Forty‐eight MCI‐AD patients at baseline in whom the diagnosis of MCI‐AD with CSF Aβ42 and p‐tau181 levels consistent with a diagnosis of AD and consensus evaluation of two neurologists and a neuropsychologist the details of which have been published previously.17 , 18 The study was approved by the Cleveland Clinic Institutional Review Board. Eight patients did not complete the longitudinal evaluations due to nonmedical reasons by their personal choice. The ADmark® Alzheimer's evaluation uses sandwich Enzyme Linked Immunosorbant Assay(ELISA) kits [Innotest β‐amyloid[1‐42], Innotest hTAU‐Ag, Innotest Phospho‐Tau[181P], Innogenetics, Ghent, Belgium]. APOE status was determined by blood samples(10 ng per subject) dispensed into 96‐well plates for TaqMan allelic discrimination detection of single nucleotide polymorphisms that discriminate the APOE alleles (rs429358, rs7412) (Life Technologies). Table 1 provides data on the Discovery cohort demographics. Inclusion, exclusion criteria are specified in Data S1 . Additional clinical and environmental factors have been documented in Table S1 (Fig. 1 ).
Innotest phospho tau 181p
Manufactured by Fujirebio
Sourced in Belgium, United States, Czechia, Spain
The INNOTEST® PHOSPHO-TAU(181P) is a quantitative in vitro diagnostic test used to measure the concentration of phosphorylated tau protein at threonine 181 (P-tau181) in human cerebrospinal fluid. The test is designed to provide analytical data for clinical laboratory use.
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Lab products found in correlation
Innotest htau ag, by Fujirebio (58 mentions)
Innotest β amyloid1 42, by Fujirebio (36 mentions)
Innotest amyloid 1 42, by Fujirebio (7 mentions)
Vilip 1 human elisa, by BioVendor (4 mentions)
Innotest β amyloid, by Fujirebio (3 mentions)
Ms6000 human ab ultra sensitive kit, by Mesoscale (3 mentions)
Innotest tau ag, by Fujirebio (3 mentions)
V plex aβ peptide panel 1 6e10 kit, by Mesoscale (3 mentions)
Polypropylene tube, by Sarstedt (2 mentions)
Innotest β amyloid 42, by Fujirebio (2 mentions)
Innotest aβ 1 42, by Fujirebio (2 mentions)
V plex human total tau kit k151lae, by Mesoscale (2 mentions)
Rs429358, by Thermo Fisher Scientific (1 mentions)
Rs7412, by Thermo Fisher Scientific (1 mentions)
Lightcycler apoe mutation detection method, by Roche (1 mentions)
Innotest htau antigen kit, by Fujirebio (1 mentions)
Pasw statistics 21, by IBM (1 mentions)
Beckman immage immunochemistry system, by Beckman Coulter (1 mentions)
Microvue ykl 40 eia elisa kit, by Quidel (1 mentions)
Lumipulse g600ii platform, by Fujirebio (1 mentions)
78 protocols using innotest phospho tau 181p
1
Alzheimer's Biomarker Discovery Cohort
2
CSF and Serum Biomarker Measurement
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Each sample, CSF or serum, was collected in the morning in fasting subjects who underwent lumbar puncture and venipuncture according to standard clinical and operating procedures. Each sample was received by the adjacent Neurology Laboratory within 30 min from collection and was centrifuged at 2500× g for 10 min at controlled room temperature. After centrifugation, samples were aliquoted into polypropylene sterile tubes and stored at −80 °C awaiting testing. CSF amyloid beta 1-42 (Aβ1-42), phosphorylated (p-tau181) and total tau protein (t-tau) were determined with the enzyme-linked immunosorbent assay (ELISA) method (INNOTEST® β-AMYLOID(1-42), INNOTEST® hTAU-Ag and INNOTEST® PHOSPHO-TAU(181P), Innogenetics, Ghent, Belgium) following the manufacturers’ instructions. For the purpose of selenoprotein P determination, the remaining aliquots of each sample were then transported deep frozen in dry ice by air courier to the Molecular Biology and Metabolism Laboratory of Tohoku University (Sendai, Japan).
3
Lumbar CSF Biomarker Analysis Protocol
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We obtained lumbar CSF before and after the LPS procedure. All CSF samples were aliquoted and stored in polypropylene tubes at –80°C until biochemical analysis.28 (link) Shunt reservoir and lumbar CSF biomarkers were also compared.9 (link),10 (link) CSF biomarkers included sAPPα (Human sAPPα Assay Kit; IBL No. 27719; Immuno-Biological Laboratories Co, Ltd, Takasaki, Japan), Aβ38 (Human Amyloid β1-38 Assay Kit, IBL No. 27717; Immuno-Biological Laboratories Co, Ltd), Aβ42 (Innotest β amyloid 1–42; Innogenetics, Ghent, Belgium), and p-tau (Innotest phospho tau-181p; Innogenetics). Immunosorbent assays were used for the rest of the biomarker measurements (Table 1 ).
4
Quantifying CSF tau and p-tau levels
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CSF levels of tau were determined by using an ELISA kit from INNOTEST® hTAU Ag (Innogenetics) with a detection range between 50-2500 pg/mL (detection limit: 34 pg/mL). Concentration of phosphorylated tau at threonine-181 (p-tau) was measured by using an ELISA kit named INNOTEST®PHOSPHO-TAU (181 P) (Inno-genetics) with a detection range between 15.6-1000 pg/mL (detection limit: 3 pg/mL).
