All of the CSF samples were collected by the time of diagnoses and reserved in the refrigerator at −80°C until we performed the assays. The Nf‐L (IBL) and phosphorylated neurofilament (pNf‐H; Biovendor) were quantified by Sandwich ELISA immunosorbent assay according to the manufacturer's instructions. The concentrations of IL‐1β, IL‐6, and IL‐17A were determined by enzyme‐linked immunosorbent assays (Bender MedSystems GmbH Campus Vienna).
Pnf h
Manufactured by BioVendor
Sourced in Germany
PNF-H is a laboratory equipment product that functions as a precise and reliable power supply. It provides a stable and adjustable source of electrical power to support various laboratory applications and experiments.
Automatically generated - may contain errors
6 protocols using pnf h
1
Quantitative Biomarker Analysis in CSF
2
Neurodegenerative Biomarkers Measurement
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Serum NF-L and serum tau protein were measured with ultrasensitive Single molecule array (Simoa) assays23 (link)24 (link)25 (link).
Samples were analyzed for S100B with electrochemiluminescence immunoassay (Elecsys S100B, Roche, Penzberg, Germany)8 (link), and with ELISAs for NF-L (IBL, Hamburg, Germany), pNF-H (Biovendor, Heidelberg, Germany), and Tau (Fujirebio, Hanover, Germany) according to manufacturer’s specifications. Mean inter-assay CV for the ELISAs was <20%14 (link).
Real-time quaking-induced conversion (RT-QuIC) was carried out as described elsewhere21 (link).
Samples were analyzed for S100B with electrochemiluminescence immunoassay (Elecsys S100B, Roche, Penzberg, Germany)8 (link), and with ELISAs for NF-L (IBL, Hamburg, Germany), pNF-H (Biovendor, Heidelberg, Germany), and Tau (Fujirebio, Hanover, Germany) according to manufacturer’s specifications. Mean inter-assay CV for the ELISAs was <20%14 (link).
Real-time quaking-induced conversion (RT-QuIC) was carried out as described elsewhere21 (link).
3
Biomarker Quantification by ELISA
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The plasma levels of biomarkers were measured using the enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions: pNF-H (BioVendor, Brno, Czech Republic) with limit of detection (LD) 23.5 pg/ml, intra- assay variability (CV) 4.5%), vWF (Life Technologies, Carlsbad, CA), with LD 0.4 ng/mL and interassay CV <10% and fibronectin (ThermoFisher, Waltham, MA) with LD 0.06 ng/mL and interassay CV <10%. The analyses were performed in a blinded fashion to MRI results and biosamples from the 3 groups were distributed between plates to minimize batch effects.
4
Dynamic Evaluation of Brain Biomarkers
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Dynamic evaluation of brain and global biomarkers was performed at D1 and D2 post-injury in the PBBI model. Serum concentrations of brain and global biomarkers pNF-H (cat no. RD191138300R; Biovendor), NF-L (item 103345; Quanterix), HSP70 (cat no. ab133060; abcam), and inflammation markers HMGB1 (ABIN416082; Antibodies-online.com ) and αII-spectrin (cat no. ABIN1572517; Antibodies-online) were determined with sandwich enzyme-linked immunosorbent assays (ELISA) at the University of Florida. Sandwich ELISAs were conducted using standard 96-well, flat-bottom, Nunc Immuno Maxisorp plates (Fisher, Pittsburgh, PA, USA), according to the manufacturer's protocol. Selected capture and detection antibody pairs were used.
Briefly, ELISA plates were passively coated overnight at 4°C with capture antibody, then washed and blocked. Serum samples were added and incubated at room temperature with shaking. After washing, peroxidase-conjugated detection antibody or HRP-conjugated streptavidin were added, which catalyzed the reaction with a colorimetric substrate (TMB; Pierce). The product was quantified by absorbance at 450 nm in a microplate spectrophotometer. Standard curves were generated using recombinant proteins corresponding to the biomarker measured in each assay. Four parameter-fit non-linear regression analyses were applied to determine biomarker quantities.
Briefly, ELISA plates were passively coated overnight at 4°C with capture antibody, then washed and blocked. Serum samples were added and incubated at room temperature with shaking. After washing, peroxidase-conjugated detection antibody or HRP-conjugated streptavidin were added, which catalyzed the reaction with a colorimetric substrate (TMB; Pierce). The product was quantified by absorbance at 450 nm in a microplate spectrophotometer. Standard curves were generated using recombinant proteins corresponding to the biomarker measured in each assay. Four parameter-fit non-linear regression analyses were applied to determine biomarker quantities.
5
Neurodegeneration Marker Analysis in SMA
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CSF was collected in polypropylene tubes and was immediately analyzed for routine CSF parameters within 1 hour after lumbar puncture. Cell count and analysis of routine parameters was done as earlier described.18 (link) For measurement of neurodegeneration markers, CSF was frozen within 1 hour after lumbar puncture and stored at −80° until analysis. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to determine the concentrations of CSF NfL (IBL, Hamburg, Germany) and pNfH (Biovendor, Heidelberg, Germany).11 (link),12 A total of 50 CSF and 50 serum samples of 25 SMA patients, and 25 CSF and 25 serum samples of 25 controls were analyzed.
6
Biomarker Quantification in CSF Samples
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CSF was centrifuged, aliquoted in polypropylene tubes and stored within two hours at -80°C until analysis. Samples were measured blinded to any clinical data. Commercially available ELISA kits were used for CHIT1 (MBL, Diegem, Belgium) with a limit of detection of 48 pg/mL. CSF was prediluted 1:5 to 1:50 and the limit of quantitation (LOQ was set to 281 pg/mL. NfL (IBL, Hamburg, Germany) and pNfH (Biovendor, Heidelberg, Germany) were measured according to the manufacturers' instructions. (17) Except for samples from Ulm, only one NfL measurement could be performed due to lack of sample with an upper LOQ of 10000 pg/mL. The mean inter-assay CV was <20% for the CHIT1, NfL, and pNfH ELISAs.
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