All CSF analyzes were performed in the neurochemistry lab of the Göttingen University Medical Center before conceptualization of this study during the diagnostic process; the technicians were blind to the final diagnosis. T-tau was measured using INNOTEST hTAU Ag ELISA Kit from Fujirebio. Tau phosphorylated at Thr181 was analyzed using INNOTEST ELISA kit PHOSPHO-TAU (181 P) from Fujirebio.
Innotest htau ag elisa kit
Manufactured by Fujirebio
Sourced in Belgium
The INNOTEST hTAU Ag ELISA kit is a quantitative enzyme-linked immunosorbent assay (ELISA) designed for the measurement of total tau protein in human cerebrospinal fluid (CSF) samples.
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7 protocols using innotest htau ag elisa kit
1
Cerebrospinal Fluid Biomarker Analysis
2
Measurement of CSF T-tau by ELISA
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CSF T-tau was measured using the INNOTEST hTau Ag ELISA kit (Fujirebio, Malvern, PA, USA, and Ghent, Belgium) according to the manufacturer’s instructions. Study samples were diluted 1:4; positive control symptomatic prion disease samples were diluted 1:10. All samples were assayed in duplicate with samples from the same individual co-located on the same plate to facilitate comparison. Following termination of the colorimetric development reaction, absorbance per well was measured at 450 nm as well as at 620 nm for background subtraction using a FLUOStar Optima absorbance plate reader, then fit to an internal standard curve. The operator was blinded to mutation status.
3
Biomarker Analysis in Adult SMA Patients
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CSFs from adult SMA patients were analyzed, in parallel with samples from healthy non-age-matched control patients, for neurodegeneration biomarkers using the appropriate ELISA kit test. Total tau (t-Tau) protein was quantified using the INNOTEST hTAU AG ELISA kit from Fujirebio (Cat nº 81572, Fujirebio, Les Ulis, France); pNF-H (phosphorylated neurofilament heavy) levels were quantified using the phosphorylated Neurofilament heavy (pNF-H) Sandwich ELISA kit from MerckMillipore (Cat nº NS170, MerckMillipore, Burlington, MA, USA); NFL (neurofilament light) levels were detected using the NF-Light ELISA Assay kit from UmanDiagnostics (Cat nº 10-7001, UmanDiagnostics, Umea, Sweden); sAPPβ (soluble Amyloid Precursor Protein Beta) was determined using the sAPP-β wild type high sensitive ELISA kit from Creative Diagnostic (Cat nº DEIA6295, Creative Diagnostic, Upton, NY, USA), and YKL-40 was analyzed using MicroVue YKL-40 EIA from Quidel (Cat nº 8020, Quidel, San Diego, CA, USA). YKL-40 levels in plasma samples were also evaluated using the same kit MicroVue YKL-40 EIA from Quidel (Cat nº 8020, San Diego, Quidel, CA, USA).
4
Quantitative CSF 14-3-3 and Tau Protein Analysis
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CSF 14-3-3 was detected by immunoblot. For each sample, the equivalent of 25 μL of CSF was loaded onto a 13% polyacrylamide gel and transferred to polyvinylidene fluoride membranes, as previously described [26 (link)]. Thee membranes were incubated with anti-14-3-3 rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), and immunoblot was revealed using an enhanced chemiluminescence system. The 14-3-3 testing was judged to be positive (+) or negative (−) compared with the positive control.
The CSF tau protein concentrations were measured in duplicate by sandwich ELISA, using the INNOTEST® hTAU Ag ELISA kit (Fujirebio Europe, Gent, Belgium), according to the manufacturer’s instructions. The absorbance values were obtained with a microplate reader and the tau concentrations were estimated from standard curves made for each assay.
The CSF tau protein concentrations were measured in duplicate by sandwich ELISA, using the INNOTEST® hTAU Ag ELISA kit (Fujirebio Europe, Gent, Belgium), according to the manufacturer’s instructions. The absorbance values were obtained with a microplate reader and the tau concentrations were estimated from standard curves made for each assay.
5
Diagnostic CSF Biomarkers in Neurodegenerative Diseases
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Lumbar punctures were performed for diagnostic purposes at the time point of first clinical workup (UCSF was at subject’s first research visit). CSF samples were stored in polypropylene tubes at −80°C until analysis. CSF was analysed in the diagnostic, research and neurochemistry laboratories of the Clinical Dementia Center, Göttingen, except 14-3-3 western blot, which was locally analysed through each national CJD surveillance unit. The presence of 14-3-3 protein was analysed by western blot as described previously.21 (link) Quantification of 14-3-3 was performed using the CircuLex 14-3-3 gamma ELISA kit as reported before.22 (link) T-tau was quantitatively measured using the INNOTEST® hTAU-Ag ELISA kit from Fujirebio. α-Syn was quantified using an electrochemiluminiscent ELISA platform as described before.13 (link) RT-QuIC was performed following an established protocol.23 (link)
6
CSF Biomarker Measurement Protocol
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CSF was sampled for T-tau, p-tau and Aβ1-42 during the reservoir tap test. CSF samples were collected under sterile conditions into 15 mL*2 centrifuge tubes. CSF sample collection and storage methods were all in accordance with the consensus guidelines for CSF biobanking [21] . Samples were sent for enzymelinked immunosorbent assay (ELISA) analysis to measure concentrations of T-tau (INNOTEST hTAU Ag ELISA kit, Fujirebio Europe N.V., Belgium), 181 p-tau (INNOTEST PHOSPHO-Tau (181p) ELISA kit, Fujirebio Europe N.V., Belgium), and Aβ-42 [INNOTEST β-AMYLOID (1-42) ELISA kit, Fujirebio Europe N.V., Belgium] and total protein. A technician, blinded to the clinical results, prospectively recorded levels of T-tau, 181 ptau, and Aβ1-42. Longitudinal stability in the measurements was ascertained using an elaborate program of internal quality control (QC) samples. Intra-and inter-assay coe cients of variation were 1.37 and 5.8% for Aβ1-42, 2.51 and 1.46% for T-tau, 3.36 and 1.52% for p-tau, respectively.
7
Cerebrospinal Fluid Biomarker Analysis
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CSF was sampled to measure T-tau, p-tau, and Aβ1–42 levels during the reservoir tap test. The CSF samples were collected under sterile conditions into two 15 ml centrifuge tubes. The CSF sample collection and storage methods were all performed in accordance with the consensus guidelines for CSF biobanking. The samples were sent to measure the concentrations of T-tau (INNOTEST hTAU Ag ELISA kit, Fujirebio Europe N.V., Belgium), 181p-tau (INNOTEST PHOSPHO-Tau (181p) ELISA kit, Fujirebio Europe N.V., Belgium), and Aβ–42 [INNOTEST β-AMYLOID (1–42) ELISA kit, Fujirebio Europe N.V., Belgium] and total proteins for enzyme-linked immunosorbent assays (ELISAs). A technician who was blinded to the clinical results prospectively recorded the levels of T-tau, 181p-tau, and Aβ1–42. The longitudinal stability of the measurements was ascertained using an elaborate program of internal quality control (QC) samples. Intra-and inter-assay coefficients of variation were 1.37 and 5.8% for Aβ1–42, 2.51 and 1.46% for T-tau, 3.36 and 1.52% for p-tau, respectively.
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