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WO2015175769A1 - Methods for the detection of amyloid beta oligomers in biological samples - Google Patents

Methods for the detection of amyloid beta oligomers in biological samples Download PDF

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Publication number
WO2015175769A1
WO2015175769A1 PCT/US2015/030753 US2015030753W WO2015175769A1 WO 2015175769 A1 WO2015175769 A1 WO 2015175769A1 US 2015030753 W US2015030753 W US 2015030753W WO 2015175769 A1 WO2015175769 A1 WO 2015175769A1
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WIPO (PCT)
Prior art keywords
antibody
oligonucleotide
detection antibody
sample
binding moiety
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PCT/US2015/030753
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French (fr)
Inventor
Sreeja GOPAL
Alvydas Mikulskis
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Biogen Ma Inc.
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Publication of WO2015175769A1 publication Critical patent/WO2015175769A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the disclosure relates to methods for detecting amyloid ⁇ oligomers in biological samples and diagnostic, prognostic, and therapeutic uses thereof.
  • the disclosure relates to highly sensitive immuno-polymerase chain reaction (immunoPCR) methods for detecting N-terminal truncated pyroglutamate amyloid ⁇ oligomers.
  • Amyloidosis refers to a diverse group of diseases (e.g., Alzheimer's disease, Down syndrome, type 2 diabetes) characterized by the deposition of insoluble amyloids (Blancas-Mejia and Ramirez- Alvarado, 2013; Hazenberg 2013). As these amyloids continue to accumulate, they can interfere with the normal functions of cells, tissues, and organs (Blancas-Mejia and Ramirez- Alvarado, 2013; Hazenberg 2013; Haass and Selkoe, 2007).
  • Amyloid aggregates in each disease share similar structural and functional features. Amyloid aggregates can become insoluble, toxic, and important to disease progression (Blancas- Mejia and Ramirez-Alvarado, 2013; Hazenberg 2013; WO2011076854; US8512677).
  • AD amyloid ⁇
  • ⁇ amyloid ⁇
  • AD Alzheimer's disease
  • ⁇ plaque formation a neurodegenerative disease characterized by ⁇ plaque formation, neuron death, and debilitating memory loss.
  • Disease progression is relatively slow, and the first symptoms of AD only manifest after several decades of neuron loss (Blennow et al., 2006).
  • AD is impossible to treat and extremely difficult to diagnose in early stages.
  • ⁇ plaques in the AD brain comprise ⁇ oligomers (Haass and Selkoe, 2007), and understanding ⁇ accumulation is believed to be critical for the development of early diagnostic tests and eventual therapeutic agents for AD.
  • ⁇ is derived from the proteolytic cleavage of amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • APP can be cleaved at multiple sites and into fragments of many different lengths.
  • ⁇ in the brain is not a single distinct species, but rather a heterogeneous collection of 30-43 amino acid-long peptides, with different C- and N-terminal sequences (Gunn et al., 2010; Haass and Selkoe, 2007). These different species vary in toxicity and aggregation propensity.
  • ⁇ - ⁇ Pyroglutamate ⁇
  • ⁇ 3- ⁇ is recognized in the art as one of the most toxic and aggregation-prone forms of ⁇ (Mori et al., 1992; Russo et al., 2002; Schilling et al, 2006; Acero et al, 2009; Wirths et al., 2009; Jawhar et al., 2011).
  • ⁇ 3- ⁇ is formed by a multi-step process that begins with the removal of the first two amino acids of ⁇ to expose a glutamate at position three. The enzyme glutaminyl cyclase then catalyzes pyroglutamate formation by the dehydration and cyclization of glutamate (Schilling et al., 2004).
  • ⁇ 3- ⁇ exhibits increased
  • ⁇ 3- ⁇ exhibits "up to 250-fold accelerated initial formation of aggregates compared to unmodified ⁇ " and is "crucial for the initiation of the disease" (Schilling et al., 2006). Not only does ⁇ 3- ⁇ precede the deposition of unmodified ⁇ in the brain, ⁇ 3- ⁇ exceeds the deposition of unmodified ⁇ and "may account for more than 50% of ⁇ accumulated in plaques" (Russo et al., 2002). pE3- ⁇ is more toxic than unmodified ⁇ to neurons in AD and Down syndrome (Acero et al., 2009; Russo et al., 2001).
  • transgenic mice In transgenic mice, expression of ⁇ 3- ⁇ causes "massive neurological impairments” starting in early adulthood (Wirths et al., 2009). By contrast, transgenic mice that only express unmodified ⁇ exhibit relatively “minimal neurofibrillary pathology and neuronal loss” (Kawarabayashi et al., 2001).
  • the ⁇ 3- ⁇ oligomer is considered to be a promising target for AD diagnosis, prognosis, and therapy (DeMattos et al., 201 1).
  • ⁇ 3- ⁇ demonstrates tremendous diagnostic, prognostic, and therapeutic potential
  • detection of ⁇ 3- ⁇ currently poses many technical challenges.
  • ⁇ concentrations in healthy tissues can be nearly nine-fold lower than in AD tissues (0.7 ⁇ g/g tissue compared to 6.1 ⁇ g/g tissue) (Harigaya et al., 2000), making detection at early stages of the disease difficult.
  • early detection may be critical, as ⁇ 3- ⁇ can oligomerize at concentrations as low as 1 ⁇ g/mL, compared to 2.5 g/mL for unmodified ⁇ (Harigaya et al., 2000).
  • ⁇ 3- ⁇ primarily in biological samples containing relatively high levels of ⁇ 3- ⁇ , such as ⁇ plaques and post-mortem AD brains, but detection remains challenging in samples containing relatively low levels of ⁇ 3- ⁇ , such as cerebrospinal fluid.
  • ⁇ 3- ⁇ has been detected in plaques from AD and Down syndrome patients (Frost et al., 2013) and AD mice (WO2012021469) by immunohistochemistry.
  • AD patients by immunohistochemistry (De Kimpe et al., 2013; WO2011151076); AD patients by ELISA (Morawski et al., 2013); AD mice by ELISA (Wu et al, 2013; US8058405); AD mice by immunohistochemistry (US8512677); AD patients, AD mice, and aged rhesus macaques by immunohistochemistry (Wirths et al, 2013); AD patients by sandwich ELISA (Wirths et al., 2010); and AD patients at different disease stages by
  • ⁇ 3- ⁇ has been detected in murine AD brains by immunoprecipitation combined with mass spectrometry (Wittnam et al, 2012) and by one- and two- dimensional gel electrophoresis combined with immunoblotting and mass spectrometry (Bibl et al., 2012).
  • ⁇ 3- ⁇ has been detected in brain tissues from patients with AD, sporadic Alzheimer's disease (SAD), and familial Alzheimer's dementias (FAD) by immunoprecipitation combined with mass spectrometry (Portelius et al., 2010).
  • CSF cerebrospinal fluid
  • a highly sensitive method for the detection of ⁇ 3- ⁇ in CSF may allow not only early detection of disease, but may provide an accessible biological sample for use in drug development, a process for which it is crucial to identify and monitor the effects of drugs in patients.
  • CSF contains particularly low concentrations of ⁇ , especially in AD patients (Mehta et al., 2000; Kawarabayashi et al, 2001 ; Blennow and Hampel, 2003; Fagan et al., 2006; Strozyk et al., 2003), which poses a significant technical challenge for detection.
  • AD patients Mehta et al., 2000; Kawarabayashi et al, 2001 ; Blennow and Hampel, 2003; Fagan et al., 2006; Strozyk et al., 2003
  • the invention provides methods for the detection of amyloid ⁇ oligomers in biological samples and diagnostic, prognostic, and therapeutic uses thereof.
  • the invention provides highly sensitive and reliable immunoPCR methods for detecting and determining levels of ⁇ 3- ⁇ .
  • the methods of the invention may be used to detect and determine levels of ⁇ 3- ⁇ in biological samples containing low levels of ⁇ 3- ⁇ , such as samples from early stages of disease, samples that are very dilute, and/or samples of very limited volume.
  • the inventive assays described herein may be used to detect and determine levels of ⁇ 3- ⁇ in cerebrospinal fluid.
  • the invention may be used to determine relative levels of ⁇ 3- ⁇ in cerebrospinal fluid.
  • the methods of the invention may be used for the diagnosis and prognosis of diseases characterized by ⁇ 3- ⁇ .
  • the disease is AD, in particular the early stages of AD.
  • the methods of the invention may be used to stratify disease states, monitor disease progression, and/or assess responsiveness to treatment.
  • the methods of the invention may be used to tailor and direct therapies.
  • FIG. 1 shows a schematic diagram of the steps involved in one embodiment of the invention.
  • a biological sample that potentially contains ⁇ 3- ⁇ is contacted by a capture antibody immobilized on an ELISA plate, a biotinylated detection antibody, and a streptavidin-oligonucleotide complex.
  • the resulting complex is released from the ELISA plate and amplified by PCR.
  • FIG. 2 shows a schematic diagram of the steps involved in an alternate embodiment of the invention.
  • a biological sample that potentially contains ⁇ 3- ⁇ is contacted by a capture antibody immobilized on an ELISA plate and an oligonucleotide- conjugated detection antibody.
  • the resulting complex is released from the ELISA plate and amplified by PCR.
  • Fig. 3 shows a hypothetical PCR amplification plot, which does not necessarily reflect a typical amplification plot for ⁇ 3- ⁇ .
  • a lower threshold cycle (CT) indicates a higher starting analyte concentration.
  • Fig. 4 shows a hypothetical standard curve, which does not necessarily reflect a typical standard curve for ⁇ 3- ⁇ .
  • Fig. 5 shows optimization of streptavidin-oligonucleotide parameters using one of two capture antibodies, 9D5 or AB 100-11, and a biotinylated detection antibody AB 100-11.
  • Fig. 6 shows optimization of immunoPCR parameters in a ⁇ 3- ⁇ depletion assay using antibodies 2F02, AB 100-11, HO-1, or buffer.
  • Fig. 7 shows normalized AC T results using 1 ⁇ g/mL or 2 ⁇ g/mL of capture antibody 9D5 or AB 100-11 and 1 g/mL or 0.2 ⁇ g/mL of detection antibody ABlOO-1 1.
  • Figs. 8A-B show the detection of ⁇ 3- ⁇ in plasma (Fig. 8 A) and cerebrospinal fluid (Fig. 8B). DESCRIPTION OF EMBODIMENTS
  • Amyloid ⁇ and ⁇ are used interchangeably to refer to amyloid ⁇ peptide and modifications, including pyroglutamate ⁇ , fragments, and/or equivalents thereof.
  • ⁇ refers to any fragment produced by the proteolytic cleavage of amyloid precursor protein (APP).
  • ⁇ oligomers is used to refer to multimeric species of ⁇ that result from the association of monomeric ⁇ species.
  • amyloidosis refers to a group of diseases characterized by the deposition of insoluble amyloid plaques, including but not limited to, Alzheimer's disease, sporadic Alzheimer's disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA), Down syndrome (DS), mild cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Parkinson-Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creutzfeld Jacob disease, Parkinson's disease, HIV-associated dementia (HAD), amyotropic lateral sclerosis (ALS), type 2 diabetes, and secondary amyloidosis resulting from other diseases, including but not limited to, tuberculosis, osteomyelitis, rheumatoi
  • ⁇ 3- ⁇ or " ⁇ ⁇ 3 " as used herein refers to N-terminally modified pyroglutamate amyloid ⁇ peptides that start at the glutamic acid (Glu) residue at position 3 of the amino acid sequence of ⁇ , wherein the Glu residue is cyclized to form a pyroglutamic acid residue.
  • oligomers include but are not limited to ⁇ ⁇ ⁇ 3 - 38 , ⁇ ⁇ ⁇ 3-4 ⁇ , and ⁇ ⁇ ⁇ 3-42 .
  • the amino acid sequences of these exemplary N-terminally modified forms of ⁇ peptides are:
  • Unmodified ⁇ is used to refer to amyloid ⁇ peptides in which the N terminus is not modified post-translationally, including but not limited to ⁇ -38 , ⁇ -4 ⁇ , and ⁇ 1 -4 2.
