distribution. Correlations between serum Tau values and the rest of the variables studied were analysed using Linear Regression. Data was expressed as mean±standard error of the mean and differences were considered significant at a p level of 0.05 or lower.

(48) Results

(49) ELISA Technical Specifications

(50) The antibody with best native reactivity, affinity and stability in the assay was chosen from the antibody-producing clones generated after the fusion of spleen- and myeloma cells. The clone chosen for antibody purification and the subsequent development of the ELISA was NB191-3C4, raised against TPRGAAPPGQ (SEQ ID NO 246).

(51) Standard Curve and Recovery

(52) A typical standard curve is presented in FIG. 1, showing chosen standards and the 4-parametric fit equation for determination of sample concentration, based on peptide concentrations of 0, 0.782, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 ng/mL. In addition the specificity of the antibody is shown by comparison to the same concentrations of the elongated peptide giving no reaction at all.

(53) Determination of the linearity or recovery by dilution in different samples resulted in following. The average determined recoveries back-calculated from samples diluted 1+1, 1+2, 1+3, 1+4, 1+5, 1+6 and 1+7 to undiluted sample were close to 100% and within the recommended ±10% (data not shown).

(54) Separation of Brains from Alzheimer's Mice and their Controls

(55) The extracted brains from the Tg4510 mice and their corresponding controls were evaluated by measuring them in the NB191-3C4 ELISA setup. As seen in FIG. 2, a very clear separation was seen, with the Tg4510 showing 10-fold higher levels of Tau-A, when compared to the corresponding controls. In addition, a western blot was performed, and as seen in FIGS. 3A-3B the Tg4510 mice showed very high levels of Tau-A, while the control mice showed very little, even if equal amounts of protein were loaded (data not shown). On the other re-probing the blot with an antibody against total Tau showed almost equal levels of total Tau, although the intensity of the high molecular weight bands was higher in the Tg4510 correlating with Tau aggregation in this pathology.

(56) Correlation of CSF and Serum Levels of Tau-A

(57) In 33 paired samples of CSF and serum we investigated whether there was a correlation between Tau-A levels in the two analytes. As seen in FIG. 4 useful levels of Tau-A were detected in both analytes, and a modest, albeit not significant, correlation was observed.

(58) Tau-A Correlates to Age, but not Body Mass Index

(59) In the serum samples from the OA study, we performed an evaluation of Tau-A levels as a function of age and body mass index. Here we found a trend towards a positive correlation with age (FIG. 5A), but no correlation with BMI (FIG. 5B). These data indicate that Tau-A increases with age as a marker of Alzheimer's should.

(60) Indication of Disease Progression

(61) A subset of the serum samples from the Alzheimer's cohort was samples collected at baseline and 18 months later. Although these data only are from women, FIG. 6 shows a strong indication that in these 7 women, the levels of Tau-A increase from baseline to follow-up, hence indicating that Tau-A increases with increasing disease, as it is well-known that development of Alzheimer's is a continuous process.

EXAMPLE 2

(62) Samples for Testing Native Reactivity of the Antibodies

(63) For assay development and validation, serum and plasma from 15 healthy adult volunteers aged 23-45 years and of both genders were used. We also tested serum samples from mice and rats to determine the level of interspecies cross reactivity.

(64) Animal Samples

(65) Tissues including brain, liver, muscle, colon, kidney, lung, skin and pancreas isolated from 5 six-month-old Sprague Dawley rats and 5 brains from each of either the wildtype or Tg4510 mice were flash-frozen in liquid nitrogen and pulverized using a Bessman pulverizer. The “powder” was transferred to a vial and weighed. Extraction buffer (50 mM Tris-HCl, 50 mM HEPES, 1 mM EDTA, 0.5% sodium deoxycholate, 15% glycerol, protease inhibitor cocktail (Roche cat#05056489001), pH8.3) was added at 1 mL buffer/250 mg tissue. The lysate was cleared by sonication. After sonication the debris was spun at 4° C./5 min/10000 rpm and the supernatants were collected and stored at −80° C. until further use. Protein concentrations were determined using the DC Protein Assay (Biorad).

(66) In Vitro Cleavage of Tissues

(67) Protease cleavage was performed by mixing 100 μg of tissue extract and 1 μg of ADAM10 in MMP buffer (100 mM Tris-HCl, 100 mM NaCl, 10 mM CaCl.sub.2, 2 mM Zn acetate, pH 8.0) and incubating for 7 days. Finally, the cleavage was verified by western blotting and ELISA analysis.

