Development and Validation of a High Sensitivity Assay for Measuring p217 + tau in Cerebrospinal Fluid

Background: Early and accurate detection and staging is critical to managing Alzheimer’s disease (AD) and supporting clinical trials. Cerebrospinal fluid (CSF) biomarkers for amyloid-β peptides, tau species, and various neurodegenerative and inflammatory analytes are leading the way in this regard, yet there is room for improved sensitivity and specificity. In particular tau is known to be present in many different fragments, conformations, and post-translationally modified forms. While the exact tau species that might best reflect AD pathology is unknown, a growing body of evidence suggests that forms with high levels of phosphorylation in the mid-region may be especially enriched in AD. Objective: Develop an assay for measuring p217tau in CSF. Methods: Here we describe the development and validation of a novel sELISA for measuring CSF tau species containing phosphorylation at threonines 212 & 217, aka p217 + tau, using the PT3 antibody. Results: While the analyte is present at extremely low levels the assay is sufficiently sensitive and specific to quantitate p217 + tau with excellent precision, accuracy, and dilution linearity, allowing good differentiation between diagnostic subgroups. The p217 + tau measurements appear to track AD pathology better than the commonly used p181tau epitope, suggesting superior diagnostic and staging performance. Finally, the assay can also be configured to differentiate antibody-bound versus antibody-free tau, and therefore can be used to measure target engagement by p217 + tau-targeting immunotherapeutics. Conclusion: The assay for measuring p217 + tau described here is highly sensitive, accurate, precise, dilution linear, and shows good potential for identifying and staging AD.

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Quantitative Measurement of Cerebrospinal Fluid Amyloid-β Species by Mass Spectrometry

Journal of Alzheimer s Disease ◽

10.3233/jad-200987 ◽

2020 ◽

pp. 1-12

Author(s):

Yusuke Seino ◽

Takumi Nakamura ◽

Tomoo Harada ◽

Naoko Nakahata ◽

Takeshi Kawarabayashi ◽

...

Keyword(s):

Mass Spectrometry ◽

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cerebrospinal Fluid ◽

Quantitative Measurement ◽

Amyloid Β ◽

High Sensitivity ◽

Csf Biomarkers ◽

Strongly Correlated ◽

Csf Levels

Background: High sensitivity liquid chromatography mass spectrometry (LC-MS/MS) was recently introduced to measure amyloid-β (Aβ) species, allowing for a simultaneous assay that is superior to ELISA, which requires more assay steps with multiple antibodies. Objective: We validated the Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-43 assay by LC-MS/MS and compared it with ELISA using cerebrospinal fluid (CSF) samples to investigate its feasibility for clinical application. Methods: CSF samples from 120 subjects [8 Alzheimer’s disease (AD) with dementia (ADD), 2 mild cognitive dementia due to Alzheimer’s disease (ADMCI), 14 cognitively unimpaired (CU), and 96 neurological disease subjects] were analyzed. Aβ species were separated using the Shimadzu Nexera X2 system and quantitated using a Qtrap 5500 LC-MS/MS system. Aβ1-40 and Aβ1-42 levels were validated using ELISA. Results: CSF levels in CU were 666±249 pmol/L in Aβ1-38, 2199±725 pmol/L in Aβ1-40, 153.7±79.7 pmol/L in Aβ1-42, and 9.78±4.58 pmol/L in Aβ1-43. The ratio of the amounts of Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-43 was approximately 68:225:16:1. Linear regression analyses showed correlations among the respective Aβ species. Both Aβ1-40 and Aβ1-42 values were strongly correlated with ELISA measurements. No significant differences were observed in Aβ1-38 or Aβ1-40 levels between AD and CU. Aβ1-42 and Aβ1-43 levels were significantly lower, whereas the Aβ1-38/1-42, Aβ1-38/1-43, and Aβ1-40/Aβ1-43 ratios were significantly higher in AD than in CU. The basic assay profiles of the respective Aβ species were adequate for clinical usage. Conclusion: A quantitative LC-MS/MS assay of CSF Aβ species is as reliable as specific ELISA for clinical evaluation of CSF biomarkers for AD.

