- Email: [email protected]
P3-042 Detection of different tau isoforms in cerebrospinal fluid from patients with Alzheimer's disease using immunoaffinity chromatography-mass spectrometry
Poster Session P3: Diagnosis and Disease Progression - CSF Markers A[3 and the associated neurotoxicity in the brains of affected individuals. The level of A[340 is about 50 or 100 time higher than the that of A[342 in various samples of cerebral fluids (CSF), tissue culture superuatants and brain extracts. Specific assays and precise measurement for human AIM2 without cross-reactivity with A[~40 is critical for interpreting results from biologic, clinical or drug discovery research. There are only two amino acid differences between the natural forms of A[340 and A[342, which create an enormous challenge in developing reagents that have the sensitivity sufficient to detect relevant levels with the necessary specificity. We observed that some commercially available 'A1342' reagents show crossreactivity with A[340 and, thus may be interpreted inappropriately. In this study, we developed rabbit polyclonal antibodies specific to human AIM2 using sequential Iigand affinity chromatography steps. The AIM2 specific assay was developed using a high affinity mouse monoclonal antibody, clone 7N22, to the NH2-terminus of human AI3 and a polyclonal antibodies specific to A[342. The assay is sensitive to 5 pg/mL and shows no cross reactivity with either APP or A[340 (at levels up to 50 ng/mL). Using this highly specific assay, consistent and reliable quantitation of AIM2 in CSE plasma and tissue homogenates is shown.
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PROTEOMIC ANALYSIS OF ANTEMORTEM CEREBROSPINAL FLUID FOR BIOMARKER IDENTIFICATION
a more specific marker for AD than total tau and addition of other posttranslational markers on tau may increase this specificity. Objective(s): Here we describe the set-up and use of matrix-assisted laser desorptionionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting in combination with immunoaffinity chromatography to identify and characterize different tan isoforms in CSF from AD patients. Methods: Anti-tan polyclonal antibodies, which recognize a peptide sequence most likely shared by all tan isoforms, were used in immunoaffinity chromatography (IA) to catch and elute different tan isoforms directly from CSE After tryptic digest and desalting on reversed phase media, the peptide mixture were mass analysed using MALDI-TOF MS. Results: The limit of detection of trypsinated tan in model systems was estimated to be 10 fmole and the IA media was shown to bind tan that could subsequently be eluted and detected by MALDI-TOF MS. The system was applied to patient and control samples and we here show the first results illustrating the capability of the combination of immunobinding and mass separation to unequivocably discriminate between AD-tau and normal tau. Conclusions: The identification of different posttranslationally modified tau isoforms in csf from AD patients using immunoaffinity chromatography mass spectrometry is a promising specific method for diagnosing patients with AD.
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Kelvin Lee* ~, Erin Finehout t , Norman Relkin ~. 1Cornell University, Ithaca, NY, USA; 2 Cornell University, Weill Medical College, New York, NY,, USA. Contact e-mail: [email protected] Background: We are applying a gel-based proteomics strategy to the analysis of antemortem cerebrospinal fluid from Alzheimer patients. Objective(s): Our goal is to identify antemortem biomarkers for AD diagnosis. Methods: Retrospective samples from probable and definite Alzheimer patients as well as age-matched normal and neurological controls have been collected from a number of CSF banks in the United States. Prospective samples are being collected from a well-characterized clinical population in New York. Using a protein two-dimensional electrophoresis, fluorescence staining and laser fluorescence scanning, we are able to visualize approximately 1700 protein spots from typical CSF samples. Results: Matrix assisted laser desorption ionization tandem time of flight mass spectrometry has permitted us to characterize and sequence proteins present in femtomolar quantities, and to identify a number of very low abundance proteins never previously reported in CSE We now report the identification of a set of molecular markers that may be relevant to AD diagnosis and studies of its pathogenesis. These markers are identified using a multivariate statistical approach and represent changes in the observed quantities of at least nine low abundance proteins in AD patients relative to controls. Additional studies are in progress to further characterize these markers and assess their applicability to Alzheimer differential diagnosis. Conclusions: Proteomic analysis of CSF may provide an effective method to identify AD biomarkers.
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DETECTION OF DIFFERENT TAU ISOFORMS IN CEREBROSPINAL FLUID FROM PATIENTS WITH ALZHEIMER'S DISEASE USING IMMUNOAFFINITY CHROMATOGRAPHY-MASS SPECTROMETRY
Justyna M. Czama .1 , Gunhild Watdemar 2, Niels H. Heegaard 1.
1Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark; 2Memory Disorders Research Unit, Dept. of Neurology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark. Contact e-mail: jmc @ssi.dk Background: Despite the fact that Alzheimer's disease (AD) is the most prevalent of dementias, there is currently no satisfactory single specific biochemical marker for this condition. One possible candidate is the neuronal microtubule-associated protein tan. The CSF-levels of tan are elevated in patients with Alzheimer's disease but also in certain other types of dementias. In AD, however, tau is hyperphosphorylated and otherwise posttranslationally modified. Phosphorylated tan appears to be
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INFLAMMATORY MARKERS CRP AND IL-6 IN CEREBROSPINAL FLUID AND SERUM IN RELATION TO THE IL-6 GENOTYPE IN PATIENTS WITH ALZHEIMER'S DISEASE
Alie Schnitemaker* 1, Cees Mulder 2, Niki S. Schoonenboom 3, Miriam Ketema 2 , Evert-Jan van Elk 2, Philip Scheltens 3 , Gerard J. van K a m p 2 t VU University Medical Center, Depts of Neurology and Nuclear
Medicine and Clinical PET research, Amsterdam, Netherlands; 2 VU University Medical Center, Department of Clinical Chemistry, Amsterdam, Netherlands; SVU University Medical Center, Department of Neurology, Amsterdam, Netherlands. Contact e-mail: [email protected] Background Many lines of evidence suggest the involvement of inflammatory processes in the pathogenesis of Alzbeimer's disease (AD). Several immunopathological studies have shown that Amyloid-fl proteins within senile plaques are associated with activated microglia and astrocytes that express pro-inflammatory proteins such as interleukin-6 (IL-6) and acute phase proteins such as C-reactive protein (CRP). Studies on inflammatory proteins in cerebrospinal fluid (CSF) and serum in patients with Alzheimer's disease (AD) show conflicting results. Furthermore, the polymorphism of the gene encoding for IL-6 (-174 G/C) has been associated with an increased risk of early onset AD. Objectives To investigate serum and CSF levels of CRP and IL-6 in patients with 'probable' AD compared to non-demented controls and to relate these concentrations with the IL-6 genotypes, in both groups. Patients a n d Methods: Thirty-seven patients who fulfilled the NINCDS-ADRDA criteria for 'probable' AD and thirty-five age-matched non-demented controls were recruited. CRP and IL-6 concentrations in serum and CSF are measured by sandwich ELISA. IL-6 genotypes were determined by PCR, followed by sequencing. Results: Serum CRP concentrations were found to be lower in AD patients compared to controls (P < 0,001).However, no significant differences in levels of CRP in CSF or levels of IL-6 in serum mad CSF between AD patients and controls could be demonstrated,
Controls AD patients P-value
Median Range Median Range
Serum 1L-6 (rig/L)
CSF 1I.-6 (ng/L)
Serum CRP (rag/L)
CSF CRP (~gfL)
1. i 1 0.42-8.72 1.07 0.18-6.27 0.70
1.61 0.75-5.54 1.40 0.46-13.7 0.50
2.38 0.40-48.8 0.86 0.16-11.1 < 0.001
3.0 0.40-86.8 2.1 0-54.6 0.054
A significant positive correlation between serum and CSF levels of CRP was found (r = 0.82, P < 0.001). No correlation could be demonstrated for IL-6 sentm and CSF values. IL-6 genotypes were similarly distributed
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PROTEOMIC ANALYSIS OF ANTEMORTEM CEREBROSPINAL FLUID FOR BIOMARKER IDENTIFICATION
a more specific marker for AD than total tau and addition of other posttranslational markers on tau may increase this specificity. Objective(s): Here we describe the set-up and use of matrix-assisted laser desorptionionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting in combination with immunoaffinity chromatography to identify and characterize different tan isoforms in CSF from AD patients. Methods: Anti-tan polyclonal antibodies, which recognize a peptide sequence most likely shared by all tan isoforms, were used in immunoaffinity chromatography (IA) to catch and elute different tan isoforms directly from CSE After tryptic digest and desalting on reversed phase media, the peptide mixture were mass analysed using MALDI-TOF MS. Results: The limit of detection of trypsinated tan in model systems was estimated to be 10 fmole and the IA media was shown to bind tan that could subsequently be eluted and detected by MALDI-TOF MS. The system was applied to patient and control samples and we here show the first results illustrating the capability of the combination of immunobinding and mass separation to unequivocably discriminate between AD-tau and normal tau. Conclusions: The identification of different posttranslationally modified tau isoforms in csf from AD patients using immunoaffinity chromatography mass spectrometry is a promising specific method for diagnosing patients with AD.
