- Email: [email protected]
Beta-amyloid oligomer detection in cerebrospinal fluid and brain tissue
Oral Sessions: O2-03: Biomarkers: Novel Background: Alzheimer’s disease and other dementias are one of the major health and social care challenges for the 21st century worldwide. The global number of people with dementia was calculated at 36 million by 2010 and is estimated to increase to 66 million by 2030 and 115 million by 2050 (World Alzheimer Report 2009). The global cost is estimated at US$604 billion by 2010 (World Alzheimer Report 2010). Governments and societies are not prepared to these challenges. Therefore Alzheimer’s Disease International (ADI), the global federation of Alzheimer associations is campaigning to make dementia a global health priority with programs of World Health Organization (WHO) and United Nations (UN). Methods: ADI has started an advocacy program towards WHO and UN meetings including training of national advocates, creating an international network and visiting international and regional meetings and make statements, contact country representatives and produce data on prevalence and cost of illness. Results: Dementia was included in the Mental Health Global Action Programme of WHO as a priority area. The UN High Level Meeting on Non-Communicable Diseases in September 2011 adopted a paragraph on the importance of mental health and Alzheimer’s disease. The WHO is going to release a report Dementia: A Public Health Priority by April 2012. A resolution on ageing will be put forward at the annual WHO assembly in May 2012. Conclusions: Alzheimer’s disease and dementia have become part of the international health agenda. This needs to be implemented in national and subnational action plans. This has happened in a few countries like Australia, Korea, France and England. A plan for the USA is on its way. Monitoring and evaluation of those plans will be crucial as well as securing the funding. Countries without a plan need to be encouraged to develop. ORAL SESSIONS: O2-03 BIOMARKERS: NOVEL O2-03-01
BETA-AMYLOID-SPECIFIC CD4+ T CELL RESPONSES CHANGE WITH AGE AND ALZHEIMER’S DISEASE
Doug Ethell, Aynun Begum, Celia Cunha, Harpreet Sidhu, Tursun Alkam, Western University of Health Sciences, Pomona, California, United States. Background: Adaptive immune responses to amyloid-beta (Ab) have been of intense interest for diagnostic and therapeutic studies of Alzheimer’s disease (AD) over the past 10-12 years. Alzheimer’s subjects may have benefitted from Ab vaccination (AN1792), but the trial was halted due brain infiltration by Ab-specific Th1 cells, which likely arose during an unbridled inflammatory response to Ab. We have since reported that Ab-specific CD4+ Th2 (non-inflammatory) cells can provide beneficial effects for cognition and pathology - in an AD mouse model - without those cells infiltrating meninges. It has remained unclear if this naturally-occurring CD4+ sub-population might play a protective role against the development of Alzheimer’s disease, or if changes in their activity might presage disease onset or the course of this disease. Methods: Technical difficulty in identifying, isolating and expanding Abspecific CD4+ T cells from human blood have led to problems in determining how adaptive immune responses might affect AD progression. We have developed a platform to assess antigen-specific immune responses in human blood by engineering dendritic cells that we derive from human stem cells (hESC and iPSC). Using this methodology, we have evaluated the breadth and