(68) Western Blotting

(69) 20 μg of each rat tissue extract and 100 μg of each mouse tissue extract was loaded onto an SDS-PAGE gel. The gel was run and the samples were transferred to nitrocellulose membranes as previously described .sup.15. Ponceau Red staining was then used to verify equal protein loading on the membranes. The levels of Tau-A fragments and total Tau protein were detected by incubation with the primary antibodies diluted to 100 ng/mL in TBS-T containing skim milk powder .sup.15. A secondary antibody recognizing mouse IgG conjugated to horse-radish peroxidase was then added, and finally the blot was visualized using enhanced chemiluminescence as previously described .sup.15.

(70) Human Samples

(71) Serum samples from Alzheimer's patients (n=21) were obtained. Characteristics: Age at onset 70(+/−7), Females/Males (16/6), baseline MDRS score 112(+/−12), Intact tau levels 849 (+/−1009).

(72) Characterization of the Tau-A ELISA Assay

(73) An antibody recognizing the ADAM10 generated cleavage sequence of Tau (TPRGAAPPGQ, SEQ ID NO 246) was selected and used for development of an ELISA (Tau-A). As seen in FIG. 7A, the assay was specific for the cleavage site, as extension of the sequence by one amino acid led to loss of reactivity. Further validation of specificity using ADAM10 degraded recombinant tau or brain extracts confirmed the specificity towards cleaved tau (FIG. 7B-C). In addition, tissue profiling confirmed that Tau primarily originates from the brain. The lower limit of detection (LLOD) was determined to be 2.9 ng/mL and the upper limit of detection (ULOD) was 226.3 ng/mL. The assay was technically robust and was able to detect Tau-A levels in human serum and plasma, as well as mouse and rat serum within dilution ranges of 1+2 to 1+6 (data not shown). In addition, a linear spiking recovery was obtained within the above described dilution ranges (data not shown). The intra-assay coefficient of variation was 5.8%, while the inter-assay CV % was 12.6%. No loss of reactivity was observed following 5 consecutive freeze-thaw cycles.

(74) Biological Validation of the Tau-A ELISA

(75) Analysis using the ELISA revealed that Tg4510 mice brains had 10-fold higher levels of Tau-A than their corresponding wt. controls (FIG. 7E). Western blotting also showed that Tg4510 mice had very high levels of Tau-A, while the control mice had very little (FIG. 7F). Re-probing the blot with an antibody against total Tau showed almost equal levels of total Tau, although the intensity of the high molecular weight bands was greater in the Tg4510.

(76) Tau-A Levels Correlate with MDRS Score

(77) To investigate whether a relationship between the marker and AD disease stage could be identified, we correlated the Tau-A levels in the AD patients to scores obtained using the Mattis Dementia Rating Scale .sup.10, and found a significant (p=0.003) and inverse relationship between MDRS and Tau-A (FIG. 8). No correlations to other parameters, such as intact Tau in CSF or age could be observed in this cohort (data not shown).

(78) Discussion

(79) Potential serum and/or plasma-based markers for AD have been investigated extensively, yet a single biomarker with a correlation to cognitive function previously remained to be identified .sup.1-3. This, to our knowledge, is the first biochemical marker monitoring proteolytic processing of Tau in serum, which appears to be a key initiator of AD pathology .sup.9. It is also the first single serum biomarker of a brain specific protein which correlates to cognitive function. While neo-epitopes have been extensively investigated in AD, as measurements of Aβ42 and phosphorylated Tau have been reported as neo-epitopes formed as a consequence of disease .sup.4, selective screening of serum for in vitro generated Tau fragments had not previously been undertaken.

(80) The combination of ADAM10 and Tau selected for this work was based on the novel hypothesis that during progression of AD, Tau will be exposed to secretase-mediated cleavage either directly in the brain or as fragments generated by other brain proteases, which then become secondarily processed as they enter the circulation; however, this requires further studies. We were able to detect a specific ADAM10-generated Tau peptide fragment in serum, as well as highly elevated levels in the brains of the Tg4510 mice. These observations suggest ADAM10 processing of Tau is a relevant process in neuronal death during AD, although the exact mechanism of action still remains to be identified.

(81) In summary, we have developed the first serum-based assay detecting pathological fragments of Tau. This fragment was directly and inversely related to cognitive function. The assay provides a useful and practical tool for the diagnosis of neuronal loss, and can be used to monitor the efficacy of treatments and progression of AD.

(82) In this specification, unless expressly otherwise indicated, the word ‘or’ is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ‘exclusive or’ which requires that only one of the conditions is met. The word ‘comprising’ is used in the sense of ‘including’ rather than in to mean ‘consisting of’. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

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