  • the amino acid sequences of these exemplary unmodified ⁇ peptides are: ⁇ 1-38 (SEQ ID NO: 4):
  • An antibody that is "specific for ⁇ 3- ⁇ " or a “pE3 ⁇ -specific antibody” refers to an antibody that exhibits binding affinity for ⁇ 3- ⁇ and does not exhibit significant binding affinity for other forms of ⁇ , including other forms of pyroglutamate ⁇ , e.g., pEl l- ⁇ .
  • Examples of ⁇ 3- ⁇ -8 ⁇ antibodies include but are not limited to:
  • hE8L comprising the amino acid sequences of SEQ ID NOs: 7 (light chain) and SEQ ID NO: 10 (heavy chain):
  • ⁇ capture antibody refers to an antibody that binds an ⁇ oligomer or ⁇ oligomer complex that is being detected and/or measured in a biological sample.
  • the ⁇ capture antibody is specific for ⁇ 3- ⁇ .
  • the detection antibody is specific for ⁇ 3- ⁇ .
  • ⁇ detection antibody refers to an antibody that binds an ⁇ oligomer or ⁇ oligomer complex that is being detected and/or measured in a biological sample and is conjugated to a second complex (examples include, but are not limited to, biotin, an oligonucleotide, digoxigenin, fluorescein).
  • the ⁇ detection antibody is specific for ⁇ 3- ⁇ .
  • the capture antibody is specific for ⁇ 3- ⁇ (i.e., at least one of the capture antibody or the detection antibody is specific for ⁇ 3- ⁇ ).
  • Polypeptide “peptide”, and “protein” are used interchangeably to refer to a polymer of amino acids.
  • the invention provides methods for detecting and determining levels of ⁇ oligomers in biological samples.
  • the invention provides highly sensitive methods to detect and determine levels of ⁇ 3- ⁇ oligomers in biological samples.
  • the methods of the invention may be used for biological samples containing low levels of ⁇ 3- ⁇ oligomers, such as samples from early stages of disease, samples that are very dilute, and/or samples of very limited volume.
  • the invention may be used to detect and determine levels of ⁇ 3- ⁇ in cerebrospinal fluid.
  • a biological sample that potentially contains ⁇ 3- ⁇ is contacted by two antibodies, a capture antibody and a detection antibody, in an immunoassay.
  • the detection antibody is directly conjugated to an oligonucleotide, which is analyzed by PCR to determine the presence or absence of ⁇ 3- ⁇ in a biological sample.
  • the detection antibody is indirectly conjugated to an oligonucleotide, which is analyzed by PCR to determine the presence or absence of ⁇ 3- ⁇ in a biological sample.
  • the detection antibody is conjugated to a first binding moiety; the oligonucleotide is conjugated to a second binding moiety; and the first binding moiety and the second binding moiety are binding partners that bind together to form a detection antibody- oligonucleotide complex before, at the same time as, or after contacting the biological sample.
  • the oligonucleotide is analyzed by PCR to determine the presence or absence of ⁇ 3- ⁇ in a biological sample.
  • the first binding moiety is biotin
  • the second binding moiety is streptavidin
  • the detection antibody is biotinylated and binds to a streptavidin-oligonucleotide complex, which is analyzed by PCR to determine the presence or absence of ⁇ 3- ⁇ in a biological sample.
  • the first binding moiety is digoxigenin
  • the second binding moiety is an anti-digoxigenin antibody.
  • the first and second binding moieties are biotin and avidin; biotin and neutravidin; fluorescein and an anti- fluorescein antibody; or other first and second binding moieties that are binding partners.
  • the PCR is quantitative PCR (qPCR), and at least one normalized ACT result is generated.
  • the normalized ACT results are analyzed in comparison to a standard curve to determine ⁇ 3- ⁇ levels in a biological sample.
  • the invention may be used to determine relative levels of ⁇ 3- ⁇ in biological samples.
  • the methods of the invention are used to diagnose a disease characterized by ⁇ 3- ⁇ .
  • the methods of the invention are used to prognose and/or treat a disease characterized by ⁇ 3- ⁇ .
  • the methods of the invention may be used to stratify disease states, monitor disease progression, and/or assess responsiveness to treatment.
  • the methods of the invention may be used to tailor and direct therapies.
  • the disease is Alzheimer's disease, in particular in the early stages of the disease.
  • Biological samples may include a fluid, a cell, or a tissue sample.
  • Biological fluids may include cerebrospinal fluid, plasma, serum, blood, saliva, urine, sweat, or peritoneal fluid.
  • a cell sample or a tissue sample may include a biopsy, a tissue, a cell suspension, or other specimens and samples, such as clinical samples. Specimens and samples may be obtained, for example, for the diagnosis, prognosis, and/or treatment of a disease.
  • the disease to be diagnosed, prognosed, or treated is primary amyloidosis.
  • Diseases of primary amyloidosis include, but are not limited to, Alzheimer's disease, sporadic Alzheimer's disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA), Down syndrome (DS), mild cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Parkinson-Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creutzfeld Jacob disease, Parkinson's disease, HIV-associated dementia (HAD), amyotropic lateral sclerosis (ALS), and type 2 diabetes.
  • SAD sporadic Alzheimer's disease
  • FAD familial Alzheimer's dementias
  • BBD familial British dementia
  • FDD familial Danish dementia
  • the disease to be diagnosed, prognosed, or treated is secondary amyloidosis.
  • Diseases of secondary amyloidosis include, but are not limited to tuberculosis, osteomyelitis, rheumatoid arthritis, granulomatous ileitis, Hodgkin's lymphoma, leprosy, and familial Mediterranean fever.
  • the methods of the invention are used to determine the relative amount of ⁇ 3- ⁇ in two or more biological samples. Two or more biological samples are assayed by PC as described above, and at least one normalized ACj value is determined for each sample. Two or more normalized AC values may be compared to determine the relative amount of ⁇ 3- ⁇ in the samples.
  • the two or more biological samples are collected from different human subjects. It is known that CSF concentrations of unmodified ⁇ are lower in AD patients (Mehta et al., 2000; Blennow and Hampel, 2003), possibly due to increased ⁇ aggregation the brain (Fagan et al., 2006; Strozyk et al., 2003), but relative concentrations of ⁇ 3- ⁇ in the CSF of AD patients is unknown.
  • the methods of the invention are used to evaluate CSF samples collected from AD patients and healthy controls. The CSF samples are assayed by PCR and analyzed for normalized ACj values as described above.
  • CSF samples are collected from AD patients, healthy controls, and at least one undiagnosed subject.
  • the CSF samples are assayed by PCR as described above and analyzed for normalized ACj values.
  • the normalized ACT value(s) of at least one undiagnosed subject are compared to the normalized ACT values of AD patients and healthy controls. The analysis may be used in the diagnosis of AD.
  • the two or more biological samples are collected from the same human subject.
  • cerebrospinal fluid and plasma are collected from the same human subject, and relative levels of ⁇ 3- ⁇ are assayed by PCR. Relatively low levels of ⁇ in CSF may be indicative of increased ⁇
  • ⁇ 3- ⁇ in cerebrospinal fluid in Fig. 8B suggests that normalized ACT values may be lower in AD than in control samples. The analysis may be useful in the diagnosis, prognosis, and treatment of disorders characterized by ⁇ 3- ⁇ .
  • the methods of the invention are used to measure ⁇ 3- ⁇ levels in a biological sample.
  • one or more biological samples containing unknown amounts of ⁇ 3- ⁇ are assayed by PCR and at least one normalized ACT value is calculated for each biological sample.
  • At least three samples containing known amounts of ⁇ 3- ⁇ are also assayed by PCR, and at least one normalized ACT value is calculated for each sample.
  • an average normalized ACT value may be calculated and used as the normalized ACT value of the sample.
  • Normalized AC T values of the samples containing known amounts of ⁇ 3- ⁇ are plotted against their corresponding ⁇ 3- ⁇ concentrations to generate a standard curve.
  • the ⁇ 3- ⁇ level of the biological sample containing an unknown amount of ⁇ 3- ⁇ is calculated by comparing the normalized ACT value of the biological sample to the normalized ACj values of the standard curve.
  • normalized ACj can be calculated by subtracting the ACj of the negative control sample(s) from the ACT of the sample(s) of interest.
  • Fig. 4 shows a hypothetical standard curve using ACj values and does not necessarily reflect a typical standard curve for ⁇ 3- ⁇ .
  • the methods of the invention are used to detect, but not to determine levels of ⁇ 3- ⁇ .
  • the methods are first used to determine the presence or absence of ⁇ 3- ⁇ .
  • the sample is further evaluated by an alternate quantitative technique.
  • a second biological sample is collected from a different tissue. The second sample is analyzed for the presence or absence of ⁇ 3- ⁇ , as described above, and/or by an alternate quantitative technique.
  • the alternate quantitative technique is mass spectrometry, in particular liquid chromatography-tandem mass spectrometry. Capturing ⁇ 3- ⁇ in a biological sample
  • the methods of the invention comprise contacting a biological sample with two antibodies, a capture antibody and a detection antibody, in an immunoassay.
  • at least one of the capture antibody or the detection antibody is specific for ⁇ 3- ⁇ .
  • ⁇ 3- ⁇ -8 ⁇ antibodies disclosed in Wirths et al., 2010, WO201 1 151076, Frost et al., 2012, WO2012021469, Mandler et al., and WO2009149486, each incorporated herein by reference include: ABlOO-1 1
  • R17L (SEQ ID NOs: 7 and 9)
  • only the capture antibody is specific for ⁇ 3- ⁇ .
  • only the detection antibody is specific for ⁇ 3- ⁇ . In some embodiments, both the capture antibody and the detection antibody are specific for pE3-
  • the capture antibody and the detection antibody are selected from the group consisting of ABlOO-l l, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3,
  • the capture antibody and the detection antibody are the same antibody.
  • both the capture antibody and the detection antibody are the capture antibody and the detection antibody.
  • both the capture antibody and the detection antibody are 9D5.
  • the capture antibody and the detection antibody are different antibodies.
  • the capture antibody is AB 100-11, and the detection antibody is selected from the group consisting of 9D5, 8C4, AB 5-5-6, AB
  • the capture antibody is 9D5
  • the detection antibody is selected from the group consisting of AB 100-1 1, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3,
  • the capture antibody is selected from the group consisting of 9D5, 8C4, AB 5-5-6, AB 6-1-
  • the capture antibody is selected from the group consisting of AB 100-11, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24- 2-3, B12L, CI-C7, hE8L, R17L, and other pE3-Ap-specific antibodies, and the detection antibody is 9D5.
  • the capture antibody is AB 100-1 1 and the detection antibody is 9D5.
  • the capture antibody is 9D5 and the detection antibody is AB 100-11. In some embodiments, neither the capture antibody nor the detection antibody is AB 100-1 1.
  • the capture antibody concentration ranges from about 0.1 ⁇ g/mL to about 2 ⁇ g/mL; about 2 ⁇ g/mL to about 5 ⁇ g/mL; or about 5 to about 10 ⁇ g/mL. In certain embodiments, the capture antibody concentration is 1 ⁇ g/mL. In alternate embodiments, the capture antibody concentration is 2 ⁇ g/mL. In some embodiments, the detection antibody concentration ranges from about 0.02 ⁇ g/mL to about 1 ⁇ g/mL; about 1 ⁇ g/mL to about 3 ⁇ g/mL; or about 3 ⁇ g/mL to about 5 ⁇ g/mL. In certain embodiments, the detection antibody concentration is 0.2 ⁇ g/mL. In alternate embodiments, the detection antibody concentration is 1 ⁇ g/mL.
  • the capture antibody contacts the biological sample and binds ⁇ 3- ⁇ to form a capture antibody/pE3-Ap complex.
  • the detection antibody contacts the biological sample and binds ⁇ 3- ⁇ to form a detection antibody/pE3-A complex.
  • the detection antibody binds ⁇ 3- ⁇ , in particular the capture antibody/pE3-AP complex.
  • the capture antibody contacts the biological sample before, at the same time as, or after the detection antibody contacts the biological sample.
