- Email: [email protected]
A NOVEL COLORIMETRIC ELISA FOR QUANTIFICATION OF TAU, PHOSPHORYLATED AT THREONINE 181, IN CEREBROSPINAL FLUID
Poster Presentations: Sunday, July 16, 2017
(0.82, 0.68-0.99, p¼0.03), SAM (0.62,0.40-0.98, p¼0.04), cobalamin/B12 (0.90,0.81-1.0), p¼0.04, taurine (0.88,0.770.99, p¼0.04), and 5-MTHF (0.91,0.82-1.00, p¼0.05). Conclusions: This meta-analysis provides support for the vascular hypothesis of AD, and suggests that cardiovascular and glial mechanisms (homocysteine, 24(s)hydroxycholesterol, and YKL-40) play an important role in AD pathogenesis. Reduced perfusion due to atherosclerosis is also suggested (homocysteine and 24(s)hydroxycholersterol). Homocysteine is associated with cognitive decline and brain atrophy. It can be inferred that vitamin B supplementation, and reduction of homocysteine levels prevent cognitive loss. P1-270
A STREAMLINED PCR-BASED FRAGMENT ANALYSIS ASSAY THAT RESOLVES BOTH SINGLE NUCLEOTIDE AND POLY-T LENGTH POLYMORPHISMS AT APOE AND TOMM40 SUSCEPTIBILITY LOCI IN ALZHEIMER’S DISEASE
Bradley Hall, Sarah N. Statt, Julie R. Thibert, Jacob Wisotsky, Jon Kemppainen, Gary J. Latham, Asuragen, Austin, TX, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder that progresses slowly over time. Previous studies have identified an AD susceptibility locus in a region that includes APOE and TOMM40. Of the three common APOE alleles, ε4 is the most informative but in only w25% of Caucasians. Independently, poly-T polymorphisms at rs10524523 of the TOMM40 gene have been reported to influence both age of onset in late-onset AD (LOAD) and measures of cognitive decline. Here we describe a rapid and reliable PCR-based assay that reports both APOE genotypes and TOMM40 poly-T length in a single workflow. Methods: Cellline gDNA samples were acquired from the Coriell Institute (7 AD, 104 other) and Asuragen (11). Whole blood (72) and matched blood/buccal gDNA (14) was isolated from presumed healthy donors. Sample gDNA was PCR amplified using prototype AmplideXÒ reagents (Asuragen). Amplicons tagged with FAM (TOMM40) and HEX (APOE) were resolved by capillary electrophoresis (CE) on a 3500 xL (Thermo Fisher) after a 2.5kV injection and 20 min run. Genotypes were determined in separate color channels from peak size patterns (APOE) or the mobility of target amplicon relative to a calibration curve (TOMM40). Results: Both APOE and TOMM40 PCR amplicons could be resolved from a single CE injection, simplifying both the workflow and data analysis. All six possible APOE genotypes could be correctly identified, and results were consistent across different operators and days. Further, APOE genotypes were concordant with reference calls for 207/208 samples. The TOMM40 poly-T repeat number was determined within a single nucleotide for all samples tested to at least 40Ts, enabling the differentiation of similarly-sized alleles and accurate categorization near allele length boundaries. For both markers, inputs as low as 5 ng could be reliably genotyped. Conclusions: PCR genotyping of APOE and TOMM40 is challenged by GC- and AT-rich sequence contexts, respectively. We report the first assay that combines the analysis of both AD markers into a rapid, streamlined, and high-throughput workflow and achieves reproducible, high-resolution and accurate genotypes. This technology has the potential to
P353
advance clinical research using a standardized assay that can harmonize results across laboratories. P1-271
A NOVEL COLORIMETRIC ELISA FOR QUANTIFICATION OF TAU, PHOSPHORYLATED AT THREONINE 181, IN CEREBROSPINAL FLUID
Victor Herbst1, Britta Brix1, Stefan Busse2, Hugo Marcel Vanderstichele3, Erik Stoops3, 1Euroimmun AG, Lubeck, Germany; 2Department of Psychiatry, University of Magdeburg, Magdeburg, Germany; 3ADx NeuroSciences, Gent, Belgium. Contact e-mail: [email protected] Background: With an increasing number of demented people