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Cerebrospinal Fluid Biomarkers of Alzheimer’s Disease: Current Evidence and Future Perspectives

Brain Sciences ◽

10.3390/brainsci11020215 ◽

2021 ◽

Vol 11 (2) ◽

pp. 215

Author(s):

Donovan A. McGrowder ◽

Fabian Miller ◽

Kurt Vaz ◽

Chukwuemeka Nwokocha ◽

Cameil Wilson-Clarke ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cerebrospinal Fluid ◽

Late Onset ◽

Amyloid Β ◽

Current Evidence ◽

Csf Biomarkers ◽

Diagnostic Efficacy ◽

Neurofilament Light ◽

New Biomarkers

Alzheimer’s disease is a progressive, clinically heterogeneous, and particularly complex neurodegenerative disease characterized by a decline in cognition. Over the last two decades, there has been significant growth in the investigation of cerebrospinal fluid (CSF) biomarkers for Alzheimer’s disease. This review presents current evidence from many clinical neurochemical studies, with findings that attest to the efficacy of existing core CSF biomarkers such as total tau, phosphorylated tau, and amyloid-β (Aβ42), which diagnose Alzheimer’s disease in the early and dementia stages of the disorder. The heterogeneity of the pathophysiology of the late-onset disease warrants the growth of the Alzheimer’s disease CSF biomarker toolbox; more biomarkers showing other aspects of the disease mechanism are needed. This review focuses on new biomarkers that track Alzheimer’s disease pathology, such as those that assess neuronal injury (VILIP-1 and neurofilament light), neuroinflammation (sTREM2, YKL-40, osteopontin, GFAP, progranulin, and MCP-1), synaptic dysfunction (SNAP-25 and GAP-43), vascular dysregulation (hFABP), as well as CSF α-synuclein levels and TDP-43 pathology. Some of these biomarkers are promising candidates as they are specific and predict future rates of cognitive decline. Findings from the combinations of subclasses of new Alzheimer’s disease biomarkers that improve their diagnostic efficacy in detecting associated pathological changes are also presented.

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A rapid and accurate method for screening T-2 toxin in food and feed using competitive AlphaLISA

FEMS Microbiology Letters ◽

10.1093/femsle/fnab029 ◽

2021 ◽

Vol 368 (6) ◽

Author(s):

Liwen Zhang ◽

Qingyu Lv ◽

Yuling Zheng ◽

Xuan Chen ◽

Decong Kong ◽

...

Keyword(s):

High Sensitivity ◽

Accurate Method ◽

Detection Methods ◽

Cereal Crops ◽

Detection Range ◽

Highly Sensitive ◽

Food And Feed ◽

Good Repeatability ◽

Feed Samples ◽

Better Than

ABSTRACT T-2 is a common mycotoxin contaminating cereal crops. Chronic consumption of food contaminated with T-2 toxin can lead to death, so simple and accurate detection methods in food and feed are necessary. In this paper, we establish a highly sensitive and accurate method for detecting T-2 toxin using AlphaLISA. The system consists of acceptor beads labeled with T-2-bovine serum albumin (BSA), streptavidin-labeled donor beads and biotinylated T-2 antibodies. T-2 in the sample matrix competes with T-2-BSA for antibodies. Adding biotinylated antibodies to the test well followed by T-2 and T-2-BSA acceptor beads yielded a detection range of 0.03–500 ng/mL. The half-maximal inhibitory concentration was 2.28 ng/mL and the coefficient of variation was <10%. In addition, this method had no cross-reaction with other related mycotoxins. This optimized method for extracting T-2 from food and feed samples achieved a recovery rate of approximately 90% in T-2 concentrations as low as 1 ng/mL, better than the performance of a commercial ELISA kit. This competitive AlphaLISA method offers high sensitivity, good specificity, good repeatability and simple operation for detecting T-2 toxin in food and feed.