  • the capture antibody and the detection antibody are the same antibody, and the ⁇ 3- ⁇ that is detected is multimeric (i.e., more than one ⁇ 3- ⁇ monomer is present, because the capture antibody and detection antibody bind to the same epitope of ⁇ 3- ⁇ ).
  • the capture antibody is immobilized on a surface.
  • the capture antibody may be immobilized on a plate (e.g., Corning Costar® or NUNC MaxiSorp®), a channel, or a column.
  • the plate is a streptavidin-coated plate, and the capture antibody is biotinylated.
  • pEl l- ⁇ or other ⁇ - ⁇ species could be detected by exchanging the pE3-Ap-specific antibody for a pEl 1- ⁇ - specific antibody (see, e.g., Perez-Garmendia et al., 2010; commercially available NBP 1-44070) or other ⁇ - ⁇ -specific antibodies.
  • the detection antibody contacts the biological sample and binds ⁇ 3- ⁇ to form a detection antibody/pE3-A ⁇ complex. In certain embodiments, the detection antibody binds ⁇ 3- ⁇ , in particular the capture
  • the detection antibody is directly or indirectly conjugated to an oligonucleotide.
  • the oligonucleotide is synthetic, custom designed, and comprises no homology to known human sequences.
  • the oligonucleotide is at least 20 bases in length.
  • the oligonucleotide has minimal homopolymers.
  • the oligonucleotide has minimal G quadruplexes.
  • the oligonucleotide is directly conjugated to the detection antibody, e.g., using commercially available kits. In alternate embodiments, the oligonucleotide is indirectly conjugated to the detection antibody before, at the same time as, or after the detection antibody contacts the biological sample and binds pE3- ⁇ .
  • the detection antibody is conjugated to a first binding moiety
  • the oligonucleotide is conjugated to a second binding moiety; the first binding moiety and the second binding moiety are binding partners that bind together to form a detection antibody-oligonucleotide complex before, at the same time as, or after contacting the biological sample.
  • the first and/or the second binding moieties are capable of multimeric binding.
  • the detection antibody is added to the sample to bind ⁇ 3- ⁇ before, at the same time as, or after the capture antibody.
  • the first and second binding moieties form a detection antibody-oligonucleotide complex after the detection antibody contacts the biological sample and binds ⁇ 3- ⁇ .
  • a capture antibody and a detection antibody conjugated to a first binding moiety contact the biological sample and each binds ⁇ 3- ⁇ , and then a second binding moiety conjugated to an oligonucleotide binds the first binding moiety.
  • the first and second binding moieties form a detection antibody-oligonucleotide complex at the same time as or before the detection antibody contacts the biological sample.
  • a capture antibody and a detection antibody-oligonucleotide complex contact the biological sample and each binds ⁇ 3- ⁇ .
  • the first binding moiety is biotin
  • the biotin- conjugated detection antibody is generated using commercially available kits (e.g., EZ- LinkTM).
  • the second binding moiety is streptavidin and the streptavidin-conjugated oligonucleotide is generated using commercially available kits (e.g., TurboLinkTM).
  • the streptavidin-oligonucleotide is generated using commercially available kits (e.g., TurboLinkTM).
  • the streptavidin- oligonucleotide concentration ranges from about 0.01 ng/mL to about 2 ng/mL; about 2 ng/mL to about 5 ng/mL; or about 5 ng/mL to about 10 ng/mL.
  • the streptavidin- oligonucleotide concentration is 1 ng/mL.
  • the first binding moiety is digoxigenin
  • the second binding moiety is an anti-digoxigenin antibody.
  • the first and second binding moieties are biotin and avidin; biotin and neutravidin; fluorescein and an anti-fluorescein antibody; or other first and second binding moieties that are binding partners.
  • the sample is transferred to a PCR plate.
  • a release buffer is added before transferring the sample.
  • the PCR plate comprises a reaction mixture that comprises primers, probes, polymerase, and free nucleotides.
  • the reaction mixture comprises primers, probes, DNA
  • the DNA polymerase is Taq polymerase.
  • the reaction mixture is heated and cooled to amplify the oligonucleotide, and the amplification activity is analyzed.
  • the primers and probes are a custom primer-probe mix comprising a primer to the oligonucleotide which is conjugated to a probe (e.g., reporter and quencher moieties).
  • the sample is analyzed by quantitative PCR (qPCR).
  • qPCR quantitative PCR
  • qPCR may be used to amplify, detect, and/or quantify oligonucleotide sequences.
  • a fluorophore is added to the reaction mixture, and a thermal cycler is used to illuminate the reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore.
  • the fluorophore is the probe in a custom primer probe comprising a fluorescent reporter dye conjugated to a primer custom designed based on the sequence of the
  • the reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100°C, 60-70°C, and 30-50°C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97°C, 55-65°C, and 35-45°C for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT).
  • CT threshold cycle
  • AC T can be calculated by subtracting the CT from the maximum cycle number, e.g., 40.
  • ACT ranges from 10-40 cycles.
  • Normalized ACT can be calculated by subtracting the ACT of the negative control sample(s) from the ACT of the sample(s) of interest.
  • normalized ACT results are used to determine the presence or absence of ⁇ 3- ⁇ in biological samples. Absence may be defined as concentrations of ⁇ 3- ⁇ that are not detectable (i.e., CT beyond the limit of detection, for example, >40 threshold cycles). Presence may be defined as concentrations of ⁇ 3- ⁇ that are detectable (i.e., within the limit of detection, for example, CT between 10-40 threshold cycles).
  • the antibodies and reagents useful in the methods of the invention can be provided in a kit (i.e., a packaged combination of reagents with instructions for performing the assay).
  • the kit may include antibodies, antibody complexes, substrates, and/or cofactors.
  • additives such as stabilizers, buffers, and other solutions may be included.
  • the relative amounts of the reagents may be varied to provide for concentrations that optimize the sensitivity of the assay.
  • the kit is a diagnostic kit.
  • the diagnostic kit may be used for the detection and diagnosis of ⁇ 3- ⁇ diseases and conditions, particularly Alzheimer's disease. LIST OF EMBODIMENTS
  • a method of detecting ⁇ 3- ⁇ in a biological sample comprising
  • the detection antibody is indirectly conjugated to an oligonucleotide before, at the same time as, or after contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody- oligonucleotide complex before, at the same time as, or after contacting the sample;
  • the detection antibody is directly conjugated to an oligonucleotide to form a detection antibody-oligonucleotide complex
  • the capture antibody is selected from the group consisting of AB 100-1 1, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17- 4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L, and wherein the detection antibody is ABlOO-11.
  • the capture antibody is selected from the group consisting of ABlOO-11, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17- 4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L, and wherein the detection antibody is 9D5.
  • the disease of embodiment 35 wherein the disease is selected from the group consisting of Alzheimer's disease, sporadic Alzheimer's disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA), Down syndrome (DS), mild cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Parkinson-Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creutzfeld Jacob disease, Parkinson's disease, HIV-associated dementia (HAD), amyotropic lateral sclerosis (ALS), type 2 diabetes, tuberculosis, osteomyelitis, rheumatoid arthritis, granulomatous ileitis, Hodgkin's lymphoma, leprosy, and familial Mediterranean fever.
  • SAD sporadic Alzheimer'
  • any one of embodiments 1 -5 wherein the biological sample is selected from the group consisting of cerebrospinal fluid, plasma, serum, blood, saliva, urine, sweat, peritoneal fluid, a biopsy, a tissue sample, a clinical specimen, a cell sample, and a cell suspension.
  • kits for assaying a biological sample for the presence, absence, or level of ⁇ 3- ⁇ comprising
  • the detection antibody is indirectly conjugated to an oligonucleotide before, at the same time as, or after contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody- oligonucleotide complex before, at the same time as, or after contacting the sample; or
  • the detection antibody is directly conjugated to an oligonucleotide to form a detection antibody-oligonucleotide complex
  • Alzheimer's amyloid beta-peptide Nat Rev Mol Cell Biol. 8(2): 101-12 (2007).
  • Amyloid beta protein starting pyroglutamate at position 3 is a major component of the amyloid deposits in the Alzheimer's disease brain. Biochem Biophys Res Commun. 24;276(2):422-7 (2000).
  • the 9D5 or AB 100-1 1 capture antibody was immobilized on a plate.
  • a NUNC Maxisorp® plate was coated with capture antibody at a dilution of 1 g/mL or 2 ⁇ g/mL.
  • the plate was sealed and incubated for 18-24 hours at 2-8° C while shaking on a microtiter plate shaker.
  • the plate was washed eight times with PBS-Tween20 Wash Buffer.
  • Example 2 ⁇ 3- ⁇ capture and detection using a bio tiny lated detection antibody
  • Blocking solution comprising 1.00 ⁇ g/mL of yeast tRNA in PBS/Casein was added to the capture antibody plate.
  • the plate was washed four times with PBS- Tween20 Wash Buffer. Cerebrospinal fluid samples were added to the plate.
  • the plate was sealed and incubated for 18-24 hours at 2-8° C while shaking on a microtiter plate shaker. The plate was washed eight times with PBS-Tween20 Wash Buffer.
  • the AB 100-11 detection antibody was conjugated to biotin using commercially available methods (EZ-LinkTM). Detection antibody was added to the plate at a dilution of 0.2 ⁇ g/mL or 1 ⁇ g/mL. The plate was sealed and incubated for 55- 65 minutes at 22-26°C while shaking on a microtiter plate shaker. The plate was washed eight times with PBS-Tween20 Wash Buffer.
  • Streptavidin-oligonucleotide was added to the plate at a concentration of 1 ng/mL. The plate was sealed and incubated for 55-65 minutes at 22-26°C while shaking on a microtiter plate shaker. The plate was washed eight times with PBS Tween20 wash buffer, followed by four washes with Tris-EDTA wash buffer (pH 8.0).
  • Release buffer comprising DTT and 1 ⁇ g/mL yeast tRNA was added to the plate.
  • the plate was sealed and incubated for 45-55 minutes at 37°C, then shaken for 5-15 minutes at 22-26°C on a microtiter plate shaker.
  • a portion of the sample was transferred to a PCR plate containing a mixture of PCR master mix as well as custom primer probe sets.
  • PCR amplification was performed using a Roche LightCycler 480 instrument. Optimized antibody concentrations are shown in Table 1, and normalized ACt results are shown in Fig. 7. Table 1. Exemplary antibody conditions using biotinylated detection antibodies
  • Threshold cycle (CT) results were obtained from the PCR reaction.
  • a low CT value indicates a high concentration of initial analyte.
  • a hypothetical amplification plot is shown in Fig. 3, which does not necessarily reflect a typical amplification plot for ⁇ 3- ⁇ .
  • streptavidin-oligonucleotide conjugate concentration 1 ng/mL was used for subsequent assessments.
  • plasma samples were first depleted of analyte using: the same antibody as the capture antibody; the ⁇ 3- ⁇ specific antibody 2F02; the non-specific antibody HO- 1 ; or a buffer control.
  • a reduction in measured analyte levels can be seen when depleted with AB 100- 1 1 or 2F02, but the reduction was lower in buffer-depleted or HO- 1 depleted samples (Fig. 6).
  • Example 6 Detection in cerebrospinal fluid (CSF) samples
  • the methods of the invention may be used to detect ⁇ 3- ⁇ in cerebrospinal fluid, which is challenging using current methods.
  • ⁇ 3- ⁇ in CSF samples was captured and detected using a biotinylated detection antibody as described in Example 2.
  • Analysis of ⁇ 3- ⁇ in plasma for AD and control samples is shown in Fig. 8A.
  • Analysis of ⁇ 3- ⁇ in cerebrospinal fluid in Fig. 8B suggests that normalized ACT values may be lower in AD than in control samples.

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Abstract

Methods for detecting and measuring levels of ρΕ3-Αβ oligomers and uses of the methods to diagnose and/or prognose diseases characterized by ρΕ3-Αβ are disclosed. Uses of the methods to develop, tailor, and direct therapies are also disclosed.