worldwide, measurement of biomarkers in cerebrospinal fluid (CSF) will be increasingly used as a routine diagnostic tool for diagnosis of Alzheimer’s disease (AD). The integration of a novel phospho-tau(181P) ELISAs in routine lab environments requires assays with good analytical performance, robustness, user-friendliness and possibilities of automation. There is a need for different P-Tau assays to extend the clinical utility for this biomarker in the near future. Methods: A one-day P-Tau181 ELISA protocol was developed (including ready-to-use components) and qualified for manual and automated testing on any open ELISA platform, the Euroimmun Analyzer I. Protocols were optimized to allow parallel analysis of a completed biomarker profile ((Amyloid Beta 142 (Aß1-42), Aß1-40, (phospho)tau, synapse proteins). Analytical performance evaluation included intra-assay, inter-assay, working ranges, and interference studies. Clinically characterized samples from the University of Magdeburg were used to for comparison with a reference test, the INNOTEST Phospho-Tau(181P). Results: The assay design of the Euroimmun P-Tau181 was compatible with the automated open ELISA platform. The automated protocol showed a linear regression of Analyzer I ¼ 0,95x Manual incubation -0,5 pg/ml with a regression coefficient of R2¼0,99. Analytical performance: Intra-Assay CV ¼ 1,3%-5,0% (at 19-127pg/ml), Inter-Assay CV ¼ 3,1% – 4,9% (at 26-119pg/ml) and showed no whole blood interference (up to 1% v/v) and no hook effect (up to 100ng/ml) was observed. Compared to Innotest the EUROIMMUN P-Tau181 showed increased sensitivity and specificity in n¼107 clinically characterized samples (52 AD patients, 55 disease/healthy controls) with a sensitivity of 93,2% and specificity of 85,4% while Fuji showed a sensitivity of 66,1% and specificity of 91,7% (both using the same Cut-Off of 61pg/ml; ROC AUC Euroimmun ¼ 0.94, AUC Fuji ¼ 0.92). Conclusions: The Euroimmun P-Tau181 ELISA can be easily run on an open ELISA platform, using the same protocol and in parallel to assays for Aß(1-40), Aß(1-42), and total tau. The assay was qualified at the analytical and clinical level. The novel assay shows strong correlation in protein concentrations with a reference method, while its clinical performance was confirmed. P1-272
MEASUREMENT OF CSF HYPOTHALAMIC PEPTIDES IN FRONTOTEMPORAL DEMENTIA
Carolin Heller1, Wendy E. Heywood2, Antonia Theodoridi1, Ione O. C. Woollacott3, Jason D. Warren3, Kevin Mills4, Jonathan D. Rohrer3, 1Institute of Neurology, University College London, London, United Kingdom; 2Institute of Child Health and Great Ormond Street Hospital, London, United Kingdom; 3 Dementia Research Centre, Institute of Neurology, University
(0.82, 0.68-0.99, p¼0.03), SAM (0.62,0.40-0.98, p¼0.04), cobalamin/B12 (0.90,0.81-1.0), p¼0.04, taurine (0.88,0.770.99, p¼0.04), and 5-MTHF (0.91,0.82-1.00, p¼0.05). Conclusions: This meta-analysis provides support for the vascular hypothesis of AD, and suggests that cardiovascular and glial mechanisms (homocysteine, 24(s)hydroxycholesterol, and YKL-40) play an important role in AD pathogenesis. Reduced perfusion due to atherosclerosis is also suggested (homocysteine and 24(s)hydroxycholersterol). Homocysteine is associated with cognitive decline and brain atrophy. It can be inferred that vitamin B supplementation, and reduction of homocysteine levels prevent cognitive loss. P1-270
A STREAMLINED PCR-BASED FRAGMENT ANALYSIS ASSAY THAT RESOLVES BOTH SINGLE NUCLEOTIDE AND POLY-T LENGTH POLYMORPHISMS AT APOE AND TOMM40 SUSCEPTIBILITY LOCI IN ALZHEIMER’S DISEASE
Bradley Hall, Sarah N. Statt, Julie R. Thibert, Jacob Wisotsky, Jon Kemppainen, Gary J. Latham, Asuragen, Austin, TX, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder that progresses slowly over time. Previous studies have identified an AD susceptibility locus in a region that includes APOE and TOMM40. Of the three common APOE alleles, ε4 is the most informative but in only w25% of Caucasians. Independently, poly-T polymorphisms at rs10524523 of the TOMM40 gene have been reported to influence both age of onset in late-onset AD (LOAD) and measures of cognitive decline. Here we describe a rapid and reliable PCR-based assay that reports both APOE genotypes and TOMM40 poly-T length in a single workflow. Methods: Cellline gDNA samples were acquired from the Coriell Institute (7 AD, 104 other) and Asuragen (11). Whole blood (72) and matched blood/buccal gDNA (14) was isolated from presumed healthy donors. Sample gDNA was PCR amplified using prototype AmplideXÒ reagents (Asuragen). Amplicons tagged with FAM (TOMM40) and HEX (APOE) were resolved by capillary electrophoresis (CE) on a 3500 xL (Thermo Fisher) after a 2.5kV injection and 20 min run. Genotypes were determined in separate color channels from peak