strength of CD4+ T cell responses to Ab in over 60 human subjects, including several with Alzheimer’s disease. Results: Early stage Alzheimer’s patients showed broad CD4+ T cell responses to Ab. A similar but reduced profile was observed in advanced AD. We also examined response profiles in control non-AD subjects from 20 to 88 years of age. Subjects under 50 years of age had small or narrow responses to Ab1-42. However, in the 5th and 6th decade of life some subjects, not all, displayed broad and robust responses. This increase was most common in subjects who carried at least one copy of ApoE4. An interesting subgroup in this study is an 88 year old Alzheimer’s patient and 7 children who ranged in age from 44 to 64. Conclusions: Ab-specific immune pro-
P239
filing may provide an associative diagnostic tool for changes in precede cognitive impairment and AD pathology. O2-03-02
AMORFIX EPITOPE PROTECTIONALZHEIMER’S DISEASE (EP-AD) DIAGNOSTIC CEREBROSPINAL FLUID TEST FOR ACCURATE IDENTIFICATION OF ALZHEIMER’S DISEASE AND MILD COGNITIVE IMPAIRMENT
Louise Scrocchi1, Elizabeth Karaskov1, Hui Chen1, Vivian Lee1, Ryan Demers1, Masoud Yousefi2, Marni Uger1, Neil Cashman1, 1Amorfix Life Sciences, Ltd., Mississauga, Ontario, Canada; 2University of British Columbia, Vancouver, British Columbia, Canada. Background: More than 35 million people worldwide have Alzheimer’s disease (AD). A major stumbling block to treatment is the absence of robust biomarkers for early detection and monitoring during clinical trials. Amorfix Life Sciences has developed the EPAD Diagnostic CSF Test as an ultra-sensitive biomarker assay for detection of oligomeric Ab, a known biomarker for AD, while minimizing the signal from monomeric Aß present at high concentrations in human CSF. Methods: The Test makes use of proprietary methodology to detect oligomeric Ab: CSF samples were treated using Epitope Protection (EP) technology to chemically modify epitopes present on the surface of monomeric Aß, which prevents their detection. This was followed by affinity-based enrichment of oligomeric Ab, and detection using the Amorfix ultra-sensitive dual-bead immunoassay. The concentration of monomeric Ab 1-42 was measured directly with the dual-bead immunoassay. All testing was performed with the samples blinded to the operator. Results: Two hundred (200) CSF samples were tested: seventy nine (79) AD, forty nine (49) MCI, and seventy two (72) controls. ROC analysis demonstrated diagnostic sensitivity and specificity of 82.0% and 81.9% respectively. Signals from the Test were higher in MCI CSF than AD CSF. S/N values: MCI (3.22+/- 0.3), AD (2.37+/0.14), and control (1.31+/-0.04). Monomeric Aß 1-42: MCI (1.81+/0.17) controls (1.59+/-0.13) and AD (0.86+/-0.09). The positive predictive value (PPV) for oligomeric Aß was 82% for both AD and MCI CSF, whereas the PPV for monomeric Ab 1-42 alone was 78% (AD) or 39% (MCI). The negative predictive value (NPV) for oligomeric Aß was 78% for AD and 89% for MCI, whereas the NPV for monomeric Ab 1-42 was 66% for AD and 49% for MCI. Conclusions: We have developed a unique system for the detection of oligomeric Ab, a recognized biomarker for AD. The EP-AD CSF Test can be used to identify AD or MCI patients when compared to non-AD controls. A comparison of monomeric Aß 1-42 can be then be used to further distinguish between AD and MCI patients. Our data demonstrate that the EP-AD CSF Diagnostic Test has a higher predictive value than measurements of Aß1-42 alone for identification of patients with MCI.