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Cerebrospinal fluid p-tau217 performs better than p-tau181 as a biomarker of Alzheimer’s disease

Nature Communications ◽

10.1038/s41467-020-15436-0 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 30

Author(s):

Shorena Janelidze ◽

Erik Stomrud ◽

Ruben Smith ◽

Sebastian Palmqvist ◽

Niklas Mattsson ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cerebrospinal Fluid ◽

Amyloid Β ◽

Pet Tracer ◽

Positron Emission ◽

Diagnostic Work ◽

Work Up ◽

The Brain ◽

Better Than

AbstractCerebrospinal fluid (CSF) p-tau181 (tau phosphorylated at threonine 181) is an established biomarker of Alzheimer’s disease (AD), reflecting abnormal tau metabolism in the brain. Here we investigate the performance of CSF p-tau217 as a biomarker of AD in comparison to p-tau181. In the Swedish BioFINDER cohort (n = 194), p-tau217 shows stronger correlations with the tau positron emission tomography (PET) tracer [18F]flortaucipir, and more accurately identifies individuals with abnormally increased [18F]flortaucipir retention. Furthermore, longitudinal increases in p-tau217 are higher compared to p-tau181 and better correlate with [18F]flortaucipir uptake. P-tau217 correlates better than p-tau181 with CSF and PET measures of neocortical amyloid-β burden and more accurately distinguishes AD dementia from non-AD neurodegenerative disorders. Higher correlations between p-tau217 and [18F]flortaucipir are corroborated in an independent EXPEDITION3 trial cohort (n = 32). The main results are validated using a different p-tau217 immunoassay. These findings suggest that p-tau217 might be more useful than p-tau181 in the diagnostic work up of AD.

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Diagnostic Significance of Methemoglobin Determination in Colorless Cerebrospinal Fluid

Clinical Chemistry ◽

10.1093/clinchem/38.8.1404 ◽

1992 ◽

Vol 38 (8) ◽

pp. 1404-1408 ◽

Cited By ~ 5

Author(s):

M Trbojević-Cepe ◽

Z Vogrinc ◽

V Brinar

Keyword(s):

Cerebrospinal Fluid ◽

Neurological Diseases ◽

Disease Process ◽

High Sensitivity ◽

Soret Band ◽

Spectrophotometric Analysis ◽

Diagnostic Significance ◽

Highly Sensitive ◽

Subarachnoid Hemorrhages ◽

Sensitive Spectrophotometric Method

Abstract The presence of various heme derivatives can be demonstrated spectrophotometrically in colorless cerebrospinal fluid (CSF). Because of the high sensitivity of the method, it may detect compounds that reflect a "traumatic tap" rather than a disease process. However, the presence of methemoglobin excludes the possibility of a hemorrhagic CSF being caused by traumatic lumbar puncture. Here we describe a highly sensitive spectrophotometric method involving measurement at the Soret band (400-420 nm) to detect methemoglobin (greater than or equal to 15%) in trace amounts of hemoglobin mixture (less than 0.3 mumol/L). We demonstrated methemoglobin in colorless CSF samples in 9% of 454 patients with cerebrovascular pathology and in 4% of 449 patients with other neurological diseases (n = 449). In a group of 21 patients with verified acute cerebral hematomas, methemoglobin was confirmed in 66% of colorless CSF samples after ultrafiltration. We conclude that routine spectrophotometric analysis of all CSF samples is very useful, allowing detection of xanthochromic compounds in patients with small cerebral and subdural hematomas as well as in those with minimal subarachnoid hemorrhages, hemorrhagic infarctions, or bleedings from aneurysms and neoplasms.

