To map the epitopes for Nbα‐syn01, we performed alanine scanning, a widely used site‐directed mutagenesis approach. Synthetic 14 amino acid long peptides spanning residues 43–56 (Table S1) of α‐syn were used based on the epitope mapped in previous study (Hmila et al., 2022 (link)). The exact epitope region was identified by two methods. In the first approach, a 384‐well black MaxiSorb plate (Nunc) was coated with 500 ng/well of the peptides in NaHCO3 and incubated overnight at 37°C under dry conditions. The following day, the plate was blocked with blocking buffer (PBST containing 2.25% gelatin) for 1 h at RT and then washed three times with PBST followed by addition of Nbα‐syn01 (100 ng/mL) for 1 h at RT. In the second approach, peptides were preincubated with Nbα‐syn01 for 2 h at RT. This was followed by the addition of the complex to a 384‐well black MaxiSorb plate coated with full length α‐syn for 1 h at RT and blocked as before. In both approaches, the bound Nbα‐syn01 was detected by addition of mouse anti‐His (1/3000) antibody followed by the addition of secondary antibody (goat anti‐mouse IgG‐HRP). After washing, enhanced chemiluminescent substrate (Super Signal ELISA Femto, Pierce Biotechnology) was added for detection. The chemiluminescence, expressed in relative light units (RLU/s), was immediately measured using Perkin‐Elmer spectrophotometer.
Supersignal elisa femto
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal ELISA Femto is a chemiluminescent substrate that provides high sensitivity detection for enzyme-linked immunosorbent assays (ELISA). It generates a bright, stable luminescent signal that can be measured using a luminometer or other light detection equipment.
Automatically generated - may contain errors
Lab products found in correlation
Nunc maxisorb, by Thermo Fisher Scientific (3 mentions)
Victor x3 multilabel plate reader, by PerkinElmer (3 mentions)
Donkey anti mouse igg hrp, by Jackson ImmunoResearch (2 mentions)
Maxisorb plate, by Thermo Fisher Scientific (1 mentions)
Fl 140, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Jackson ImmunoResearch (1 mentions)
Extravidin peroxidase, by Merck Group (1 mentions)
White 96 well plate, by Greiner (1 mentions)
Nano glo luciferase assay reagent, by Promega (1 mentions)
Glomax, by Promega (1 mentions)
Cellulose adsorbent pad, by Merck Group (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Polyethylene glycol 10000, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Jetprime, by Avantor (1 mentions)
Glomax 20 20, by Promega (1 mentions)
Anti rabbit hrp antibody, by Abcam (1 mentions)
A300 688, by Fortis Life Sciences (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Southern Biotech (1 mentions)
12 protocols using supersignal elisa femto
1
Epitope Mapping of Nbα-syn01 via Alanine Scanning
2
Quantifying α-Synuclein in Cerebrospinal Fluid
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A 384-well ELISA microplate (Nunc MaxiSorb, Nunc) was coated by overnight incubation at 4 °C with Syn-O2 (0.2 μg/ml) in 200 mM NaHCO3, pH 9.6 (50 μl/well). The plate was then washed with PBST and incubated with 100 μl/well of blocking buffer for 2 h at 37 °C. After washing, 50 μl of the CSF samples (thawed on ice and Tween-20 was added to a final concentration of 0.05 %) was added to each well, and plates were incubated at 37 °C for 2.5 h. FL-140 (rabbit polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted in blocking buffer at 1:1 K, was added to the appropriate wells, and incubated at 37 °C for 2 h. Next, the plate was washed and incubated for 2 h at 37 °C with 50 μl/well of goat anti-rabbit IgG HRP (Jackson ImmunoResearch) diluted in blocking buffer (1:15 K). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate (SuperSignal ELISA Femto, Pierce Biotechnology, Rockford, IL). The chemiluminescence, expressed in relative light units, was immediately measured using VICTOR™ X3 multilabel plate reader (PerkinElmer). The standard curve for the ELISA assay was obtained using serial dilutions of recombinant human o-α-syn in artificial CSF (50 μl/well).