O2-03-03
BETA-AMYLOID OLIGOMER DETECTION IN CEREBROSPINAL FLUID AND BRAIN TISSUE
Kim Bruggink1, H. Bea Kuiperij1, Wesley Jongbloed2, Rob Veerhuis2, Marcel M. Verbeek1, 1Radboud University Medical Centre Nijmegen, Nijmegen, Netherlands; 2VU University Medical Center, Amsterdam, Netherlands. Background: Alzheimer’s Disease (AD) is the most common cause of dementia worldwide. Presently used CSF biomarkers can discriminate fairly well between AD and healthy controls, but are insufficiently able to distinguish between different forms of dementia. New biomarkers for early detection are urgently needed. Pathological hallmarks of AD are extracellular deposits of aggregated amyloid b (Ab). Ab is known to aggregate from monomers, via oligomers to fibrils. Oligomeric Ab is believed to be the most neurotoxic form. Therefore, Ab oligomers might be a specific diagnostic marker for AD. Methods: In this study we developed an ELISA specific for Ab oligomers, by using the same monoclonal capture and (labeled) detection antibody. The specificity of the ELISA was tested with synthetic
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Oral Sessions: O2-03: Biomarkers: Novel
Ab. A mutant form of Ab, that shows significantly reduced aggregation, was used as a negative control. Furthermore, we describe a method to abolish interference from human-anti-mouse heterophilic antibodies in human biological samples. Finally, oligomers were measured in CSF samples from AD (n ¼ 25) and non-demented control (n ¼ 25) patients and brain tissue of both humans (10 AD, 6 controls) and APP/PS1 transgenic mice. Results: First, we showed that oligomers can be measured in biological samples. In transgenic murine tissue, Ab oligomer levels in the cortex and hippocampus increased with age. In human hippocampi, Ab oligomers were significantly increased in AD compared to controls. Ab oligomer levels were similar in CSF of AD and control patients. Characterization of the assay by size-exclusion-chromatography analysis demonstrated that low molecular weight Ab oligomers, rather than larger Ab oligmers, are particularly detected by this ELISA. Conclusions: We developed an ELISA that is specific for low molecular weight Ab oligomers and is able to measure oligomeric Ab in biological samples. Our analysis confirmed that Ab oligomer concentrations are increased in human hippocampal AD tissue compared to controls and increased in Tg mice brain tissue. However CSF concentrations of Ab oligomers, as detected by this assay, may not serve as a biomarker for AD. Future studies will have to reveal if high molecular weight Ab oligomers may serve as such biomarkers. O2-03-04
UTILIZATION OF A PROTEASE-GENERATED FRAGMENT OF TAU AS A BIOMARKER OF ALZHEIMER’S DISEASE
Mette Sørensen1, Yaguo Wang2, Monika Pajak3, Kevin Duffin4, AnneChristine Bay-Jensen3, Natasha Barascuk3, Morten Karsdal3, Kim Henriksen3, 1Nordic Bioscience, Herlev, Denmark; 2Nordic Bioscience A/S, Bejiing, China; 3Nordic Bioscience A/S, Herlev, Denmark; 4Eli Lily and Company, Indianapolis, Indiana, United States. Background: Biomarkers of neurodegeneration in Alzheimer’s disease (AD) have long been restricted to CSF. Serological biomarkers for detection of proteins from the brain are hampered by the blood-brain barrier where only small protein fragments can cross. During AD pathological enzymatic processing of brain proteins, such as Tau, is known to happen, and we speculate that small protein fragments containing pathologically relevant information will cross the blood-brain barrier in AD patients. Hence, we aimed to develop an early serological marker for AD, focusing on specific fragments of Tau. Methods: Tau was cleaved with ADAM10 and fragments identified by Mass spectrometry. NB191, a monoclonal antibody was raised against one of the Tau cleavage fragments and used to develop an ELISA assay (Tau-A ELISA). Brains from Tg4510 and wild-type mice were extracted together with a series of rat organs. Serum samples from AD patients were investigated and compared to healthy age- and sex-matched controls. Results: NB191 recognizes a cleavage site generated by ADAM10 but not an elongated sequence with one more amino acid, and the Tau-A ELISA showed through dilution and spike recovery tests in human serum to be technically robust, with an LDL of 2.932ng/ml and intra- and inter-assay CVs of 5.84% and 12.6%. Higher levels of the Tau fragment were detected both when human recombinant Tau protein was in vitro cleaved with ADAM10 compared to non-cleaved Tau, and when rat brain, but not other tissues, was digested using ADAM10. These data were further supported by the finding that intact Tau primarily was found in brain. In brain extracts from Tg4510 mice the Tau-A level was 10-fold higher than in wild-type mice (P <0.001). Importantly, in 32 serum samples from AD patients and 70 from healthy controls a trend (P ¼ 0.09) towards increased levels was observed in the AD samples. For AD patients an inverse correlation between MDRS and Tau-A was observed (R ¼ 0.4 and P ¼ 0.002) Conclusions: We have developed an ELISA assay detecting a pathologically relevant fragment of Tau in serum samples, speculating that the assay could be a future tool for prognosis and diagnosis of Alzheimer’s disease. Clinically important an inverse correlation between MDRS and Tau-A was detected.