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Development and Application of Novel Chemiluminescence Immunoassays for Highly Sensitive Detection of Anisakis simplex Proteins in Thermally Processed Seafood

Pathogens ◽

10.3390/pathogens9100777 ◽

2020 ◽

Vol 9 (10) ◽

pp. 777

Author(s):

Maciej Kochanowski ◽

Mirosław Różycki ◽

Joanna Dąbrowska ◽

Jacek Karamon ◽

Jacek Sroka ◽

...

Keyword(s):

Sandwich Elisa ◽

High Sensitivity ◽

Anisakis Simplex ◽

Processed Food ◽

Highly Sensitive ◽

Thermally Processed ◽

Third Stage ◽

Seafood Products ◽

Highly Sensitive Detection ◽

Development And Validation

The third-stage larvae (L3) of Anisakis simplex are the most important source of hidden allergens in seafood products. However, there exist no commercial methods for detecting Anisakis proteins in food. Furthermore, only a few methods have been validated for the detection of A. simplex in thermally processed food. The aims of our study are (i) the development and validation of high-sensitivity chemiluminescent (CL) immunoassays for the detection of A. simplex proteins in processed seafood, (ii) and A. simplex antigen detection in common seafood products from Polish markets. We developed and validated CL sandwich ELISA (S-ELISA) and CL competitive ELISA (C-ELISA) methods for A. simplex proteins detection in food, with respective detection limits of 0.5 and 5 ng/mL. The usefulness of the assays for detecting A. simplex proteins in highly processed food was evaluated by examination of autoclaved canned fish spiked with A. simplex larvae (1–8 larvae/200 g). Commercial real-time PCR was unable to detect A. simplex in autoclaved samples at all levels of enrichment with Anisakis larvae. CL-S-ELISA was used to test various types of seafood products from Polish markets. Among all tested products (n = 259), 28% were positive. A. simplex antigens were found mostly (n = 39) in smoked fish products: mackerel, herring, cod, and hake. Other positive samples were found in marinated herrings, canned cod livers, canned mackerels, and surimi sticks. In tuna, Atlantic argentine, anchovy, sardine, sprat, and squid products, A. simplex antigens were not detected. This study provides novel effective tools for the detection of A. simplex proteins in processed food and highlights the potential allergic hazards for Anisakis-sensitized Polish consumers of seafood.

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Cerebrospinal fluid biomarkers of β-amyloid metabolism in multiple sclerosis

Multiple Sclerosis Journal ◽

10.1177/1352458512460603 ◽

2012 ◽

Vol 19 (5) ◽

pp. 543-552 ◽

Cited By ~ 32

Author(s):

Kristin Augutis ◽

Markus Axelsson ◽

Erik Portelius ◽

Gunnar Brinkmalm ◽

Ulf Andreasson ◽

...

Keyword(s):

Multiple Sclerosis ◽

Cerebrospinal Fluid ◽

Inflammatory Diseases ◽

Amyloid Β ◽

Csf Biomarkers ◽

Aβ Peptide ◽

Aβ Peptides ◽

App Metabolism ◽

Disease Modifying

Background: Amyloid precursor protein (APP) and amyloid β (Aβ) peptides are intensely studied in neuroscience and their cerebrospinal fluid (CSF) measurements may be used to track the metabolic pathways of APP in vivo. Reduced CSF levels of Aβ and soluble APP (sAPP) fragments are reported in inflammatory diseases, including multiple sclerosis (MS); but in MS, the precise pathway of APP metabolism and whether it can be affected by disease-modifying treatments remains unclear. Objective: To characterize the CSF biomarkers of APP degradation in MS, including the effects of disease-modifying therapy. Methods: CSF samples from 87 MS patients (54 relapsing–remitting (RR) MS; 33 secondary progressive (SP) MS and 28 controls were analyzed for sAPP and Aβ peptides by immunoassays, plus a subset of samples was analyzed by immunoprecipitation and mass spectrometry (IP-MS). Patients treated with natalizumab or mitoxantrone were examined at baseline, and after 1–2 years of treatment. Results: CSF sAPP and Aβ peptide levels were reduced in MS patients; but they increased again towards normal, after natalizumab treatment. A multivariate model of IP-MS-measured Aβ species separated the SPMS patients from controls, with RRMS patients having intermediate levels. Conclusions: We confirmed and extended our previous observations of altered CSF sAPP and Aβ peptide levels in MS patients. We found that natalizumab therapy may be able to counteract the altered APP metabolism in MS. The CSF Aβ isoform distribution was found to be distinct in SPMS patients, as compared to the controls.