size patterns (APOE) or the mobility of target amplicon relative to a calibration curve (TOMM40). Results: Both APOE and TOMM40 PCR amplicons could be resolved from a single CE injection, simplifying both the workflow and data analysis. All six possible APOE genotypes could be correctly identified, and results were consistent across different operators and days. Further, APOE genotypes were concordant with reference calls for 207/208 samples. The TOMM40 poly-T repeat number was determined within a single nucleotide for all samples tested to at least 40Ts, enabling the differentiation of similarly-sized alleles and accurate categorization near allele length boundaries. For both markers, inputs as low as 5 ng could be reliably genotyped. Conclusions: PCR genotyping of APOE and TOMM40 is challenged by GC- and AT-rich sequence contexts, respectively. We report the first assay that combines the analysis of both AD markers into a rapid, streamlined, and high-throughput workflow and achieves reproducible, high-resolution and accurate genotypes. This technology has the potential to
P353
advance clinical research using a standardized assay that can harmonize results across laboratories. P1-271
A NOVEL COLORIMETRIC ELISA FOR QUANTIFICATION OF TAU, PHOSPHORYLATED AT THREONINE 181, IN CEREBROSPINAL FLUID
Victor Herbst1, Britta Brix1, Stefan Busse2, Hugo Marcel Vanderstichele3, Erik Stoops3, 1Euroimmun AG, Lubeck, Germany; 2Department of Psychiatry, University of Magdeburg, Magdeburg, Germany; 3ADx NeuroSciences, Gent, Belgium. Contact e-mail: [email protected] Background: With an increasing number of demented people
worldwide, measurement of biomarkers in cerebrospinal fluid (CSF) will be increasingly used as a routine diagnostic tool for diagnosis of Alzheimer’s disease (AD). The integration of a novel phospho-tau(181P) ELISAs in routine lab environments requires assays with good analytical performance, robustness, user-friendliness and possibilities of automation. There is a need for different P-Tau assays to extend the clinical utility for this biomarker in the near future. Methods: A one-day P-Tau181 ELISA protocol was developed (including ready-to-use components) and qualified for manual and automated testing on any open ELISA platform, the Euroimmun Analyzer I. Protocols were optimized to allow parallel analysis of a completed biomarker profile ((Amyloid Beta 142 (Aß1-42), Aß1-40, (phospho)tau, synapse proteins). Analytical performance evaluation included intra-assay, inter-assay, working ranges, and interference studies. Clinically characterized samples from the University of Magdeburg were used to for comparison with a reference test, the INNOTEST Phospho-Tau(181P). Results: The assay design of the Euroimmun P-Tau181 was compatible with the automated open ELISA platform. The automated protocol showed a linear regression of Analyzer I ¼ 0,95x Manual incubation -0,5 pg/ml with a regression coefficient of R2¼0,99. Analytical performance: Intra-Assay CV ¼ 1,3%-5,0% (at 19-127pg/ml), Inter-Assay CV ¼ 3,1% – 4,9% (at 26-119pg/ml) and showed no whole blood interference (up to 1% v/v) and no hook effect (up to 100ng/ml) was observed. Compared to Innotest the EUROIMMUN P-Tau181 showed increased sensitivity and specificity in n¼107 clinically characterized samples (52 AD patients, 55 disease/healthy controls) with a sensitivity of 93,2% and specificity of 85,4% while Fuji showed a sensitivity of 66,1% and specificity of 91,7% (both using the same Cut-Off of 61pg/ml; ROC AUC Euroimmun ¼ 0.94, AUC Fuji ¼ 0.92). Conclusions: The Euroimmun P-Tau181 ELISA can be easily run on an open ELISA platform, using the same protocol and in parallel to assays for Aß(1-40), Aß(1-42), and total tau. The assay was qualified at the analytical and clinical level. The novel assay shows strong correlation in protein concentrations with a reference method, while its clinical performance was confirmed. P1-272
MEASUREMENT OF CSF HYPOTHALAMIC PEPTIDES IN FRONTOTEMPORAL DEMENTIA
Carolin Heller1, Wendy E. Heywood2, Antonia Theodoridi1, Ione O. C. Woollacott3, Jason D. Warren3, Kevin Mills4, Jonathan D. Rohrer3, 1Institute of Neurology, University College London, London, United Kingdom; 2Institute of Child Health and Great Ormond Street Hospital, London, United Kingdom; 3 Dementia Research Centre, Institute of Neurology, University