O2-03-05
BETA-AMYLOID-17 IS A MAJOR BETA-AMYLOID FRAGMENT ISOFORM IN CEREBROSPINAL FLUID AND BLOOD THAT SHOWS DIAGNOSTIC VALUE
Virginia Perez1, Leticia Sarasa1, Jose A. Allue1, Diego Casabona1, Maria Montanes1, Daniel Insua1, Hassnae Badi1, Inmaculada Monleon1, Ana M. Lacosta1, Itziar San Jose1, Lluis Tarraga2, Merce Boada-Rovira2, Pedro Pesini1, Manuel Sarasa1, 1Araclon Biotech Ltd., Logro~no and Zaragoza, Spain; 2Fundacio ACE. Institut Catala de Neurociencies Aplicades, Barcelona, Spain. Background: In addition to the main Ab40 and Ab42 isoforms produced by the activity of beta- and gamma-secretases, there are other Ab isoforms or Ab fragments produced by the activity of other proteases, which might be implicated in the pathogenesis of Alzheimer disease (AD). Methods: By immunoprecipitation and mass spectrometry we have analized the Ab isoforms present in CSF and blood. We have developed an antibody specific for Abx-17 and by ELISA sandwichwe have measured the amount of Ab1-17 that is free in plasma (FP), its total amount in plasma (TP) and that that is bound to blood cells (CB) in groups over 65 years old of healthy control (HC), probable (prodromal) and possible (non-prodromal) mild cognitive impairment (MCI) and mild-AD patients (n ¼ 64; 16 per group). By immunohistochemistry we have studied the presence of Ab17 in the senile plaques of the brain. Results: We have discovered thatAb1-17, an Ab fragment produced by the activity of beta- and alpha-secretases, is a predominant Ab fragment isoform in blood and CSF. It is the most abundant Ab fragment in CSF and blood after Ab1-40. The ratio FP/CB of Ab17 in blood discriminated between HC and probable-MCI or mild-AD patients with high sensitivity and specificity, or between possible-MCI and mild-AD patients (discrimination between HC and AD: sensitivity ¼ 0.88; specificity ¼ 0.79; AUC ¼ 0.86; p<0.001; and discrimination between possible-MCI and AD: sensitivity ¼ 0.88; specificity ¼ 0.71; AUC ¼ 0.83; P <0.01). Ab17 is present in the senile plaques of AD brains. Conclusions: These results reinforce the interest of blood Ab markers for early diagnosis of Ab pathology, screening of participants, and assessment of efficacy in clinical trials of Ab-targeted drugs.