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Cerebrospinal fluid profiles with increasing number of cerebral microbleeds in a continuum of cognitive impairment

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x15606141 ◽

2015 ◽

Vol 36 (3) ◽

pp. 621-628 ◽

Cited By ~ 23

Author(s):

Sara Shams ◽

Tobias Granberg ◽

Juha Martola ◽

Xiaozhen Li ◽

Mana Shams ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cerebrospinal Fluid ◽

Cognitive Impairment ◽

Serum Albumin ◽

Amyloid Β ◽

Cerebral Microbleeds ◽

Csf Biomarkers ◽

Barrier Dysfunction ◽

T Tau

Cerebral microbleeds (CMBs) are hypothesised to have an important yet unknown role in the dementia disease pathology. In this study we analysed increasing number of CMBs and their independent associations with routine cerebrospinal fluid (CSF) biomarkers in a continuum of cognitive impairment. A total of 1039 patients undergoing dementia investigation were analysed and underwent lumbar puncture, and an MRI scan. CSF samples were analysed for amyloid β (Aβ) 42, total tau (T-tau), tau phosphorylated at threonine 18 (P-tau) and CSF/serum albumin ratios. Increasing number of CMBs were independently associated with low Aβ42 levels, in the whole cohort, Alzheimer’s disease and mild cognitive impairment ( p < 0.05). CSF/serum albumin ratios were high with multiple CMBs ( p < 0.001), reflecting accompanying blood–brain barrier dysfunction. T-tau and P-tau levels were lower in Alzheimer’s patients with multiple CMBs when compared to zero CMBs, but did not change in the rest of the cohort. White matter hyperintensities were associated with low Aβ42 in the whole cohort and Alzheimer’s disease ( p < 0.05). Aβ42 is the routine CSF-biomarker mainly associated with CMBs in cognitive impairment, and there is an accumulative effect with increasing number of CMBs.

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Markers of vitamin B12 status in relation to cerebrospinal fluid biomarkers and cognitive performance

Proceedings of The Nutrition Society ◽

10.1017/s0029665120006333 ◽

2020 ◽

Vol 79 (OCE2) ◽

Author(s):

Babak Hooshmand ◽

Franziska Koch ◽

Patrik Fissler ◽

Markus Otto ◽

Hayrettin Tumani ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Vitamin B12 ◽