Description

METHODS FOR THE DETECTION OF AMYLOID β OLIGOMERS
IN BIOLOGICAL SAMPLES
[001] This application claims the benefit of U.S. Provisional Application No. 61/993,853, filed May 15, 2014, which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[002] The disclosure relates to methods for detecting amyloid β oligomers in biological samples and diagnostic, prognostic, and therapeutic uses thereof. In a particular aspect, the disclosure relates to highly sensitive immuno-polymerase chain reaction (immunoPCR) methods for detecting N-terminal truncated pyroglutamate amyloid β oligomers.
[003] Peptides that are normally soluble can aggregate into insoluble configurations, such as amyloid and amyloid-like structures, that contribute to disease. Amyloidosis refers to a diverse group of diseases (e.g., Alzheimer's disease, Down syndrome, type 2 diabetes) characterized by the deposition of insoluble amyloids (Blancas-Mejia and Ramirez- Alvarado, 2013; Hazenberg 2013). As these amyloids continue to accumulate, they can interfere with the normal functions of cells, tissues, and organs (Blancas-Mejia and Ramirez- Alvarado, 2013; Hazenberg 2013; Haass and Selkoe, 2007). Although the precursor molecules for these diseases differ, the amyloid aggregates in each disease share similar structural and functional features. Amyloid aggregates can become insoluble, toxic, and important to disease progression (Blancas- Mejia and Ramirez-Alvarado, 2013; Hazenberg 2013; WO2011076854; US8512677).
[004] In particular, amyloid β (Αβ) is thought to play an important role in the development of Alzheimer's disease (AD) (Haass and Selkoe, 2007). AD is a neurodegenerative disease characterized by Αβ plaque formation, neuron death, and debilitating memory loss. Disease progression is relatively slow, and the first symptoms of AD only manifest after several decades of neuron loss (Blennow et al., 2006). At present, AD is impossible to treat and extremely difficult to diagnose in early stages.
[005] Αβ plaques in the AD brain comprise Αβ oligomers (Haass and Selkoe, 2007), and understanding Αβ accumulation is believed to be critical for the development of early diagnostic tests and eventual therapeutic agents for AD. Αβ is derived from the proteolytic cleavage of amyloid precursor protein (APP). APP can be cleaved at multiple sites and into fragments of many different lengths. Thus, Αβ in the brain is not a single distinct species, but rather a heterogeneous collection of 30-43 amino acid-long peptides, with different C- and N-terminal sequences (Gunn et al., 2010; Haass and Selkoe, 2007). These different species vary in toxicity and aggregation propensity.
[006] Pyroglutamate Αβ (ρΕ-Αβ), and in particular ρΕ3-Αβ, is recognized in the art as one of the most toxic and aggregation-prone forms of Αβ (Mori et al., 1992; Russo et al., 2002; Schilling et al, 2006; Acero et al, 2009; Wirths et al., 2009; Jawhar et al., 2011). ρΕ3-Αβ is formed by a multi-step process that begins with the removal of the first two amino acids of Αβ to expose a glutamate at position three. The enzyme glutaminyl cyclase then catalyzes pyroglutamate formation by the dehydration and cyclization of glutamate (Schilling et al., 2004). ρΕ3-Αβ exhibits increased
hydrophobicity and stability and is particularly resistant to degradation by peptidases compared to unmodified Αβ (Saido et al., 1996; Jawhar et al., 201 1).
[007] As a result, ρΕ3-Αβ exhibits "up to 250-fold accelerated initial formation of aggregates compared to unmodified Αβ" and is "crucial for the initiation of the disease" (Schilling et al., 2006). Not only does ρΕ3-Αβ precede the deposition of unmodified Αβ in the brain, ρΕ3-Αβ exceeds the deposition of unmodified Αβ and "may account for more than 50% of Αβ accumulated in plaques" (Russo et al., 2002). pE3- Αβ is more toxic than unmodified Αβ to neurons in AD and Down syndrome (Acero et al., 2009; Russo et al., 2001). In transgenic mice, expression of ρΕ3-Αβ causes "massive neurological impairments" starting in early adulthood (Wirths et al., 2009). By contrast, transgenic mice that only express unmodified Αβ exhibit relatively "minimal neurofibrillary pathology and neuronal loss" (Kawarabayashi et al., 2001). The ρΕ3-Αβ oligomer is considered to be a promising target for AD diagnosis, prognosis, and therapy (DeMattos et al., 201 1). Treating diseased neurons in vitro with antibodies to ρΕ3-Αβ reduces toxicity, and in mouse models, "reduce[s] Αβ plaque load and normalize[s] behavioral deficits" (Frost et al., 2012; Wirths et al, 2010;
WO2011151076; US8283517).
[008] Although ρΕ3-Αβ demonstrates tremendous diagnostic, prognostic, and therapeutic potential, detection of ρΕ3-Αβ currently poses many technical challenges. For example, Αβ concentrations in healthy tissues can be nearly nine-fold lower than in AD tissues (0.7 μg/g tissue compared to 6.1 μg/g tissue) (Harigaya et al., 2000), making detection at early stages of the disease difficult. Yet early detection may be critical, as ρΕ3-Αβ can oligomerize at concentrations as low as 1 μg/mL, compared to 2.5 g/mL for unmodified Αβ (Harigaya et al., 2000).
[009] As a result, current methods are able to detect ρΕ3-Αβ primarily in biological samples containing relatively high levels of ρΕ3-Αβ, such as Αβ plaques and post-mortem AD brains, but detection remains challenging in samples containing relatively low levels of ρΕ3-Αβ, such as cerebrospinal fluid. For example, ρΕ3-Αβ has been detected in plaques from AD and Down syndrome patients (Frost et al., 2013) and AD mice (WO2012021469) by immunohistochemistry. Several antibodies have been developed to detect ρΕ3-Αβ in post-mortem brain tissue from: AD patients by immunohistochemistry (De Kimpe et al., 2013; WO2011151076); AD patients by ELISA (Morawski et al., 2013); AD mice by ELISA (Wu et al, 2013; US8058405); AD mice by immunohistochemistry (US8512677); AD patients, AD mice, and aged rhesus macaques by immunohistochemistry (Wirths et al, 2013); AD patients by sandwich ELISA (Wirths et al., 2010); and AD patients at different disease stages by
immunohistochemistry (Mandler et al, 2012). ρΕ3-Αβ has been detected in plasma from AD patients by sandwich ELISA (WO201 1 151076). Unmodified Αβ has been detected in CSF by ELISA (WO2013009703) and by immunoprecipitation (Bibl et al., 2012).
[0010] ρΕ3-Αβ has been detected in murine AD brains by immunoprecipitation combined with mass spectrometry (Wittnam et al, 2012) and by one- and two- dimensional gel electrophoresis combined with immunoblotting and mass spectrometry (Bibl et al., 2012). ρΕ3-Αβ has been detected in brain tissues from patients with AD, sporadic Alzheimer's disease (SAD), and familial Alzheimer's dementias (FAD) by immunoprecipitation combined with mass spectrometry (Portelius et al., 2010).
[0011] During early stages of AD, however, biomarkers are present at much lower concentrations than during late stages of AD, and detection may be difficult using current methods. Yet early detection is critical for therapeutic intervention, as neuron loss begins several decades before the onset of AD symptoms. In particular, cerebrospinal fluid (CSF) provides a more rapid and sensitive readout of changes in the brain compared to other biological samples, and it is known that AD even in its earliest stages can cause changes in CSF (Blennow et al., 2009; Mehta et al., 2000; Blennow and Hampel, 2003). Thus, a highly sensitive method for the detection of ρΕ3-Αβ in CSF may allow not only early detection of disease, but may provide an accessible biological sample for use in drug development, a process for which it is crucial to identify and monitor the effects of drugs in patients. However, CSF contains particularly low concentrations of Αβ, especially in AD patients (Mehta et al., 2000; Kawarabayashi et al, 2001 ; Blennow and Hampel, 2003; Fagan et al., 2006; Strozyk et al., 2003), which poses a significant technical challenge for detection. Thus, there is a significant unmet need for highly sensitive assays for the detection of ρΕ3-Αβ, particularly for early disease stages and/or in biological samples containing low levels οΐρΕ3-Αβ.
[0012] The invention provides methods for the detection of amyloid β oligomers in biological samples and diagnostic, prognostic, and therapeutic uses thereof. In specific embodiments, the invention provides highly sensitive and reliable immunoPCR methods for detecting and determining levels of ρΕ3-Αβ. The methods of the invention may be used to detect and determine levels of ρΕ3-Αβ in biological samples containing low levels of ρΕ3-Αβ, such as samples from early stages of disease, samples that are very dilute, and/or samples of very limited volume. In particular, the inventive assays described herein may be used to detect and determine levels of ρΕ3-Αβ in cerebrospinal fluid. In some embodiments, the invention may be used to determine relative levels of ρΕ3-Αβ in cerebrospinal fluid.
[0013] In some embodiments, the methods of the invention may be used for the diagnosis and prognosis of diseases characterized by ρΕ3-Αβ. In some embodiments, the disease is AD, in particular the early stages of AD. In further embodiments, the methods of the invention may be used to stratify disease states, monitor disease progression, and/or assess responsiveness to treatment. In addition, the methods of the invention may be used to tailor and direct therapies.
BRIEF DESCRIPTION OF THE FIGURES
[0014] Fig. 1 shows a schematic diagram of the steps involved in one embodiment of the invention. A biological sample that potentially contains ρΕ3-Αβ is contacted by a capture antibody immobilized on an ELISA plate, a biotinylated detection antibody, and a streptavidin-oligonucleotide complex. The resulting complex is released from the ELISA plate and amplified by PCR.
[0015] Fig. 2 shows a schematic diagram of the steps involved in an alternate embodiment of the invention. A biological sample that potentially contains ρΕ3-Αβ is contacted by a capture antibody immobilized on an ELISA plate and an oligonucleotide- conjugated detection antibody. The resulting complex is released from the ELISA plate and amplified by PCR.
[0016] Fig. 3 shows a hypothetical PCR amplification plot, which does not necessarily reflect a typical amplification plot for ρΕ3-Αβ. A lower threshold cycle (CT) indicates a higher starting analyte concentration.
[0017] Fig. 4 shows a hypothetical standard curve, which does not necessarily reflect a typical standard curve for ρΕ3-Αβ.
[0018] Fig. 5 shows optimization of streptavidin-oligonucleotide parameters using one of two capture antibodies, 9D5 or AB 100-11, and a biotinylated detection antibody AB 100-11.
[0019] Fig. 6 shows optimization of immunoPCR parameters in a ρΕ3-Αβ depletion assay using antibodies 2F02, AB 100-11, HO-1, or buffer.
[0020] Fig. 7 shows normalized ACT results using 1 μg/mL or 2 μg/mL of capture antibody 9D5 or AB 100-11 and 1 g/mL or 0.2 μg/mL of detection antibody ABlOO-1 1.
[0021] Figs. 8A-B show the detection of ρΕ3-Αβ in plasma (Fig. 8 A) and cerebrospinal fluid (Fig. 8B). DESCRIPTION OF EMBODIMENTS
[0022] In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.
[0023] "Amyloid β" and "Αβ" are used interchangeably to refer to amyloid β peptide and modifications, including pyroglutamate Αβ, fragments, and/or equivalents thereof. In particular, "Αβ" as used herein refers to any fragment produced by the proteolytic cleavage of amyloid precursor protein (APP).
[0024] "Αβ oligomers" is used to refer to multimeric species of Αβ that result from the association of monomeric Αβ species.