O2-03-06
AN LC-MS/MS-BASED METHOD FOR QUANTIFICATION OF BETA-AMYLOID-1-38, BETA-AMYLOID-1-40 AND BETA-AMYLOID-1-42 IN THE CEREBROSPINAL FLUID OF ALZHEIMER’S PATIENTS AND HEALTHY CONTROLS
Josef Pannee1, Johan Gobom1, Madalina Opperman2, Alan Atkins2, Henrik Zetterberg3, Lennart Minthon4, Kaj Blennow1, Oskar Hansson5, Erik Portelius1, 1Institute of Neuroscience and Physiology, M€olndal, Sweden; 2Thermo Scientific, Hemel Hempstead, United Kingdom; 3 University of Gothenburg, M€olndal, Sweden; 4Clinical Memory Research Unit, Malm€o, Sweden; 5Lund University, Malm€o, Sweden. Background: There is a major need in the Alzheimer’s Disease (AD) community for an assay that with high sensitivity, specificity and reproducibly can measure biomarkers with an excellent stability over time. The assays used today are antibody-based and struggling with lot-to-lot variability and elusive matrix effects that influence analyte quantitation depending on sample handling. To overcome the variability and the possible matrix effects that appear with antibody-based amyloid b (Ab) assays, we have developed a selected reaction monitoring (SRM) assay for Ab1-38, Ab1-40 and Ab1-42 in cerebrospinal fluid (CSF). Methods: The CSF samples were extracted for Ab using solid-phase-extraction (SPE) in a 96-well format. The SRM quantifications of Ab isoforms were performed on a TSQ Vantage mass spectrometer. The absolute concentrations of Ab1-38, Ab1-40 and Ab1-42 were determined by adding stable isotope-labeled variants of the peptides to the CSF before SPE purification. Results: The method has been validated for specificity, recovery, intra- and inter-assay precision,
BETA-AMYLOID-SPECIFIC CD4+ T CELL RESPONSES CHANGE WITH AGE AND ALZHEIMER’S DISEASE
Doug Ethell, Aynun Begum, Celia Cunha, Harpreet Sidhu, Tursun Alkam, Western University of Health Sciences, Pomona, California, United States. Background: Adaptive immune responses to amyloid-beta (Ab) have been of intense interest for diagnostic and therapeutic studies of Alzheimer’s disease (AD) over the past 10-12 years. Alzheimer’s subjects may have benefitted from Ab vaccination (AN1792), but the trial was halted due brain infiltration by Ab-specific Th1 cells, which likely arose during an unbridled inflammatory response to Ab. We have since reported that Ab-specific CD4+ Th2 (non-inflammatory) cells can provide beneficial effects for cognition and pathology - in an AD mouse model - without those cells infiltrating meninges. It has remained unclear if this naturally-occurring CD4+ sub-population might play a protective role against the development of Alzheimer’s disease, or if changes in their activity might presage disease onset or the course of this disease. Methods: Technical difficulty in identifying, isolating and expanding Abspecific CD4+ T cells from human blood have led to problems in determining how adaptive immune responses might affect AD progression. We have developed a platform to assess antigen-specific immune responses in human blood by engineering dendritic cells that we derive from human stem cells (hESC and iPSC). Using this methodology, we have evaluated the breadth and strength of CD4+ T cell responses to Ab in over 60 human subjects, including several with Alzheimer’s disease. Results: Early stage Alzheimer’s patients showed broad CD4+ T cell responses to Ab. A similar but reduced profile was observed in advanced AD. We also examined response profiles in control non-AD subjects from 20 to 88 years of age. Subjects under 50 years of age had small or narrow responses to Ab1-42. However, in the 5th and 6th decade of life some subjects, not all, displayed broad and robust responses. This increase was most common in subjects who carried at least one copy of ApoE4. An interesting subgroup in this study is an 88 year old Alzheimer’s patient and 7 children who ranged in age from 44 to 64. Conclusions: Ab-specific immune pro-
P239
filing may provide an associative diagnostic tool for changes in precede cognitive impairment and AD pathology. O2-03-02