3
Quantitative α-Synuclein ELISA in CSF
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A 384-well ELISA microplate (Nunc MaxiSorb, NUNC) was coated by overnight incubation at 4 °C with 0.1 μg/ml Syn-140 (sheep anti-α-syn polyclonal antibody) in 200 mM NaHCO3, pH 9.6 (50 μl/well). The plate was then washed with PBST and incubated with 100 μl/well of blocking buffer (PBST containing 2.5 % gelatin) for 2 h at 37 °C. After washing, 50 μl of the CSF samples (thawed on ice and Tween-20 was added to a final concentration of 0.05 %) was added to each well, and plates were incubated at 37 °C for 2.5 h. 11D12 (mouse anti-α-syn monoclonal antibody), diluted in blocking buffer at 1:5 K was added to the appropriate wells, and incubated at 37 °C for 2 h. Next, the plate was washed and incubated for 2 h at 37 °C with 50 μl/well of species-appropriate secondary antibody (donkey anti-mouse IgG HRP, Jackson ImmunoResearch) diluted in blocking buffer (1:20 K). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate (SuperSignal ELISA Femto, Pierce Biotechnology, Rockford, IL). The chemiluminescence, expressed in relative light units, was immediately measured using VICTOR™ X3 multilabel plate reader (PerkinElmer). The standard curve for the ELISA assay was carried out using serial dilutions of recombinant human α-syn in artificial CSF.
4
Quantifying Protein Biomarkers in CSF
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A 384-well ELISA microplate was coated by overnight incubation at 4°C with 1 μg/ml mAb 211 (Santa Cruz Biotechnology, USA) in 200 mM NaHCO3, pH 9.6 (50 μl/well). The plate was washed with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST) and incubated with 100 μl/well of blocking buffer (PBS containing 2.5% gelatin and 0.05% Tween-20) for 2 hours at 37°C. After washing, 50 μl of the CSF samples (thawed on ice before Tween-20 was added to a final concentration of 0.05%) was added to each well, and then the plate was incubated at 37°C for another 3 hours. Biotinylated 211 diluted to 1 μg/ml in blocking buffer was added, and the plate was incubated at 37°C for 2 hours. The plate was washed and then incubated for 1 hour at 37°C with 50 μl/well of ExtrAvidin-Peroxidase (Sigma-Aldrich, Dorset, UK). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate (SuperSignal ELISA Femto; Pierce Biotechnology, Rockford, IL, USA). Then the chemiluminescence in relative light units was immediately measured with a Victor3 1420 (Wallac) microplate reader [30 (link),31 ]. The samples were screened in a blind fashion and tested randomly. The case and control samples were run on a single plate to avoid plate-to-plate variations, and the results were confirmed with at least two independent experiments.
5
Quantifying Phosphorylated α-Synuclein in CSF
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A 384-well ELISA microplate (Nunc MaxiSorb, Nunc) was coated by overnight incubation at 4 °C with 0.5 μg/ml Syn-140 (sheep anti-α-syn polyclonal antibody) in 200 mM NaHCO3, pH 9.6 (50 μl/well). The plate was then washed with PBST and incubated with 100 μl/well of blocking buffer for 2 h at 37 °C. After washing, 50 μl of the CSF samples (thawed on ice and Tween-20 was added to a final concentration of 0.05 %) was added to each well, and plates were incubated at 37 °C for 2.5 h. PS129 (mouse anti-pS129-α-syn monoclonal antibody) diluted in blocking buffer (1:1 K) were added to the appropriate wells, and incubated at 37 °C for 2 h. Next, the plate was washed and incubated for 2 h at 37 °C with 50 μl/well of species-appropriate secondary antibody (donkey anti-mouse IgG HRP (Jackson ImmunoResearch)) diluted in blocking buffer (1:20 K). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate (SuperSignal ELISA Femto, Pierce Biotechnology, Rockford, IL). The chemiluminescence, expressed in relative light units, was immediately measured using VICTOR™ X3 multilabel plate reader (PerkinElmer). The standard curve for the ELISA assay was obtained using serial dilutions of recombinant human p-S129-α-syn in artificial CSF (50 μl/well).