[0025] The term "amyloidosis" refers to a group of diseases characterized by the deposition of insoluble amyloid plaques, including but not limited to, Alzheimer's disease, sporadic Alzheimer's disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA), Down syndrome (DS), mild cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Parkinson-Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creutzfeld Jacob disease, Parkinson's disease, HIV-associated dementia (HAD), amyotropic lateral sclerosis (ALS), type 2 diabetes, and secondary amyloidosis resulting from other diseases, including but not limited to, tuberculosis, osteomyelitis, rheumatoid arthritis, granulomatous ileitis, Hodgkin's lymphoma, leprosy, and familial Mediterranean fever. [0026] "ρΕ3-Αβ" or "ΑβρΕ3" as used herein refers to N-terminally modified pyroglutamate amyloid β peptides that start at the glutamic acid (Glu) residue at position 3 of the amino acid sequence of Αβ, wherein the Glu residue is cyclized to form a pyroglutamic acid residue. Examples of such oligomers include but are not limited to ΑβΡΕ3-38, ΑβΡΕ3-4ο, and ΑβΡΕ3-42. The amino acid sequences of these exemplary N-terminally modified forms of Αβ peptides are:
Αβ 3-38 (SEQ ID NO: 1):
Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-
Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-
Gly
Αβ 3-40 (SEQ ID NO: 2):
Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe- Phe-Ala-
Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly- Val
Αβ 3-42 (SEQ ID NO: 3):
Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-
Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-
Gly-Val-Val-Ile-Ala
[0027] "Unmodified Αβ" is used to refer to amyloid β peptides in which the N terminus is not modified post-translationally, including but not limited to Αβι-38, Αβι-4ο, and Αβ1 -42. The amino acid sequences of these exemplary unmodified Αβ peptides are: Αβ 1-38 (SEQ ID NO: 4):
Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-
Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-
Val-Gly-Gly
Αβ 1-40 (SEQ ID NO: 5):
Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-
Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-
Val-Gly-Gly-Val-Val
Αβ 1-42 (SEQ ID NO: 6):
Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-
Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-
Val-Gly-Gly-Val-Val-Ile-Ala
[0028] An antibody that is "specific for ρΕ3-Αβ" or a "pE3^-specific antibody" refers to an antibody that exhibits binding affinity for ρΕ3-Αβ and does not exhibit significant binding affinity for other forms of Αβ, including other forms of pyroglutamate Αβ, e.g., pEl l-Αβ. Examples of ρΕ3-Αβ-8ρεϋϊβΰ antibodies include but are not limited to:
ABlOO-11 ;
9D5 (DSM ACC3056);
8C4 (DSM ACC3066);
AB 5-5-6 (DSM ACC2923);
AB 6-1-6 (DSM ACC2924);
AB 17-4-3 (DSM ACC2925); AB 24-2-3 (DSM ACC2926);
B12L, comprising the amino acid sequences of
SEQ ID NO: 7 (light chain):
DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPG
QSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY
CVQGTHYPFTFGQGT LEI RTVAAPSVFIFPPSDEQLKSGTASVV
CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
and
SEQ ID NO: 8 (heavy chain):
QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQG LEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSE DTAVYYCAREGITVYWGQGTTVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LS S V VTVP S S S LGTQT YICNVNHKP SNTKVDK VEPKS CDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKA GQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVD SRWQQGNVFSCSVMHEALHNHYTQ SLSLSPG; R17L, comprising the amino acid sequences of SEQ ID NO: 7 (light chain) and SEQ ID NO: 9 (heavy chain):
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTRYYINWVRQAPGQG LEWMGWINPGSGNTKYNEKF GRVTITADESTSTAYMELSSLRSE DTAVYYCAREGTTVYWGQGTTVTVSSAST GPSVFPLAPSSKSTS GGT A ALGCL VKD YFPEP VTV S WNS G ALTS G VHTFP A VLQ S S GL YS L S SWT VP S S SLGTQT YICN VNHKP SNTKVDKKVEPKS CDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNA TKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENYKTTPPVLDSDGSFFLYS KLTVD SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;
hE8L, comprising the amino acid sequences of SEQ ID NOs: 7 (light chain) and SEQ ID NO: 10 (heavy chain):
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYINWVRQAPGQG LEWMGWINPGSGNT YNEKFKGRVTITADESTSTAYMELSSLRSE DTAVYYCAREGETVYWGQGTTVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS L S SWT VP S S SLGTQT YICN VNHKP SNTKVDKKVEPKS CDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG; CI-C7, comprising the amino acid sequences of
SEQ ID NO: 11 (light chain):
DIQMTQSPSSLSASVGDRVTITCKSTRSLLYSRSKTYLNWYQQKP GKAPKLLIYAVSKLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCVQGTHYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
and
SEQ ID NO: 12 (heavy chain):
EVQLVQSGAEVK PGESLKISCKGSGYTFTDYYINWVRQMPGKG
LEWMGWINPGSGNTKYNEKFKGQVTISADKSISTAYLQWSSLKA
SDTAMYYCAREGVTVYWGQGTLVTVSSASTKGPSVFPLAPSSKS
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
L YSLS S V VT VP S S S LGTQT YICNVNHKP SNTKVD KVEPKS CDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKT PREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
G.
[0029] "Αβ capture antibody" refers to an antibody that binds an Αβ oligomer or Αβ oligomer complex that is being detected and/or measured in a biological sample. In some embodiments, the Αβ capture antibody is specific for ρΕ3-Αβ. In embodiments wherein the capture antibody is not specific for ρΕ3-Αβ, then the detection antibody is specific for ρΕ3-Αβ.
[0030] "Αβ detection antibody" refers to an antibody that binds an Αβ oligomer or Αβ oligomer complex that is being detected and/or measured in a biological sample and is conjugated to a second complex (examples include, but are not limited to, biotin, an oligonucleotide, digoxigenin, fluorescein). In some embodiments, the Αβ detection antibody is specific for ρΕ3-Αβ. In embodiments wherein the detection antibody is not specific for ρΕ3-Αβ, then the capture antibody is specific for ρΕ3-Αβ (i.e., at least one of the capture antibody or the detection antibody is specific for ρΕ3-Αβ).
[0031] "Polypeptide", "peptide", and "protein" are used interchangeably to refer to a polymer of amino acids.
[0032] The articles "a" and "an," as used herein, should be understood to mean "at least one," unless clearly indicated to the contrary.
[0033] The phrase "and/or," when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, "A, B, and/or C" indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase "and/or" may be used interchangeably with "at least one of or "one or more of the elements in a list.
[0034] Previously, Frost et al. had used immunostaining to detect ρΕ3-Αβ in plaques. Bayer et al. (WO201 1151076) had used an ELISA to detect ρΕ3-Αβ in plasma. Morawski et al. (2013), Wu et al. (2013), Probiodrug AG (US8058405), and Wirths et al. (2010) had used ELISAs to detect ρΕ3-Αβ in the brain. De Kimpe et al. (2013), Bayer et al. (WO2011151076), Eli Lilly & Co. (WO2012021469), Wirths et al. (2013), Dealwis (US8512677), and Mandler et al. (2012) had used immunostaining to detect ρΕ3-Αβ in the brain. In addition, Wittnam et al., (2012), Bibl et al. (2012), and
Portelius et al. (2010) had used mass spectrometry to detect ρΕ3-Αβ in the brain.
However, detection of ρΕ3-Αβ remains challenging in samples containing relatively low levels of ρΕ3-Αβ, such as cerebrospinal fluid.
[0035] The invention provides methods for detecting and determining levels of Αβ oligomers in biological samples. In particular, the invention provides highly sensitive methods to detect and determine levels of ρΕ3-Αβ oligomers in biological samples. The methods of the invention may be used for biological samples containing low levels of ρΕ3-Αβ oligomers, such as samples from early stages of disease, samples that are very dilute, and/or samples of very limited volume. In particular, the invention may be used to detect and determine levels of ρΕ3-Αβ in cerebrospinal fluid.
[0036] In one aspect of these embodiments, a biological sample that potentially contains ρΕ3-Αβ is contacted by two antibodies, a capture antibody and a detection antibody, in an immunoassay. In some embodiments, the detection antibody is directly conjugated to an oligonucleotide, which is analyzed by PCR to determine the presence or absence of ρΕ3-Αβ in a biological sample. In alternate embodiments, the detection antibody is indirectly conjugated to an oligonucleotide, which is analyzed by PCR to determine the presence or absence of ρΕ3-Αβ in a biological sample. For example, the detection antibody is conjugated to a first binding moiety; the oligonucleotide is conjugated to a second binding moiety; and the first binding moiety and the second binding moiety are binding partners that bind together to form a detection antibody- oligonucleotide complex before, at the same time as, or after contacting the biological sample. The oligonucleotide is analyzed by PCR to determine the presence or absence of ρΕ3-Αβ in a biological sample. In certain embodiments, the first binding moiety is biotin, and the second binding moiety is streptavidin, i.e., the detection antibody is biotinylated and binds to a streptavidin-oligonucleotide complex, which is analyzed by PCR to determine the presence or absence of ρΕ3-Αβ in a biological sample. In certain embodiments, the first binding moiety is digoxigenin, and the second binding moiety is an anti-digoxigenin antibody. In alternate embodiments, the first and second binding moieties are biotin and avidin; biotin and neutravidin; fluorescein and an anti- fluorescein antibody; or other first and second binding moieties that are binding partners. [0037] In some embodiments, the PCR is quantitative PCR (qPCR), and at least one normalized ACT result is generated. Optionally, the normalized ACT results are analyzed in comparison to a standard curve to determine ρΕ3-Αβ levels in a biological sample. In an alternate embodiment, the invention may be used to determine relative levels of ρΕ3-Αβ in biological samples.
[0038] In one embodiment, the methods of the invention are used to diagnose a disease characterized by ρΕ3-Αβ. In a further embodiment, the methods of the invention are used to prognose and/or treat a disease characterized by ρΕ3-Αβ. The methods of the invention may be used to stratify disease states, monitor disease progression, and/or assess responsiveness to treatment. In addition, the methods of the invention may be used to tailor and direct therapies. In some embodiments, the disease is Alzheimer's disease, in particular in the early stages of the disease.
[0039] The invention provides methods for detecting and determining levels of Αβ oligomers in biological samples. Biological samples may include a fluid, a cell, or a tissue sample. Biological fluids may include cerebrospinal fluid, plasma, serum, blood, saliva, urine, sweat, or peritoneal fluid. A cell sample or a tissue sample may include a biopsy, a tissue, a cell suspension, or other specimens and samples, such as clinical samples. Specimens and samples may be obtained, for example, for the diagnosis, prognosis, and/or treatment of a disease.
[0040] In some embodiments, the disease to be diagnosed, prognosed, or treated is primary amyloidosis. Diseases of primary amyloidosis include, but are not limited to, Alzheimer's disease, sporadic Alzheimer's disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA), Down syndrome (DS), mild cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Parkinson-Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creutzfeld Jacob disease, Parkinson's disease, HIV-associated dementia (HAD), amyotropic lateral sclerosis (ALS), and type 2 diabetes.
[0041] In some embodiments, the disease to be diagnosed, prognosed, or treated is secondary amyloidosis. Diseases of secondary amyloidosis include, but are not limited to tuberculosis, osteomyelitis, rheumatoid arthritis, granulomatous ileitis, Hodgkin's lymphoma, leprosy, and familial Mediterranean fever.
[0042] In some embodiments, the methods of the invention are used to determine the relative amount of ρΕ3-Αβ in two or more biological samples. Two or more biological samples are assayed by PC as described above, and at least one normalized ACj value is determined for each sample. Two or more normalized AC values may be compared to determine the relative amount of ρΕ3-Αβ in the samples.
[0043] In certain embodiments, the two or more biological samples are collected from different human subjects. It is known that CSF concentrations of unmodified Αβ are lower in AD patients (Mehta et al., 2000; Blennow and Hampel, 2003), possibly due to increased Αβ aggregation the brain (Fagan et al., 2006; Strozyk et al., 2003), but relative concentrations of ρΕ3-Αβ in the CSF of AD patients is unknown. In one embodiment, the methods of the invention are used to evaluate CSF samples collected from AD patients and healthy controls. The CSF samples are assayed by PCR and analyzed for normalized ACj values as described above. The normalized ACT values are compared to determine the relative amount of ρΕ3-Αβ in the samples. Analysis of ρΕ3-Αβ in cerebrospinal fluid in Fig. 8B suggests that normalized ACj values may be lower in AD than in control samples, although the difference is not statistically significant at this sample size. [0044] In certain embodiments, CSF samples are collected from AD patients, healthy controls, and at least one undiagnosed subject. The CSF samples are assayed by PCR as described above and analyzed for normalized ACj values. The normalized ACT value(s) of at least one undiagnosed subject are compared to the normalized ACT values of AD patients and healthy controls. The analysis may be used in the diagnosis of AD.