AMORFIX EPITOPE PROTECTIONALZHEIMER’S DISEASE (EP-AD) DIAGNOSTIC CEREBROSPINAL FLUID TEST FOR ACCURATE IDENTIFICATION OF ALZHEIMER’S DISEASE AND MILD COGNITIVE IMPAIRMENT
Louise Scrocchi1, Elizabeth Karaskov1, Hui Chen1, Vivian Lee1, Ryan Demers1, Masoud Yousefi2, Marni Uger1, Neil Cashman1, 1Amorfix Life Sciences, Ltd., Mississauga, Ontario, Canada; 2University of British Columbia, Vancouver, British Columbia, Canada. Background: More than 35 million people worldwide have Alzheimer’s disease (AD). A major stumbling block to treatment is the absence of robust biomarkers for early detection and monitoring during clinical trials. Amorfix Life Sciences has developed the EPAD Diagnostic CSF Test as an ultra-sensitive biomarker assay for detection of oligomeric Ab, a known biomarker for AD, while minimizing the signal from monomeric Aß present at high concentrations in human CSF. Methods: The Test makes use of proprietary methodology to detect oligomeric Ab: CSF samples were treated using Epitope Protection (EP) technology to chemically modify epitopes present on the surface of monomeric Aß, which prevents their detection. This was followed by affinity-based enrichment of oligomeric Ab, and detection using the Amorfix ultra-sensitive dual-bead immunoassay. The concentration of monomeric Ab 1-42 was measured directly with the dual-bead immunoassay. All testing was performed with the samples blinded to the operator. Results: Two hundred (200) CSF samples were tested: seventy nine (79) AD, forty nine (49) MCI, and seventy two (72) controls. ROC analysis demonstrated diagnostic sensitivity and specificity of 82.0% and 81.9% respectively. Signals from the Test were higher in MCI CSF than AD CSF. S/N values: MCI (3.22+/- 0.3), AD (2.37+/0.14), and control (1.31+/-0.04). Monomeric Aß 1-42: MCI (1.81+/0.17) controls (1.59+/-0.13) and AD (0.86+/-0.09). The positive predictive value (PPV) for oligomeric Aß was 82% for both AD and MCI CSF, whereas the PPV for monomeric Ab 1-42 alone was 78% (AD) or 39% (MCI). The negative predictive value (NPV) for oligomeric Aß was 78% for AD and 89% for MCI, whereas the NPV for monomeric Ab 1-42 was 66% for AD and 49% for MCI. Conclusions: We have developed a unique system for the detection of oligomeric Ab, a recognized biomarker for AD. The EP-AD CSF Test can be used to identify AD or MCI patients when compared to non-AD controls. A comparison of monomeric Aß 1-42 can be then be used to further distinguish between AD and MCI patients. Our data demonstrate that the EP-AD CSF Diagnostic Test has a higher predictive value than measurements of Aß1-42 alone for identification of patients with MCI.
O2-03-03
BETA-AMYLOID OLIGOMER DETECTION IN CEREBROSPINAL FLUID AND BRAIN TISSUE
Kim Bruggink1, H. Bea Kuiperij1, Wesley Jongbloed2, Rob Veerhuis2, Marcel M. Verbeek1, 1Radboud University Medical Centre Nijmegen, Nijmegen, Netherlands; 2VU University Medical Center, Amsterdam, Netherlands. Background: Alzheimer’s Disease (AD) is the most common cause of dementia worldwide. Presently used CSF biomarkers can discriminate fairly well between AD and healthy controls, but are insufficiently able to distinguish between different forms of dementia. New biomarkers for early detection are urgently needed. Pathological hallmarks of AD are extracellular deposits of aggregated amyloid b (Ab). Ab is known to aggregate from monomers, via oligomers to fibrils. Oligomeric Ab is believed to be the most neurotoxic form. Therefore, Ab oligomers might be a specific diagnostic marker for AD. Methods: In this study we developed an ELISA specific for Ab oligomers, by using the same monoclonal capture and (labeled) detection antibody. The specificity of the ELISA was tested with synthetic
P240
Oral Sessions: O2-03: Biomarkers: Novel
