Streptavidin poly hrp

For the sandwich ELISA (Figure 1C), the plates were coated with 100 µl of 1.1 µg/ml antibodies in carbonate buffer, and incubated overnight at 4°C. The blocking of the wells was performed the same way as for the indirect ELISA. Afterwards, various concentrations of vasoinhibin, PRL, human serum (diluted up to 1 to 20 in TBST) and BSA as blank were dissolved in 100 µl of TBST, added to the wells and incubated for 1 h at RT while shaking. The bound antigen was detected by the biotinylated antibodies, which were diluted 1 to 3000 in 100 µl TBST overnight at 4°C. Streptavidin-poly-HRP (Thermo Fisher Scientific, 21140) was added diluted 1 to 3000 in 100 µl TBST for 1 h while shaking. The detection was performed the same way as for the indirect ELISA. The standard curves were fitted on the basis of the 4-parameter Rodbard equation. Cross-titration was performed to determine the most efficient caption and detection antibody concentration. Therefore, a sandwich ELISA with a constant antigen concentration of 5 µg/ml, various caption antibody concentrations (1, 2, 3, 4, 5, 6 µg/ml), and various detection antibody dilutions (1 to 1000; 1 to 2000, 1 to 3000, 1 to 4000) was performed. PRL in human sera was determined by a commercial ELISA kit according to the instructions of the manufacturer (cat. no. EIA-1291, DRG Diagnostics, Marburg, Germany).

An in-house ELISA assay was developed to measure total α-syn in CSF samples. A 96-well Polysorp NUNC microplate (Thermo Fischer Scientific, USA) was coated with the capture antibody (4B12, BioLegend) in coating buffer overnight at 4 °C. The plate was then washed three times with phosphate-buffered saline with 0.05% Tween-20 (PBST) and incubated with 300 μL/well of blocking buffer for 1 h at 37 °C. After three washes with PBST, serially diluted recombinant α-syn and thawed CSF (diluted 3:1 with sample dilution buffer) were applied to each well and incubated for 2.5 h at room temperature (RT). Subsequently, the plate was washed five times and incubated with the biotinylated detection antibody (4D6, BioLegend) in the reaction solution for 1 h at RT. After washing, the plate was incubated with streptavidin poly-HRP (Thermo Fischer Scientific, USA) in the reaction solution for 0.5 h at 37 °C. Finally, the plate was washed five times and reacted with TMB substrate (Thermo Fischer Scientific, USA) for 0.5 h at RT in the dark. The optical density was measured after adding the stop solution. Reagents (AGMIG-0100, Arista biologicals) to mitigate heterophilic antibody interference was included in all assays to remove false signals.

To evaluate PK, three C57BL/6 mice for each time point were administered a single intravenous (IV) dose of 5 µg mIL12-F_HAB-His, hIL15-F_HAB-His, mIL-12-His, or hIL-15-His. Quantitation of each compound in serum was performed with an ELISA, based on consistent detection of a histidine (His) tag to avoid potential interference from the native cytokines. Plates were coated overnight with 6x-His Epitope Tag Antibody (Pierce Cat#His.H8) at 2-8°C, then washed, and sample dilutions added for 1 h incubation at room temperature. Rabbit anti-6-His biotinylated secondary antibody (Bethyl Cat# A190-114B) was then added to the wells for 1 h at room temperature, followed by streptavidin poly-HRP (ThermoScientific Cat# 21140) for 30 minutes at 37°C. The reaction was stopped with H₂SO₄ and the plates were read at 450nm.

IgE measurements were performed following the protocol by Kobayashi et al., 2009 [68 (link)]. Briefly, ELISA plates (Immulon 4 HBX; Thermo Labsystems, Philadelphia, PA, USA) were coated with 5 μL/mL rat anti-mouse IgE mAb (LO-ME-3; Bio-Rad, Hercules, CA) in 0.1 M carbonate buffer (pH 9.5) for 2 h at 37 °C. Blocking was performed with PBS containing 1% BSA (MilliporeSigma, Burlington, MA, USA) overnight at 4 °C. After blocking, plasma samples diluted with PBS containing 1% BSA and 0.05% Tween 20 were added to the plates, and incubated for 2 h at room temperature. Afterwards, plates were treated with 1 μg/mL OVA or OVA + PEI, which had been biotinylated using a microbiotinylation kit (MilliporeSigma,), for 1 h at room temperature, followed by 1/5000 streptavidin-poly-HRP (ThermoFisher) for 30 min at room temperature. After the final washing, peroxidase substrate (TMB substrate kit) was added and the reaction stopped 20 min later with 1 M HCl. After each step, plates were washed with PBS containing 0.05% Tween 20. A microplate autoreader (Thermomax; Molecular Devices, San Jose, CA, USA) was used to measure absorbance at 450 nm.

APCs were cultured overnight with T-cell hybridomas (6.9-11, 6.9-51, and 6.9-61) recognizing the 6.9HIP. The T-cell responses were tested for IL-2 production measured by IL-2 ELISA (Bectin Dickinson) coupled with streptavidin-poly HRP (Thermo Fisher). The responses were quantified using the OptEIA TMB Substrate (Bectin Dickinson), and the data (OD at 450 nm) were collected using an iMark Microplate Reader (Bio-Rad Laboratories).

ELISA plates (# 650‐061, Greiner Bio One) were coated overnight with 1.0 μg/mL Polyclonal goat anti human IgA (Southern Biotech, 2050‐01). The next day, plates were washed (0.05% tween in PBS) once and blocked with 5% non‐fat dry milk powder (Friesche Vlag). After blocking, plates were washed three times and the 50 μL undiluted cell culture supernatants were added to 50 μL blocking buffer (non‐fat dry milk) per well. The IgA1 and IgA2 recombinant proteins (16‐16‐090701‐1M and 16‐16‐090701‐2M, Athens Research) were serially diluted in blocking buffer to obtain a standard curve. Plates were incubated for 90 min at 37°C. Plates were washed and incubated with 1:5000 biotinylated anti‐IgA1 (#9130‐08, Southern Biotech) or 1:2000 biotinylated anti‐IgA2 (#9140‐08, Southern Biotech) for 1 hour at room temperature while shaking (180 rpm). Plates were washed 6 times and 1:10 000 streptavidin poly HRP (#21140, Thermo Fisher Scientific) was added and incubated for 1 h at room temperature while shaking (180 rpm). 100 μL TMB (#37574 Thermo Fisher Scientific) was added. Plates were developed, subsequently stopped with 2% HCL and measured at 450 nm with 620 nm as reference.