[0045] In some embodiments, the two or more biological samples are collected from the same human subject. For example, cerebrospinal fluid and plasma are collected from the same human subject, and relative levels of ρΕ3-Αβ are assayed by PCR. Relatively low levels of Αβ in CSF may be indicative of increased Αβ
aggregation in the Αβ plaques of the brain (Fagan et al., 2006; Strozyk et al., 2003). Analysis of ρΕ3-Αβ in cerebrospinal fluid in Fig. 8B suggests that normalized ACT values may be lower in AD than in control samples. The analysis may be useful in the diagnosis, prognosis, and treatment of disorders characterized by ρΕ3-Αβ.
[0046] In some embodiments, the methods of the invention are used to measure ρΕ3-Αβ levels in a biological sample. As described above, one or more biological samples containing unknown amounts of ρΕ3-Αβ are assayed by PCR and at least one normalized ACT value is calculated for each biological sample. At least three samples containing known amounts of ρΕ3-Αβ are also assayed by PCR, and at least one normalized ACT value is calculated for each sample. In certain embodiments wherein two or more normalized ACT values are determined for a sample, an average normalized ACT value may be calculated and used as the normalized ACT value of the sample. Normalized ACT values of the samples containing known amounts of ρΕ3-Αβ are plotted against their corresponding ρΕ3-Αβ concentrations to generate a standard curve. The ρΕ3-Αβ level of the biological sample containing an unknown amount of ρΕ3-Αβ is calculated by comparing the normalized ACT value of the biological sample to the normalized ACj values of the standard curve. In certain embodiments, normalized ACj can be calculated by subtracting the ACj of the negative control sample(s) from the ACT of the sample(s) of interest. Fig. 4 shows a hypothetical standard curve using ACj values and does not necessarily reflect a typical standard curve for ρΕ3-Αβ.
[0047] In some embodiments, the methods of the invention are used to detect, but not to determine levels of ρΕ3-Αβ. For example, the methods are first used to determine the presence or absence of ρΕ3-Αβ. In biological samples wherein the presence of ρΕ3-Αβ is indicative of a disease, the sample is further evaluated by an alternate quantitative technique. In biological samples wherein the absence of ρΕ3-Αβ is indicative of a disease, a second biological sample is collected from a different tissue. The second sample is analyzed for the presence or absence of ρΕ3-Αβ, as described above, and/or by an alternate quantitative technique. In some embodiments, the alternate quantitative technique is mass spectrometry, in particular liquid chromatography-tandem mass spectrometry. Capturing ρΕ3-Αβ in a biological sample
[0048] The methods of the invention comprise contacting a biological sample with two antibodies, a capture antibody and a detection antibody, in an immunoassay. In some embodiments, at least one of the capture antibody or the detection antibody is specific for ρΕ3-Αβ. ρΕ3-Αβ-8ρεαίίϋ antibodies disclosed in Wirths et al., 2010, WO201 1 151076, Frost et al., 2012, WO2012021469, Mandler et al., and WO2009149486, each incorporated herein by reference include: ABlOO-1 1
9D5 (DSM ACC3056)
8C4 (DSM ACC3066)
AB 5-5-6 (DSM ACC2923)
AB 6-1 -6 (DSM ACC2924) AB 17-4-3 (DSM ACC2925)
AB 24-2-3 (DSM ACC2926)
B12L (SEQ ID NOs: 7 and 8)
R17L (SEQ ID NOs: 7 and 9)
hE8L (SEQ ID NOs: 7 and 10)
CI-C7 (SEQ ID NOs: 11 and 12)
[0049] In some embodiments, only the capture antibody is specific for ρΕ3-Αβ.
In some embodiments, only the detection antibody is specific for ρΕ3-Αβ. In some embodiments, both the capture antibody and the detection antibody are specific for pE3-
Αβ. For example, the capture antibody and the detection antibody are selected from the group consisting of ABlOO-l l, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3,
B12L, CI-C7, hE8L, R17L, and other pE3-A -specific antibodies. In some embodiments, the capture antibody and the detection antibody are the same antibody.
In a certain embodiment, both the capture antibody and the detection antibody are
AB 100-1 1. In an alternate embodiment, both the capture antibody and the detection antibody are 9D5. In some embodiments, the capture antibody and the detection antibody are different antibodies. For example, the capture antibody is AB 100-11, and the detection antibody is selected from the group consisting of 9D5, 8C4, AB 5-5-6, AB
6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, R17L, and other pE3-Ap-specific antibodies. Or, the capture antibody is 9D5, and the detection antibody is selected from the group consisting of AB 100-1 1, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3,
B12L, CI-C7, hE8L, R17L, and other pE3-Ap-specific antibodies. Alternatively, the capture antibody is selected from the group consisting of 9D5, 8C4, AB 5-5-6, AB 6-1-
6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, R17L, and other pE3-Ap-specific antibodies, and the detection antibody is AB 100-1 1. Or, the capture antibody is selected from the group consisting of AB 100-11, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24- 2-3, B12L, CI-C7, hE8L, R17L, and other pE3-Ap-specific antibodies, and the detection antibody is 9D5. In a certain embodiment, the capture antibody is AB 100-1 1 and the detection antibody is 9D5. In an alternate embodiment, the capture antibody is 9D5 and the detection antibody is AB 100-11. In some embodiments, neither the capture antibody nor the detection antibody is AB 100-1 1.
[0050] In some embodiments, the capture antibody concentration ranges from about 0.1 μg/mL to about 2 μg/mL; about 2 μg/mL to about 5 μg/mL; or about 5 to about 10 μg/mL. In certain embodiments, the capture antibody concentration is 1 μg/mL. In alternate embodiments, the capture antibody concentration is 2 μg/mL. In some embodiments, the detection antibody concentration ranges from about 0.02 μg/mL to about 1 μg/mL; about 1 μg/mL to about 3 μg/mL; or about 3 μg/mL to about 5 μg/mL. In certain embodiments, the detection antibody concentration is 0.2 μg/mL. In alternate embodiments, the detection antibody concentration is 1 μg/mL.
[0051] In one embodiment, the capture antibody contacts the biological sample and binds ρΕ3-Αβ to form a capture antibody/pE3-Ap complex. The detection antibody contacts the biological sample and binds ρΕ3-Αβ to form a detection antibody/pE3-A complex. In certain embodiments, the detection antibody binds ρΕ3-Αβ, in particular the capture antibody/pE3-AP complex. In some embodiments, the capture antibody contacts the biological sample before, at the same time as, or after the detection antibody contacts the biological sample. In a certain embodiment, the capture antibody and the detection antibody are the same antibody, and the ρΕ3-Αβ that is detected is multimeric (i.e., more than one ρΕ3-Αβ monomer is present, because the capture antibody and detection antibody bind to the same epitope of ρΕ3-Αβ).
[0052] In some embodiments, the capture antibody is immobilized on a surface. For example, the capture antibody may be immobilized on a plate (e.g., Corning Costar® or NUNC MaxiSorp®), a channel, or a column. In some embodiments, the plate is a streptavidin-coated plate, and the capture antibody is biotinylated.
[0053] The skilled artisan would appreciate that pEl l-Αβ or other ρΕ-Αβ species could be detected by exchanging the pE3-Ap-specific antibody for a pEl 1-Αβ- specific antibody (see, e.g., Perez-Garmendia et al., 2010; commercially available NBP 1-44070) or other ρΕ-Αβ-specific antibodies.
Detecting ρΕ3-Αβ in a biological sample
[0054] In some embodiments, the detection antibody contacts the biological sample and binds ρΕ3-Αβ to form a detection antibody/pE3-Aβ complex. In certain embodiments, the detection antibody binds ρΕ3-Αβ, in particular the capture
antibody/pE3-Aβ complex. In some embodiments, the detection antibody is directly or indirectly conjugated to an oligonucleotide. In some embodiments, the oligonucleotide is synthetic, custom designed, and comprises no homology to known human sequences. In certain embodiments, the oligonucleotide is at least 20 bases in length. In certain embodiments, the oligonucleotide has minimal homopolymers. In certain embodiments, the oligonucleotide has minimal G quadruplexes.
[0055] In certain embodiments, the oligonucleotide is directly conjugated to the detection antibody, e.g., using commercially available kits. In alternate embodiments, the oligonucleotide is indirectly conjugated to the detection antibody before, at the same time as, or after the detection antibody contacts the biological sample and binds pE3- Αβ. In these embodiments, the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety; the first binding moiety and the second binding moiety are binding partners that bind together to form a detection antibody-oligonucleotide complex before, at the same time as, or after contacting the biological sample. In some embodiments, the first and/or the second binding moieties are capable of multimeric binding. In some embodiments, the detection antibody is added to the sample to bind ρΕ3-Αβ before, at the same time as, or after the capture antibody.
[0056] In some embodiments, the first and second binding moieties form a detection antibody-oligonucleotide complex after the detection antibody contacts the biological sample and binds ρΕ3-Αβ. For example, a capture antibody and a detection antibody conjugated to a first binding moiety contact the biological sample and each binds ρΕ3-Αβ, and then a second binding moiety conjugated to an oligonucleotide binds the first binding moiety. In alternate embodiments, the first and second binding moieties form a detection antibody-oligonucleotide complex at the same time as or before the detection antibody contacts the biological sample. For example, a capture antibody and a detection antibody-oligonucleotide complex contact the biological sample and each binds ρΕ3-Αβ.
[0057] In certain embodiments, the first binding moiety is biotin, and the biotin- conjugated detection antibody is generated using commercially available kits (e.g., EZ- Link™). In certain embodiments, the second binding moiety is streptavidin and the streptavidin-conjugated oligonucleotide is generated using commercially available kits (e.g., TurboLink™). In some embodiments, the streptavidin-oligonucleotide
concentration ranges from about 0.01 ng/mL to about 2 ng/mL; about 2 ng/mL to about 5 ng/mL; or about 5 ng/mL to about 10 ng/mL. In some embodiments, the streptavidin- oligonucleotide concentration is 1 ng/mL. In certain embodiments, the first binding moiety is digoxigenin, and the second binding moiety is an anti-digoxigenin antibody. In alternate embodiments, the first and second binding moieties are biotin and avidin; biotin and neutravidin; fluorescein and an anti-fluorescein antibody; or other first and second binding moieties that are binding partners. [0058] When the detection antibody is bound to ρΕ3-Αβ, in particular to the capture antibody/pE3-Ap complex, the sample is transferred to a PCR plate. In embodiments wherein the capture antibody is immobilized, a release buffer is added before transferring the sample. In some embodiments, the PCR plate comprises a reaction mixture that comprises primers, probes, polymerase, and free nucleotides. In certain embodiments, the reaction mixture comprises primers, probes, DNA
polymerase, salts, magnesium, free nucleotides, and optimized PCR buffer. In certain embodiments, the DNA polymerase is Taq polymerase. The reaction mixture is heated and cooled to amplify the oligonucleotide, and the amplification activity is analyzed. In certain embodiments, the primers and probes are a custom primer-probe mix comprising a primer to the oligonucleotide which is conjugated to a probe (e.g., reporter and quencher moieties).
[0059] In some embodiments, the sample is analyzed by quantitative PCR (qPCR). qPCR may be used to amplify, detect, and/or quantify oligonucleotide sequences. In some embodiments, a fluorophore is added to the reaction mixture, and a thermal cycler is used to illuminate the reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. In a certain embodiment, the fluorophore is the probe in a custom primer probe comprising a fluorescent reporter dye conjugated to a primer custom designed based on the sequence of the
oligonucleotide that is being quantified. In some embodiments, the reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100°C, 60-70°C, and 30-50°C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97°C, 55-65°C, and 35-45°C for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT).