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Kelvin Lee* ~, Erin Finehout t , Norman Relkin ~. 1Cornell University, Ithaca, NY, USA; 2 Cornell University, Weill Medical College, New York, NY,, USA. Contact e-mail: [email protected] Background: We are applying a gel-based proteomics strategy to the analysis of antemortem cerebrospinal fluid from Alzheimer patients. Objective(s): Our goal is to identify antemortem biomarkers for AD diagnosis. Methods: Retrospective samples from probable and definite Alzheimer patients as well as age-matched normal and neurological controls have been collected from a number of CSF banks in the United States. Prospective samples are being collected from a well-characterized clinical population in New York. Using a protein two-dimensional electrophoresis, fluorescence staining and laser fluorescence scanning, we are able to visualize approximately 1700 protein spots from typical CSF samples. Results: Matrix assisted laser desorption ionization tandem time of flight mass spectrometry has permitted us to characterize and sequence proteins present in femtomolar quantities, and to identify a number of very low abundance proteins never previously reported in CSE We now report the identification of a set of molecular markers that may be relevant to AD diagnosis and studies of its pathogenesis. These markers are identified using a multivariate statistical approach and represent changes in the observed quantities of at least nine low abundance proteins in AD patients relative to controls. Additional studies are in progress to further characterize these markers and assess their applicability to Alzheimer differential diagnosis. Conclusions: Proteomic analysis of CSF may provide an effective method to identify AD biomarkers.
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DETECTION OF DIFFERENT TAU ISOFORMS IN CEREBROSPINAL FLUID FROM PATIENTS WITH ALZHEIMER'S DISEASE USING IMMUNOAFFINITY CHROMATOGRAPHY-MASS SPECTROMETRY
Justyna M. Czama .1 , Gunhild Watdemar 2, Niels H. Heegaard 1.
1Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark; 2Memory Disorders Research Unit, Dept. of Neurology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark. Contact e-mail: jmc @ssi.dk Background: Despite the fact that Alzheimer's disease (AD) is the most prevalent of dementias, there is currently no satisfactory single specific biochemical marker for this condition. One possible candidate is the neuronal microtubule-associated protein tan. The CSF-levels of tan are elevated in patients with Alzheimer's disease but also in certain other types of dementias. In AD, however, tau is hyperphosphorylated and otherwise posttranslationally modified. Phosphorylated tan appears to be
$363
INFLAMMATORY MARKERS CRP AND IL-6 IN CEREBROSPINAL FLUID AND SERUM IN RELATION TO THE IL-6 GENOTYPE IN PATIENTS WITH ALZHEIMER'S DISEASE
Alie Schnitemaker* 1, Cees Mulder 2, Niki S. Schoonenboom 3, Miriam Ketema 2 , Evert-Jan van Elk 2, Philip Scheltens 3 , Gerard J. van K a m p 2 t VU University Medical Center, Depts of Neurology and Nuclear
Medicine and Clinical PET research, Amsterdam, Netherlands; 2 VU University Medical Center, Department of Clinical Chemistry, Amsterdam, Netherlands; SVU University Medical Center, Department of Neurology, Amsterdam, Netherlands. Contact e-mail: [email protected] Background Many lines of evidence suggest the involvement of inflammatory processes in the pathogenesis of Alzbeimer's disease (AD). Several immunopathological studies have shown that Amyloid-fl proteins within senile plaques are associated with activated microglia and astrocytes that express pro-inflammatory proteins such as interleukin-6 (IL-6) and acute phase proteins such as C-reactive protein (CRP). Studies on inflammatory proteins in cerebrospinal fluid (CSF) and serum in patients with Alzheimer's disease (AD) show conflicting results. Furthermore, the polymorphism of the gene encoding for IL-6 (-174 G/C) has been associated with an increased risk of early onset AD. Objectives To investigate serum and CSF levels of CRP and IL-6 in patients with 'probable' AD compared to non-demented controls and to relate these concentrations with the IL-6 genotypes, in both groups. Patients a n d Methods: Thirty-seven patients who fulfilled the NINCDS-ADRDA criteria for 'probable' AD and thirty-five age-matched non-demented controls were recruited. CRP and IL-6 concentrations in serum and CSF are measured by sandwich ELISA. IL-6 genotypes were determined by PCR, followed by sequencing. Results: Serum CRP concentrations were found to be lower in AD patients compared to controls (P < 0,001).However, no significant differences in levels of CRP in CSF or levels of IL-6 in serum mad CSF between AD patients and controls could be demonstrated,
Controls AD patients P-value
Median Range Median Range
Serum 1L-6 (rig/L)
CSF 1I.-6 (ng/L)
Serum CRP (rag/L)
CSF CRP (~gfL)
1. i 1 0.42-8.72 1.07 0.18-6.27 0.70
1.61 0.75-5.54 1.40 0.46-13.7 0.50
2.38 0.40-48.8 0.86 0.16-11.1 < 0.001
3.0 0.40-86.8 2.1 0-54.6 0.054
A significant positive correlation between serum and CSF levels of CRP was found (r = 0.82, P < 0.001). No correlation could be demonstrated for IL-6 sentm and CSF values. IL-6 genotypes were similarly distributed