6
Quantifying Glucan Interactions Using ELISA-like Assays
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A 96-well white plate (Greiner Bio-one, Frickenhausen, Germany) was coated with supBGRP-LgBiT (2 μg/mL) in PBS and incubated overnight at 4 °C. The plate was washed with PBS containing 0.05% Tween 20 (wash buffer) and blocked with 1% BSA-containing wash buffer (assay buffer) at room temperature for 1 h. After washing, the plate was incubated for 1 h with various soluble and insoluble glucan samples in assay buffer, and then washed with wash buffer. For the HRP-based ELISA-like assay, the plate coated with supBGRP-LgBiT was treated with Neg1-E321Q-biotin (2 μg/mL) for 1 h, washed, and further treated with streptavidin-HRP in assay buffer for 20 min. The peroxidase substrate, SuperSignal ELISA Femto (Thermo Fisher Scientific), was added after removing the unbound HRP. For the sandwich split-NanoLuc ELISA-like assay, plates incubated with various glucan samples were washed and treated with Neg1-E321Q-SmBiT (2 μg/mL) in assay buffer for 1 h. After washing, the bioactivity of the split-enzyme complex was monitored using the Nano-Glo luciferase assay reagent (Promega, WI, USA). Luminescence signals from HRP or NanoLuc were measured using a microplate reader (GloMax; Promega).
7
Autoantibody Screening Using Plate Assays
8
Optimized Lateral Flow Immunoassay for Ochratoxin A
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The polyclonal anti-OTA antibody produced in rabbit and the HRP-OTA conjugate were kindly provided by Euroclone (Milan, Italy). Polyclonal anti-HRP antibody produced in rabbit, ovalbumin (OVA), Tween-20, and polyethylene glycol 10000 were obtained from Sigma-Aldrich Co (St. Louis, MO). The CL HRP detection substrate Supersignal ELISA Femto was purchased from Thermo Scientific Inc. (Rockford, IL). The other reagents were of analytical grade and were employed as received. Phosphate buffered saline (PBS) contained 10 mmol L -1 Na2HPO4, 2 mmol L -1 KH2PO4, 137 mmol L -1 NaCl, 2.7 mmol L -1 KCl, with pH adjusted to 7.4. The LFIA strips were produced using Whatman Standard 14 glass fibre sample pad (GE Healthcare Lifescience, Chalfont St. Giles, UK), Hi-flow plus 180 nitrocellulose membrane cards (Merck Millipore, Billerica, MA), and cellulose adsorbent pads (Merck Millipore).
9
Quantification of Nav1.5 Membrane Expression
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For the quantification of Nav1.5 expression at the plasma membrane, three copies of the hemagglutinin (HA) epitope were inserted at amino acid position 304 in the extracellular S5-S6 loop of domain I of Nav1.5. HeLa cells were transfected with the indicated constructs using jetPRIME (Peqlab). After 48 h, cells were fixed with 4% PFA (w/v) (in PBS), washed three times with PBS and blocked with 10% normal goat serum (v/v) (in PBS). Cells were stained with a monoclonal anti-HA primary antibody (HA-probe (F-7), Santa Cruz, dilution 1:100) and washed 4 times for 15 minutes with PBS. As a secondary antibody a horseradish-peroxidase (HRP)-conjugated antibody (goat anti-mouse IgG-HRP, Santa Cruz, dilution 1:5000) was used. After several washing steps with PBS (6 times for 20 minutes), surface expression was measured as relative light units (RLUs) in a luminometer (GloMax 20/20, Promega) using a luminogenic substrate (SuperSignal ELISA Femto, Thermo Scientific).
10
NEM Quantification by Optimized ELISA
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