[0060] In embodiments wherein CT results are obtained from the qPCR reaction (see Fig. 3 for a hypothetical amplification plot), ACT can be calculated by subtracting the CT from the maximum cycle number, e.g., 40. In certain embodiments, ACT ranges from 10-40 cycles. Normalized ACT can be calculated by subtracting the ACT of the negative control sample(s) from the ACT of the sample(s) of interest. In some embodiments, normalized ACT results are used to determine the presence or absence of ρΕ3-Αβ in biological samples. Absence may be defined as concentrations of ρΕ3-Αβ that are not detectable (i.e., CT beyond the limit of detection, for example, >40 threshold cycles). Presence may be defined as concentrations of ρΕ3-Αβ that are detectable (i.e., within the limit of detection, for example, CT between 10-40 threshold cycles).
Kits
[0061] The antibodies and reagents useful in the methods of the invention can be provided in a kit (i.e., a packaged combination of reagents with instructions for performing the assay). The kit may include antibodies, antibody complexes, substrates, and/or cofactors. In addition, additives such as stabilizers, buffers, and other solutions may be included. The relative amounts of the reagents may be varied to provide for concentrations that optimize the sensitivity of the assay.
[0062] In a specific embodiment, the kit is a diagnostic kit. The diagnostic kit may be used for the detection and diagnosis of ρΕ3-Αβ diseases and conditions, particularly Alzheimer's disease. LIST OF EMBODIMENTS
1. A method of detecting ρΕ3-Αβ in a biological sample comprising
(a) contacting the sample with a pE3-AP-specific capture antibody;
(b) contacting the sample with a pE3-AP-specific detection antibody, wherein
(i) the detection antibody is indirectly conjugated to an oligonucleotide before, at the same time as, or after contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody- oligonucleotide complex before, at the same time as, or after contacting the sample;
or
(ii) the detection antibody is directly conjugated to an oligonucleotide to form a detection antibody-oligonucleotide complex;
(c) providing the oligonucleotide with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d).
2. The method of embodiment 1, comprising
(a) contacting the sample with a pE3-AP-specific capture antibody;
(b) contacting the sample with a ρΕ3-Αβ-8ρεαΑϋ detection antibody, wherein the detection antibody is indirectly conjugated to an oligonucleotide after contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody-oligonucleotide complex after contacting the sample;
(c) providing the oligonucleotide with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d). 3. The method of embodiment 1, comprising
(a) contacting the sample with a pE3-AP-specific detection antibody, wherein the detection antibody is indirectly conjugated to an oligonucleotide after contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody-oligonucleotide complex after contacting the sample;
(b) contacting the sample with a pE3-A -specific capture antibody;
(c) providing the oligonucleotide with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d). 4. The method of embodiment 1 , comprising
(a) contacting the sample with a pE3-AP-specific capture antibody;
(b) contacting the sample with a pE3-AP-specific detection antibody, wherein the detection antibody is indirectly conjugated to an oligonucleotide at the same as or before contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody-oligonucleotide complex at the same as or before contacting the sample;
(c) providing the oligonucleotide with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d).
5. The method of embodiment 1, comprising
(a) contacting the sample with a pE3-AP-specific detection antibody, wherein the detection antibody is indirectly conjugated to an oligonucleotide at the same as or before contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody-oligonucleotide complex at the same as or before contacting the sample;
(b) contacting the sample with a pE3-AP-specific capture antibody; (c) providing the oligonucleotide with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d).
6. The method of any one of embodiments 1-5, wherein the first binding moiety is biotin, and the second binding moiety is streptavidin.
7. The method of any one of embodiments 1-5, wherein the first binding moiety is digoxigenin, and the second binding moiety is an anti-digoxigenin antibody.
8. The method of any one of embodiments 1-7, wherein the first binding moiety and/or the second binding moiety are capable of multimeric binding.
9. The method of any one of embodiments 1-8, wherein the capture antibody contacts the sample before, at the same time as, or after the detection antibody contacts the sample.
10. The method of any one of embodiments 1-9, wherein the capture antibody and detection antibody are selected from the group consisting of AB 100-11, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L. 11. The method of embodiment 10, wherein the capture antibody and the detection antibody are not the same antibody.
12. The method of embodiment 10, wherein the capture antibody and the detection antibody are the same antibody.
13. The method of embodiment 12, wherein the ρΕ3-Αβ that is detected is multimeric ρΕ3-Αβ.
14. The method of any one of embodiments 1-9, wherein the capture antibody is selected from the group consisting of AB 100-11 and 9D5, and wherein the detection antibody is selected from the group consisting of AB 100-11, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L.
15. The method of embodiment 14, wherein the capture antibody is AB 100-11, and wherein the detection antibody is selected from the group consisting of ABlOO-11, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI- C7, hE8L, and R17L.
16. The method of embodiment 14, wherein the capture antibody is 9D5, and wherein the detection antibody is selected from the group consisting of AB100- 11, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L. 17. The method of any one of embodiments 1-9, wherein the capture antibody is selected from the group consisting of AB 100-11, 9D5, 8C4, AB 5-5-6, AB 6-1- 6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L, and wherein the detection antibody is selected from the group consisting of AB 100-11 and 9D5.
18. The method of embodiment 17, wherein the capture antibody is selected from the group consisting of AB 100-1 1, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17- 4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L, and wherein the detection antibody is ABlOO-11.
19. The method of embodiment 17, wherein the capture antibody is selected from the group consisting of ABlOO-11, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17- 4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L, and wherein the detection antibody is 9D5.
20. The method of any one of embodiments 1-9, wherein the capture antibody and detection antibody are selected from the group consisting of AB 100-11 and 9D5.
21. The method of embodiment 20, wherein the capture antibody is 9D5, and wherein the detection antibody is 9D5.
22. The method of embodiment 20, wherein the capture antibody is AB 100-1 1, and wherein the detection antibody is AB 100-1 1. 23. The method of embodiment 20, wherein the capture antibody is AB 100-1 1 , and wherein the detection antibody is 9D5.
24. The method of embodiment 20, wherein the capture antibody is 9D5, and wherein the detection antibody is AB 100-1 1.
25. The method of any one of embodiments 1 -9, wherein the capture antibody is immobilized on a surface and released from the surface prior to amplification.
26. The method of embodiment 25, wherein the surface is a plate, a channel, or a column.
27. The method of any one of embodiments 1 -9, wherein the concentration of the capture antibody is 0.1 - 10 μg/mL.
28. The method of any one of embodiments 1 -9, wherein the concentration of the detection antibody is 0.02 μg/mL - 10 g/mL.
29. The method of embodiment 6, wherein the concentration of the streptavidin- oligonucleotide conjugate is 0.01 ng/mL - 10 ng/niL.
30. The method of any one of embodiments 1 -9, wherein at least one CT value is obtained for the biological sample. 31. The method of embodiment 30, wherein the presence of ρΕ3-Αβ in the biological sample is determined by the at least one CT value of the biological sample being within the limit of detection.
32. The method of embodiment 30, wherein the absence of ρΕ3-Αβ in the biological sample is determined by the at least one C value of the biological sample being beyond the limit of detection.
33. The method of embodiment 30, wherein the ρΕ3-Αβ level in the biological sample is measured by
(a) further obtaining at least one CT value for each of at least three known concentrations of ρΕ3-Αβ;
(b) using the CT values of (a) to generate a standard curve comprising the at least three CT values and their corresponding ρΕ3-Αβ concentrations; and
(c) calculating the concentration of ρΕ3-Αβ in the biological sample by comparing the at least one CT value of the biological sample to the CT values of the standard curve.
34. The method of embodiment 30, wherein relative ρΕ3-Αβ levels in the biological sample are measured by
(a) further obtaining at least one CT value for at least one additional biological sample; and
(b) comparing the CT values to determine relative levels of ρΕ3-Αβ in the biological samples. (a) diagnose a disease characterized by ρΕ3-Αβ;
(b) prognose a disease characterized by ρΕ3-Αβ; and/or
(c) develop, tailor, and/or direct therapies for a disease characterized by ρΕ3-Αβ.
36. The disease of embodiment 35, wherein the disease is selected from the group consisting of Alzheimer's disease, sporadic Alzheimer's disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA), Down syndrome (DS), mild cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Parkinson-Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creutzfeld Jacob disease, Parkinson's disease, HIV-associated dementia (HAD), amyotropic lateral sclerosis (ALS), type 2 diabetes, tuberculosis, osteomyelitis, rheumatoid arthritis, granulomatous ileitis, Hodgkin's lymphoma, leprosy, and familial Mediterranean fever.
37. The biological sample of any one of embodiments 1 -5, wherein the biological sample is selected from the group consisting of cerebrospinal fluid, plasma, serum, blood, saliva, urine, sweat, peritoneal fluid, a biopsy, a tissue sample, a clinical specimen, a cell sample, and a cell suspension.
38. The biological sample of any one of embodiments 1 -5, wherein the biological sample contains low levels of ρΕ3-Αβ. 39. The biological sample of embodiment 37 or 38, wherein the biological sample is cerebrospinal fluid.
40. The biological sample of embodiment 37 or 38, wherein the biological sample is plasma.
41. A kit for assaying a biological sample for the presence, absence, or level of ρΕ3-Αβ, the kit comprising
(a) instructions for assaying the biological sample for ρΕ3-Αβ;
(b) at least one capture antibody of any one of embodiments 1-5;
(c) at least one detection antibody of any one of embodiments 1-5, optionally wherein the detection antibody is complexed to a first binding moiety;
(d) optionally at least one oligonucleotide conjugated to a second binding moiety, wherein the first binding moiety and the second binding moiety are binding partners that bind to form a detection antibody-oligonucleotide complex.
42. The method of any one of embodiments 1-5 for detecting ρΕ3-Αβ in a cerebrospinal fluid sample comprising
(a) contacting the sample with a capture antibody selected from antibody 9D5 or antibody ABlOO-1 1 ;
(b) contacting the sample with a detection antibody selected from antibody 9D5 or antibody AB 100-11, wherein
(i) the detection antibody is indirectly conjugated to an oligonucleotide before, at the same time as, or after contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody- oligonucleotide complex before, at the same time as, or after contacting the sample; or
(ii) the detection antibody is directly conjugated to an oligonucleotide to form a detection antibody-oligonucleotide complex;
(c) providing the oligonucleotide of (b) with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the oligonucleotide; and
(e) analyzing the amplification activity of (d).
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[0064] The following examples provide illustrative embodiments of the disclosure. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The Examples do not in any way limit the disclosure.
EXAMPLES
[0065] The examples discussed below are intended to be purely exemplary of the invention and should not be considered to limit the invention in any way. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.), but some experimental errors and deviations should be expected.
Example 1: Capture antibody immobilization
[0066] The 9D5 or AB 100-1 1 capture antibody was immobilized on a plate. A NUNC Maxisorp® plate was coated with capture antibody at a dilution of 1 g/mL or 2 μg/mL. The plate was sealed and incubated for 18-24 hours at 2-8° C while shaking on a microtiter plate shaker. The plate was washed eight times with PBS-Tween20 Wash Buffer. Example 2: ρΕ3-Αβ capture and detection using a bio tiny lated detection antibody
[0067] Blocking solution comprising 1.00 μg/mL of yeast tRNA in PBS/Casein was added to the capture antibody plate. The plate was washed four times with PBS- Tween20 Wash Buffer. Cerebrospinal fluid samples were added to the plate. The plate was sealed and incubated for 18-24 hours at 2-8° C while shaking on a microtiter plate shaker. The plate was washed eight times with PBS-Tween20 Wash Buffer.
[0068] The AB 100-11 detection antibody was conjugated to biotin using commercially available methods (EZ-Link™). Detection antibody was added to the plate at a dilution of 0.2 μg/mL or 1 μg/mL. The plate was sealed and incubated for 55- 65 minutes at 22-26°C while shaking on a microtiter plate shaker. The plate was washed eight times with PBS-Tween20 Wash Buffer.
[0069] Streptavidin-oligonucleotide was added to the plate at a concentration of 1 ng/mL. The plate was sealed and incubated for 55-65 minutes at 22-26°C while shaking on a microtiter plate shaker. The plate was washed eight times with PBS Tween20 wash buffer, followed by four washes with Tris-EDTA wash buffer (pH 8.0).