Ab. A mutant form of Ab, that shows significantly reduced aggregation, was used as a negative control. Furthermore, we describe a method to abolish interference from human-anti-mouse heterophilic antibodies in human biological samples. Finally, oligomers were measured in CSF samples from AD (n ¼ 25) and non-demented control (n ¼ 25) patients and brain tissue of both humans (10 AD, 6 controls) and APP/PS1 transgenic mice. Results: First, we showed that oligomers can be measured in biological samples. In transgenic murine tissue, Ab oligomer levels in the cortex and hippocampus increased with age. In human hippocampi, Ab oligomers were significantly increased in AD compared to controls. Ab oligomer levels were similar in CSF of AD and control patients. Characterization of the assay by size-exclusion-chromatography analysis demonstrated that low molecular weight Ab oligomers, rather than larger Ab oligmers, are particularly detected by this ELISA. Conclusions: We developed an ELISA that is specific for low molecular weight Ab oligomers and is able to measure oligomeric Ab in biological samples. Our analysis confirmed that Ab oligomer concentrations are increased in human hippocampal AD tissue compared to controls and increased in Tg mice brain tissue. However CSF concentrations of Ab oligomers, as detected by this assay, may not serve as a biomarker for AD. Future studies will have to reveal if high molecular weight Ab oligomers may serve as such biomarkers. O2-03-04
UTILIZATION OF A PROTEASE-GENERATED FRAGMENT OF TAU AS A BIOMARKER OF ALZHEIMER’S DISEASE
Mette Sørensen1, Yaguo Wang2, Monika Pajak3, Kevin Duffin4, AnneChristine Bay-Jensen3, Natasha Barascuk3, Morten Karsdal3, Kim Henriksen3, 1Nordic Bioscience, Herlev, Denmark; 2Nordic Bioscience A/S, Bejiing, China; 3Nordic Bioscience A/S, Herlev, Denmark; 4Eli Lily and Company, Indianapolis, Indiana, United States. Background: Biomarkers of neurodegeneration in Alzheimer’s disease (AD) have long been restricted to CSF. Serological biomarkers for detection of proteins from the brain are hampered by the blood-brain barrier where only small protein fragments can cross. During AD pathological enzymatic processing of brain proteins, such as Tau, is known to happen, and we speculate that small protein fragments containing pathologically relevant information will cross the blood-brain barrier in AD patients. Hence, we aimed to develop an early serological marker for AD, focusing on specific fragments of Tau. Methods: Tau was cleaved with ADAM10 and fragments identified by Mass spectrometry. NB191, a monoclonal antibody was raised against one of the Tau cleavage fragments and used to develop an ELISA assay (Tau-A ELISA). Brains from Tg4510 and wild-type mice were extracted together with a series of rat organs. Serum samples from AD patients were investigated and compared to healthy age- and sex-matched controls. Results: NB191 recognizes a cleavage site generated by ADAM10 but not an elongated sequence with one more amino acid, and the Tau-A ELISA showed through dilution and spike recovery tests in human serum to be technically robust, with an LDL of 2.932ng/ml and intra- and inter-assay CVs of 5.84% and 12.6%. Higher levels of the Tau fragment were detected both when human recombinant Tau protein was in vitro cleaved with ADAM10 compared to non-cleaved Tau, and when rat brain, but not other tissues, was digested using ADAM10. These data were further supported by the finding that intact Tau primarily was found in brain. In brain extracts from Tg4510 mice the Tau-A level was 10-fold higher than in wild-type mice (P <0.001). Importantly, in 32 serum samples from AD patients and 70 from healthy controls a trend (P ¼ 0.09) towards increased levels was observed in the AD samples. For AD patients an inverse correlation between MDRS and Tau-A was observed (R ¼ 0.4 and P ¼ 0.002) Conclusions: We have developed an ELISA assay detecting a pathologically relevant fragment of Tau in serum samples, speculating that the assay could be a future tool for prognosis and diagnosis of Alzheimer’s disease. Clinically important an inverse correlation between MDRS and Tau-A was detected.
