Human peripheral blood mononuclear cells (PBMC), isolated from healthy donor leukocones (NHSBT) using Ficoll-Paque Plus (GE Healthcare), were cultured at 2 × 105 cells/well in 96-well plates (Corning) pre-coated with 0.5 μg/mL anti-human CD3 monoclonal antibody (OKT3, eBioscience). Cultures were supplemented with 100 ng/mL staphylococcal enterotoxin A (SEA) (Sigma), 30 μg/mL tremelimumab or isotype control (MedImmune) and selumetinib at the concentrations indicated. Following 72 h incubation at 37 °C, IL-2 release was measured by DELFIA using reagents from a human IL-2 ELISA kit (R&D systems) and europium-labelled streptavidin (Perkin-Elmer). Results were repeated in two independent experiments.
Europium labelled streptavidin
Manufactured by PerkinElmer
Sourced in Denmark
Europium-labelled streptavidin is a fluorescent protein complex composed of streptavidin, a tetrameric protein that binds to biotin, and a europium chelate label. Europium is a rare earth metal that emits long-lived fluorescence when excited, providing a highly sensitive and stable signal for various bioanalytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Opteia human elisa kit, by BD (2 mentions)
Victor x4 multilabel plate reader, by PerkinElmer (2 mentions)
Delfia assay buffer, by PerkinElmer (2 mentions)
Delfia enhancement solution, by PerkinElmer (2 mentions)
96 well plate, by Corning (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Human il 2 elisa kit, by R&D Systems (1 mentions)
Staphylococcal enterotoxin a sea, by Merck Group (1 mentions)
Fluoronunc, by Thermo Fisher Scientific (1 mentions)
B2518, by Merck Group (1 mentions)
Victor x5 fluorometer, by PerkinElmer (1 mentions)
5 protocols using europium labelled streptavidin
1
PBMC Stimulation and IL-2 Measurement
2
H-ficolin Quantification Assay Protocol
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Quantification of the H-ficolin concentration was performed blinded to subject identity as previously described using normal human serum as standard except for a few changes39 (link). In brief, serum was thawed, diluted in assay buffer, and added to microtiter wells (FluoroNunc, Thermo Scientific, Waltham, MA, USA) coated with acetylated bovine serum albumin (B2518, Sigma-Aldrich, St. Louis, MO, USA), which is recognized by H-ficolin. Standards, samples, and controls were added automatically to plates using a Janus Varispan automated work-station (PerkinElmer, Waltham, MA, USA). In-house biotinylated anti-H-ficolin antibody, europium-labelled streptavidin (PerkinElmer) and enhancement solution (Ampliqon, Odense, Denmark) were added in successive steps with triple washing in between. The europium fluorescence intensity was detected with a Victor X5 fluorometer (PerkinElmer). Intra-assay and inter-assay coefficients of variation were below 10% and 16%, respectively. H-ficolin concentration was used as a predictor as a continuous variable as well as by comparing subjects grouped according to H-ficolin quartiles.
3
Quantifying Soluble IL-2RA in SLE Patients
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Soluble IL-2RA (sCD25) concentrations were measured in plasma samples from a cohort of SLE patients (N=41) and healthy donors (N=141), which included the three donors profiled in this study, using the BD OptEIA Human ELISA Kit (BD Biosciences; cat# 559104) according to the manufacturer’s instructions. Assay readout was performed using Europium-labelled streptavidin (Perkin Elmer, cat# 1244-360) combined with time-resolved fluorescence spectroscopy using DELFIA reagents (DELFIA Assay buffer, cat# 1244-106 and DELFIA Enhancement Solution, cat# 1244-104, Perkin Elmer). Plasma samples were diluted 1:20 and measured in duplicate in Corning 96-well clear polystyrene microtiter plates (Sigma, cat# CL3795-100EA) using a VICTOR X4 multilabel plate reader (Perkin Elmer). Each 96-well plate contained a recombinant sCD25 protein standard curve with a detection range of 31.25–500pg/ml.
4
PrP Detection in CJD Diagnosis
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The CDI assay was performed and calibrated as described previously [25 (link), 26 (link)] (but omitting the NaPTA precipitation step). PrP was detected using the dissociation‐enhanced lanthanide fluorescence immunoassay technology of PerkinElmer (PerkinElmer, Cambridge, UK). The capture antibody used was MAR‐1 (0.5 μg/well), provided by CSL Behring, Marburg, Germany. The detection antibody was 0.2 μg/mL biotin‐conjugated 3F4 (BioLegend), used in combination with 0.2 μg/mL Europium‐labelled streptavidin (PerkinElmer). In CDI, senPrPSc is detected based on an increase in signal following denaturation of the sample (D) with guanidine hydrochloride (GdnHCl), compared with non‐denature (N) samples, However, brain homogenates were assayed by CDI following limited proteolytic digestion with a low concentration of PK (2.5 μg/mL, 1 h, 37°C) to minimise the background signal, without digesting senPrPSc [26 (link), 48 (link)].
Test samples were continually analysed in comparison to three non‐CJD neurological control cases and one vCJD case. A cut‐off threshold was established based on the mean [D–N] value for non‐CJD neurological control test samples plus three standard deviations of the mean.
Test samples were continually analysed in comparison to three non‐CJD neurological control cases and one vCJD case. A cut‐off threshold was established based on the mean [D–N] value for non‐CJD neurological control test samples plus three standard deviations of the mean.
5
Quantifying Soluble IL-2RA in SLE
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Soluble IL-2RA (sCD25) concentrations were measured in plasma samples from a cohort of SLE patients (N=41) and healthy donors (N=141), which included the three donors profiled in this study, using the BD OptEIA Human ELISA Kit (BD Biosciences; cat# 559104) according to the manufacturer's instructions. Assay readout was performed using Europium-labelled streptavidin (Perkin Elmer, cat# 1244-360) combined with time-resolved fluorescence spectroscopy using DELFIA reagents (DELFIA Assay buffer, cat# 1244-106 and DELFIA Enhancement Solution, cat# 1244-104, Perkin Elmer). Plasma samples were diluted 1:20 and measured in duplicate in Corning 96-well clear polystyrene microtiter plates (Sigma, cat# CL3795-100EA) using a VICTOR X4 multilabel plate reader (Perkin Elmer). Each 96-well plate contained a recombinant sCD25 protein standard curve with a detection range of 31.25-500pg/ml.
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