[0070] Release buffer comprising DTT and 1 μg/mL yeast tRNA was added to the plate. The plate was sealed and incubated for 45-55 minutes at 37°C, then shaken for 5-15 minutes at 22-26°C on a microtiter plate shaker. A portion of the sample was transferred to a PCR plate containing a mixture of PCR master mix as well as custom primer probe sets. PCR amplification was performed using a Roche LightCycler 480 instrument. Optimized antibody concentrations are shown in Table 1, and normalized ACt results are shown in Fig. 7. Table 1. Exemplary antibody conditions using biotinylated detection antibodies
Figure imgf000042_0001
Example 3: Analysis of immunoPCR results
[0071] Threshold cycle (CT) results were obtained from the PCR reaction. A low CT value indicates a high concentration of initial analyte. A hypothetical amplification plot is shown in Fig. 3, which does not necessarily reflect a typical amplification plot for ρΕ3-Αβ.
Example 4: Optimization of immunoPCR (streptavidin-oligonucleotide concentration)
[0072] Two lots of streptavidin-oligonucleotide were produced using commercially available methods (TurboLink™). Both were tested in parallel using capture antibody 9D5 or AB 100-1 1, followed by biotinylated detection antibody ABlOO-11 , as shown in Fig. 5. A streptavidin-oligonucleotide conjugate concentration of 1 ng/mL was used for subsequent assessments.
Example 5: Assay specificity assessment
[0073] In order to determine whether the analyte captured in the assay was specific for ρΕ3-Αβ, plasma samples were first depleted of analyte using: the same antibody as the capture antibody; the ρΕ3-Αβ specific antibody 2F02; the non-specific antibody HO- 1 ; or a buffer control. In three of the four samples tested, a reduction in measured analyte levels can be seen when depleted with AB 100- 1 1 or 2F02, but the reduction was lower in buffer-depleted or HO- 1 depleted samples (Fig. 6).
Example 6: Detection in cerebrospinal fluid (CSF) samples
[0074] The methods of the invention may be used to detect ρΕ3-Αβ in cerebrospinal fluid, which is challenging using current methods. ρΕ3-Αβ in CSF samples was captured and detected using a biotinylated detection antibody as described in Example 2. Analysis of ρΕ3-Αβ in plasma for AD and control samples is shown in Fig. 8A. Analysis of ρΕ3-Αβ in cerebrospinal fluid in Fig. 8B suggests that normalized ACT values may be lower in AD than in control samples.

Claims

We claim:
1. A method of detecting ρΕ3-Αβ in a biological sample comprising
(a) contacting the sample with a pE3-Ap-specific capture antibody;
(b) contacting the sample with a pE3-Ap-specific detection antibody, wherein
(i) the detection antibody is indirectly conjugated to an oligonucleotide before, at the same time as, or after contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody-oligonucleotide complex before, at the same time as, or after contacting the sample;
or
(ii) the detection antibody is directly conjugated to an oligonucleotide to form a detection antibody-oligonucleotide complex;
(c) providing the oligonucleotide of (b) with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d).
2. The method of claim 1 , comprising
(a) contacting the sample with a pE3-AP-specific capture antibody;
(b) contacting the sample with a pE3-AP-specific detection antibody, wherein the detection antibody is indirectly conjugated to an oligonucleotide after contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody-oligonucleotide complex after contacting the sample;
(c) providing the oligonucleotide with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d).
3. The method of claim 1 , comprising
(a) contacting the sample with a pE3-AP-specific detection antibody, wherein the detection antibody is indirectly conjugated to an oligonucleotide after contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody-oligonucleotide complex after contacting the sample;
(b) contacting the sample with a pE3-A -specific capture antibody;
(c) providing the oligonucleotide with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d).
4. The method of claim 1 , comprising
(a) contacting the sample with a pE3-Ap-specific capture antibody;
(b) contacting the sample with a pE3-AP-specific detection antibody, wherein the detection antibody is indirectly conjugated to an oligonucleotide at the same time as or before contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody- oligonucleotide complex at the same time as or before contacting the sample;
(c) providing the oligonucleotide with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d).
5. The method of claim 1, comprising
(a) contacting the sample with a pE3-AP-specific detection antibody, wherein the detection antibody is indirectly conjugated to an oligonucleotide at the same time as or before contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody - oligonucleotide complex at the same time as or before contacting the sample;
(b) contacting the sample with a pE3-AP-specific capture antibody; (c) providing the oligonucleotide with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d).
6. The method of any one of claims 1-5, wherein the first binding moiety is biotin, and the second binding moiety is streptavidin.
7. The method of any one of claims 1-5, wherein the first binding moiety is digoxigenin, and the second binding moiety is an anti-digoxigenin antibody.
8. The method of any one of claims 1-7, wherein the first binding moiety and/or the second binding moiety are capable of multimeric binding.
9. The method of any one of claims 1-8, wherein the capture antibody contacts the sample before, at the same time as, or after the detection antibody contacts the sample.
10. The method of any one of claims 1-9, wherein the capture antibody and detection antibody are selected from the group consisting of ABlOO-11 , 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L.
11. The method of claim 10, wherein the capture antibody and the detection antibody are not the same antibody.
12. The method of claim 10, wherein the capture antibody and the detection antibody are the same antibody.
13. The method of claim 12, wherein the ρΕ3-Αβ that is detected is multimeric ρΕ3-Αβ.
14. The method of any one of claims 1-9, wherein the capture antibody is selected from the group consisting of AB 100-11 and 9D5, and wherein the detection antibody is selected from the group consisting of ABlOO-11, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L.
15. The method of claim 14, wherein the capture antibody is AB 100-1 1, and wherein the detection antibody is selected from the group consisting of AB 100-11, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L.
16. The method of claim 14, wherein the capture antibody is 9D5, and wherein the detection antibody is selected from the group consisting of ABlOO-11 , 9D5, 8C4, AB 5- 5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L.
17. The method of any one of claims 1-9, wherein the capture antibody is selected from the group consisting of ABlOO-11, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24- 2-3, B12L, CI-C7, hE8L, and R17L, and wherein the detection antibody is selected from the group consisting of ABlOO-1 1 and 9D5.
18. The method of claim 17, wherein the capture antibody is selected from the group consisting of ABlOO-11, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L, and wherein the detection antibody is AB 100-1 1.
19. The method of claim 17, wherein the capture antibody is selected from the group consisting of ABlOO-1 1, 9D5, 8C4, AB 5-5-6, AB 6-1-6, AB 17-4-3, AB 24-2-3, B12L, CI-C7, hE8L, and R17L, and wherein the detection antibody is 9D5.
20. The method of any one of claims 1-9, wherein the capture antibody and detection antibody are selected from the group consisting of AB 100-1 1 and 9D5.
21. The method of claim 20, wherein the capture antibody is 9D5, and wherein the detection antibody is 9D5.
22. The method of claim 20, wherein the capture antibody is AB 100-11, and wherein the detection antibody is AB 100-11.
23. The method of claim 20, wherein the capture antibody is AB 100-11, and wherein the detection antibody is 9D5.
24. The method of claim 20, wherein the capture antibody is 9D5, and wherein the detection antibody is AB 100-1 1.
25. The method of any one of claims 1-9, wherein the capture antibody is immobilized on a surface and released from the surface prior to amplification.
26. The method of claim 25, wherein the surface is a plate, a channel, or a column.
27. The method of any one of claims 1-9, wherein the concentration of the capture antibody is 0.1 - 10 μg/mL.
28. The method of any one of claims 1-9, wherein the concentration of the detection antibody is 0.02 μg/mL - 10 μg/mL.
29. The method of claim 6, wherein the concentration of the streptavidin- oligonucleotide conjugate is 0.01 ng/mL - 10 ng/mL.
30. The method of any one of claims 1-9, wherein at least one Cj value is obtained for the biological sample.
31. The method of claim 30, wherein the presence of ρΕ3-Αβ in the biological sample is determined by the at least one CT value of the biological sample being within the limit of detection.
32. The method of claim 30, wherein the absence of ρΕ3-Αβ in the biological sample is determined by the at least one CT value of the biological sample being beyond the limit of detection.
33. The method of claim 30, wherein the ρΕ3-Αβ level in the biological sample is measured by (a) further obtaining at least one CT value for each of at least three known concentrations of ρΕ3-Αβ;
(b) using the Cj values of (a) to generate a standard curve comprising the at least three C values and their corresponding ρΕ3-Αβ concentrations; and
(c) calculating the concentration of ρΕ3-Αβ in the biological sample by comparing the at least one CT value of the biological sample to the C values of the standard curve.
34. The method of claim 30, wherein relative ρΕ3-Αβ levels in the biological sample are measured by
(a) further obtaining at least one C value for at least one additional biological sample; and
(b) comparing the C values to determine relative levels of ρΕ3-Αβ in the biological samples.
35. Use of the methods of any one of claims 30-34 to
(a) diagnose a disease characterized by ρΕ3-Αβ;
(b) prognose a disease characterized by ρΕ3-Αβ; and/or
(c) develop, tailor, and/or direct therapies for a disease characterized by ρΕ3-Αβ.
36. The disease of claim 35, wherein the disease is selected from the group consisting of Alzheimer's disease, sporadic Alzheimer's disease (SAD), familial Alzheimer's dementias (FAD), familial British dementia (FBD), familial Danish dementia (FDD), cerebral amyloid angiopathy (CAA), Down syndrome (DS), mild cognitive impairment (MCI), Lewy body dementia, macular degeneration, cardiac amyloidosis, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Parkinson-Dementia complex of Guam, supranuclear palsy, multiple sclerosis, prion diseases, Creutzfeld Jacob disease, Parkinson's disease, HIV-associated dementia (HAD), amyotropic lateral sclerosis (ALS), type 2 diabetes, tuberculosis, osteomyelitis, rheumatoid arthritis, granulomatous ileitis, Hodgkin's lymphoma, leprosy, and familial Mediterranean fever.
37. The biological sample of any one of claims 1-5, wherein the biological sample is selected from the group consisting of cerebrospinal fluid, plasma, serum, blood, saliva, urine, sweat, peritoneal fluid, a biopsy, a tissue sample, a clinical specimen, a cell sample, and a cell suspension.
38. The biological sample of any one of claims 1-5, wherein the biological sample contains low levels of ρΕ3-Αβ.
39. The biological sample of claim 37 or 38, wherein the biological sample is cerebrospinal fluid.
40. The biological sample of claim 37 or 38, wherein the biological sample is plasma.
41. A kit for assaying a biological sample for the presence, absence, or level of pE3- Αβ, the kit comprising
(a) instructions for assaying the biological sample for ρΕ3-Αβ;
(b) at least one capture antibody of any one of claims 1-5;
(c) at least one detection antibody of any one of claims 1-5, optionally wherein the detection antibody is complexed to a first binding moiety; (d) optionally at least one oligonucleotide conjugated to a second binding moiety, wherein the first binding moiety and the second binding moiety are binding partners that bind to form a detection antibody-oligonucleotide complex.
42. The method of any one of claims 1-5 for detecting ρΕ3-Αβ in a cerebrospinal fluid sample comprising
(a) contacting the sample with a capture antibody selected from antibody 9D5 or antibody AB 100-1 1 ;
(b) contacting the sample with a detection antibody selected from antibody 9D5 or antibody AB 100-11, wherein
(i) the detection antibody is indirectly conjugated to an oligonucleotide before, at the same time as, or after contacting the sample, wherein the detection antibody is conjugated to a first binding moiety, and the oligonucleotide is conjugated to a second binding moiety, wherein the first binding moiety and second binding moiety are binding partners that bind together to form a detection antibody-oligonucleotide complex before, at the same time as, or after contacting the sample;
or
(ii) the detection antibody is directly conjugated to an oligonucleotide to form a detection antibody-oligonucleotide complex;
(c) providing the oligonucleotide with a reaction mixture comprising primers, polymerase, and free nucleotides;
(d) heating and cooling the reaction mixture of (c) to amplify the
oligonucleotide; and
(e) analyzing the amplification activity of (d).
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