Cognitive Performance ◽

Amyloid Β ◽

Binary Logistic Regression ◽

Cognitive Status ◽

University Hospital ◽

Binary Logistic Regression Analysis ◽

Csf Biomarkers ◽

Total Tau

AbstractIntroduction: The association between markers of vitamin B12 status and cerebrospinal fluid (CSF) biomarkers of Alzheimer's type dementia which precede cognitive impairment has been investigated by only a few small studies and the results have been inconsistent.Aim: To investigate the associations of vitamin B12 related markers (vitamin B12, holotranscobalamin (HoloTC), homocysteine (tHcy), and methylmalonic acid (MMA)) with CSF total tau, Amyloid-β 42 (Aβ42) and cognitive performance.Methods: Data included 462 patients aged 40–94 years referred to the Memory Clinic at the Ulm University Hospital, Ulm, Germany between December 2009 and August 2015. Vitamin B12 and HoloTC were measured via chemiluminescence microparticle immunoassay, tHcy via chemiluminescence immunoassay and MMA via liquid chromatography mass specterometry. CERAD battery was used to examine the cognitive status, and different domains were derived. Regression models were used to investigate the associations.Results: After adjusting for age, sex, creatinine levels and APOEε4 status, higher values of vitamin B12 and lower values of MMA were associated with lower concentrations of CSF total-tau: the odds ratios (ORs) (95% confidence intervals (CI)) in a binary logistic regression analysis investigating the associations with total tau cut-off of 400 pg/ml were 0.39 (0.15–0.99) and 5.60 (1.93–16.26) for the highest quartile of B12 and MMA compared to the lowest, respectively. Furthermore, HoloTC, MMA, and tHcy were associated with several cognitive domains such as episodic memory and executive functioning. No relationships were found with Aβ42.Conclusions: Vitamin B12 and its related markers may be independent predictors of CSF biomarkers of Alzheimer's disease and cognitive status. Randomized controlled trials are needed to determine the importance of vitamin B12 supplementation on slowing structural brain changes and cognitive decline in older adults.

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Unravelling the Association Between Amyloid-PET and Cerebrospinal Fluid Biomarkers in the Alzheimer’s Disease Spectrum: Who Really Deserves an A+?

Journal of Alzheimer s Disease ◽

10.3233/jad-210593 ◽

2021 ◽

pp. 1-12

Author(s):

Luca Sacchi ◽

Tiziana Carandini ◽

Giorgio Giulio Fumagalli ◽

Anna Margherita Pietroboni ◽

Valeria Elisa Contarino ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cerebrospinal Fluid ◽

Amyloid Β ◽

Standardized Uptake Value ◽

Csf Biomarkers ◽

Amyloid Pet ◽

Linear Regression Models ◽

Csf Analysis ◽

Disease Spectrum

Background: Association between cerebrospinal fluid (CSF)-amyloid-β (Aβ)42 and amyloid-PET measures is inconstant across the Alzheimer’s disease (AD) spectrum. However, they are considered interchangeable, along with Aβ 42/40 ratio, for defining ‘Alzheimer’s Disease pathologic change’ (A+). Objective: Herein, we further characterized the association between amyloid-PET and CSF biomarkers and tested their agreement in a cohort of AD spectrum patients. Methods: We include ed 23 patients who underwent amyloid-PET, MRI, and CSF analysis showing reduced levels of Aβ 42 within a 365-days interval. Thresholds used for dichotomization were: Aβ 42 < 640 pg/mL (Aβ 42+); pTau > 61 pg/mL (pTau+); and Aβ 42/40 < 0.069 (ADratio+). Amyloid-PET scans were visually assessed and processed by four pipelines (SPMCL, SPMAAL, FSGM, FSWC). Results: Different pipelines gave highly inter-correlated standardized uptake value ratios (SUVRs) (rho = 0.93–0.99). The most significant findings were: pTau positive correlation with SPMCL SUVR (rho = 0.56, p = 0.0063) and Aβ 42/40 negative correlation with SPMCL and SPMAAL SUVRs (rho = –0.56, p = 0.0058; rho = –0.52, p = 0.0117 respectively). No correlations between CSF-Aβ 42 and global SUVRs were observed. In subregion analysis, both pTau and Aβ 42/40 values significantly correlated with cingulate SUVRs from any pipeline (R2 = 0.55–0.59, p < 0.0083), with the strongest associations observed for the posterior/isthmus cingulate areas. However, only associations observed for Aβ 42/40 ratio were still significant in linear regression models. Moreover, combining pTau with Aβ 42 or using Aβ 42/40, instead of Aβ 42 alone, increased concordance with amyloid-PET status from 74% to 91% based on visual reads and from 78% to 96% based on Centiloids. Conclusion: We confirmed that, in the AD spectrum, amyloid-PET measures show a stronger association and a better agreement with CSF-Aβ 42/40 and secondarily pTau rather than Aβ 42 levels.

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