Briefly, before antibody incubations, each sample (75 μl) was diluted 1:1 in sample diluent. The colorimetric reaction was measured at 450 nm with a 1420 Multilabel Counter Victor 2 (Wallac) (PerkinElmer). Each sample was measured in duplicate. For analysis we calculated the median.
Briefly, before antibody incubations, each sample (75 μl) was diluted 1:1 in sample diluent. The colorimetric reaction was measured at 450 nm with a 1420 Multilabel Counter Victor 2 (Wallac) (PerkinElmer). Each sample was measured in duplicate. For analysis we calculated the median.
5
Alzheimer's Biomarker Analysis in CSF
6
CSF Collection and Analysis for Alzheimer's
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A total of 12.5 mL CSF is collected in two polypropylene tubes via a lumbar puncture in intervertebral space at level L3/L4, L4/L5, or L5/S1. Part of the CSF is used for routine analyses including number of leucocytes, total protein, and glucose. Within 2 h after collection, the rest is stored at −20°C for analysis of Aβ1–42, total tau, and ptau within 2 months using sandwich enzyme-linked immunosorbent assays (Innotest β-Amyloid1–42, Innotest hTAU-Ag, and Innotest Phosphotau(181P); Innogenetics, Gent, Belgium). The remainder of the CSF is directly transferred to the Alzheimer Center Biobank for storage and future analysis (including Aβ1–40). CSF is aliquoted into 0.5 mL samples and stored at −80°C according to the standard protocols [37] (link).
7
APOE Genotyping and CSF Biomarker Measurement
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DNA was isolated from 10 ml ethylenediamine tetraacetic acid blood and the APOE genotype was determined using the Light Cycler APOE mutation detection method (Roche Diagnostics GmbH, Mannheim, Germany).
CSF Aβ1–42, tau, and ptau were measured by commercially available ELISAs (Innotest -β-amyloid(1-42), Innotest hTAU-Ag 168 and Innotest Phosphotau(181P); Innogenetics, Ghent, 169 Belgium). The performance of the assays was monitored with internal quality controls consisting of pools of surplus CSF specimens. In the study period, multiple internal quality controls with various concentrations have been used. The interassay coefficient of variation (CV) (mean ± standard deviation) was 11.3 ± 4.9% for Aβ, 9.3 ± 1.5% for tau, and 9.4 ± 2.5% for ptau [21 (link)].
CSF Aβ1–42, tau, and ptau were measured by commercially available ELISAs (Innotest -β-amyloid(1-42), Innotest hTAU-Ag 168 and Innotest Phosphotau(181P); Innogenetics, Ghent, 169 Belgium). The performance of the assays was monitored with internal quality controls consisting of pools of surplus CSF specimens. In the study period, multiple internal quality controls with various concentrations have been used. The interassay coefficient of variation (CV) (mean ± standard deviation) was 11.3 ± 4.9% for Aβ, 9.3 ± 1.5% for tau, and 9.4 ± 2.5% for ptau [21 (link)].
8
Quantifying Tau Protein Levels in CSF
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CSF levels of total Tau protein were measured using a commercially available ELISA kit (INNOTEST™ hTAU Ag, Innogenetics) with a detection range of 50 to 2,500 pg/ml (detection limit: 34 pg/ml). For the determination of Tau level, we followed the manufacturer’s instructions. Human Tau, phosphorylated at Thr181 (phosphorylated Tau) was analyzed quantitatively by the use of a commercially available ELISA kit (INNOTEST™PHOSPHO-TAU (181 P), Innogenetics) with a detection range of 15.6 to 1,000 pg/ml (detection limit: 3 pg/ml).
Briefly, before antibody incubations, each sample (75 μl) was diluted 1:1 in sample diluent. The colorimetric reaction was measured at 450 nm with a 1420 Victor2 Multilabel Counter (Wallac) (PerkinElmer). Each sample was measured in duplicate. For analysis, we calculated the median.
Briefly, before antibody incubations, each sample (75 μl) was diluted 1:1 in sample diluent. The colorimetric reaction was measured at 450 nm with a 1420 Victor2 Multilabel Counter (Wallac) (PerkinElmer). Each sample was measured in duplicate. For analysis, we calculated the median.
9
Cerebrospinal Fluid Biomarker Measurement
10
Biomarker Analysis in Cerebrospinal Fluid
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CSF samples were collected by lumbar puncture, which were performed in the morning to exclude influence on the results from possible diurnal fluctuations in biomarker levels. Twenty milliliter (mL) of CSF were collected in a polypropylene tube and immediately transported to the local laboratory for centrifugation at 2 000×g at room temperature for 10 minutes. The supernatant was collected, gently mixed to avoid possible gradient effects, and aliquoted in 0.5 mL aliquots in screw-cap polypropylene tubes that were stored at –80°C, without being thawed and refrozen, pending biochemical analyses. CSF levels of total (T)-tau, phosphorylated (P)-tau181, and amyloid-β amino acids 1 to 42 (Aβ42) were determined using sandwich enzyme-linked immunosorbent assays (INNOTEST® hTau Ag, INNOTEST® PHOSPHO-TAU (181P) , and INNOTEST® β-AMYLOID (1 –42) , respectively) from Innogenetics, Gent [25 (link)]. The CSF samples were processed at the Clinical Neurochemical Laboratory at Gothenburg University as part of clinical routine on multiple occasions during the course of the study. The analytical variability was low [26 (link)].To obtain apolipoprotein E (APOE)ɛ4 status, blood was drawn from all study participants and subsequently analysed using solid-phase minisequencing.
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