O2-03-05
BETA-AMYLOID-17 IS A MAJOR BETA-AMYLOID FRAGMENT ISOFORM IN CEREBROSPINAL FLUID AND BLOOD THAT SHOWS DIAGNOSTIC VALUE
Virginia Perez1, Leticia Sarasa1, Jose A. Allue1, Diego Casabona1, Maria Montanes1, Daniel Insua1, Hassnae Badi1, Inmaculada Monleon1, Ana M. Lacosta1, Itziar San Jose1, Lluis Tarraga2, Merce Boada-Rovira2, Pedro Pesini1, Manuel Sarasa1, 1Araclon Biotech Ltd., Logro~no and Zaragoza, Spain; 2Fundacio ACE. Institut Catala de Neurociencies Aplicades, Barcelona, Spain. Background: In addition to the main Ab40 and Ab42 isoforms produced by the activity of beta- and gamma-secretases, there are other Ab isoforms or Ab fragments produced by the activity of other proteases, which might be implicated in the pathogenesis of Alzheimer disease (AD). Methods: By immunoprecipitation and mass spectrometry we have analized the Ab isoforms present in CSF and blood. We have developed an antibody specific for Abx-17 and by ELISA sandwichwe have measured the amount of Ab1-17 that is free in plasma (FP), its total amount in plasma (TP) and that that is bound to blood cells (CB) in groups over 65 years old of healthy control (HC), probable (prodromal) and possible (non-prodromal) mild cognitive impairment (MCI) and mild-AD patients (n ¼ 64; 16 per group). By immunohistochemistry we have studied the presence of Ab17 in the senile plaques of the brain. Results: We have discovered thatAb1-17, an Ab fragment produced by the activity of beta- and alpha-secretases, is a predominant Ab fragment isoform in blood and CSF. It is the most abundant Ab fragment in CSF and blood after Ab1-40. The ratio FP/CB of Ab17 in blood discriminated between HC and probable-MCI or mild-AD patients with high sensitivity and specificity, or between possible-MCI and mild-AD patients (discrimination between HC and AD: sensitivity ¼ 0.88; specificity ¼ 0.79; AUC ¼ 0.86; p<0.001; and discrimination between possible-MCI and AD: sensitivity ¼ 0.88; specificity ¼ 0.71; AUC ¼ 0.83; P <0.01). Ab17 is present in the senile plaques of AD brains. Conclusions: These results reinforce the interest of blood Ab markers for early diagnosis of Ab pathology, screening of participants, and assessment of efficacy in clinical trials of Ab-targeted drugs.
O2-03-06
AN LC-MS/MS-BASED METHOD FOR QUANTIFICATION OF BETA-AMYLOID-1-38, BETA-AMYLOID-1-40 AND BETA-AMYLOID-1-42 IN THE CEREBROSPINAL FLUID OF ALZHEIMER’S PATIENTS AND HEALTHY CONTROLS
Josef Pannee1, Johan Gobom1, Madalina Opperman2, Alan Atkins2, Henrik Zetterberg3, Lennart Minthon4, Kaj Blennow1, Oskar Hansson5, Erik Portelius1, 1Institute of Neuroscience and Physiology, M€olndal, Sweden; 2Thermo Scientific, Hemel Hempstead, United Kingdom; 3 University of Gothenburg, M€olndal, Sweden; 4Clinical Memory Research Unit, Malm€o, Sweden; 5Lund University, Malm€o, Sweden. Background: There is a major need in the Alzheimer’s Disease (AD) community for an assay that with high sensitivity, specificity and reproducibly can measure biomarkers with an excellent stability over time. The assays used today are antibody-based and struggling with lot-to-lot variability and elusive matrix effects that influence analyte quantitation depending on sample handling. To overcome the variability and the possible matrix effects that appear with antibody-based amyloid b (Ab) assays, we have developed a selected reaction monitoring (SRM) assay for Ab1-38, Ab1-40 and Ab1-42 in cerebrospinal fluid (CSF). Methods: The CSF samples were extracted for Ab using solid-phase-extraction (SPE) in a 96-well format. The SRM quantifications of Ab isoforms were performed on a TSQ Vantage mass spectrometer. The absolute concentrations of Ab1-38, Ab1-40 and Ab1-42 were determined by adding stable isotope-labeled variants of the peptides to the CSF before SPE purification. Results: The method has been validated for specificity, recovery, intra- and inter-assay precision,