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12 protocols using bioaffy 1000 cd

1

Detection of Anti-Type I IFN Autoantibodies

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Detection of anti-type I IFN autoantibodies was performed using Gyros as described previously64 . Cytokines, recombinant human IFNα2 (Miltenyi Biotec, 130-108-984) or recombinant human IFNω (Merck, SRP3061) were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, A21445)) diluted in Rexxip F (Gyros Protein Technologies, P0004825; 1:500 dilution of the 2 mg ml−1 stock to yield a final concentration of 4 μg ml−1). Phosphate-buffered saline, 0.01% Tween-20 (PBS-T) and Gyros Wash buffer (Gyros Protein Technologies, P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1:100 in 0.01% PBS-T and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, P0004253) and the Gyrolab xPand (Gyros Protein Technologies, P0020520). Cleaning cycles were performed in 20% ethanol.
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2

Quantifying Autoantibodies to Type I Interferons

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Recombinant E. coli-derived human (rh)GM-CSF (#215-GMP, R&D Systems) was first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (#A39257, Thermo Fisher Scientific), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG (#A21445, Thermo Fisher Scientific)) diluted in Rexxip F (#P0004825, Gyros Protein Technologies; 1:500 dilution of the 2 mg/ml stock to yield a final concentration of 4 μg/ml). Buffer phosphate-buffered saline, 0.01% Tween 20 (PBS-T), and Gyros Wash buffer (#P0020087, Gyros Protein Technologies) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1:100 in 0.01% PBS-T and tested with the Bioaffy 1000 CD (#P0004253, Gyros Protein Technologies) and the Gyrolab xPand (#P0020520, Gyros Protein Technologies). Cleaning cycles were performed in 20% ethanol.
Screening of autoantibodies to type I IFN-α2a 100 pg/mL, IFN-β 10 ng/mL, and IFN-ω 100 pg/mL in plasma diluted 1 to 10 were performed as previously described using a dual interferon stimulated response element (ISRE) luciferase system in HEK293T cells [18 (link)]. Results were normalized to non-stimulated condition.
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3

Rapid Cytokine Quantification in Plasma

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Cytokines, recombinant human (rh)IFN-α2 (Milteny Biotec, ref. number 130-108-984) or rhIFN-ω (Merck, ref. number SRP3061), were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, cat. number A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, ref. number A21445) diluted in Rexip F (Gyros Protein Technologies, ref. number P0004825; 1/500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Buffer PBS-T 0.01% and Gyros Wash buffer (Gyros Protein Technologies, ref. number P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1/100 in PBS-T 0.01% and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, ref. number P0004253), and the Gyrolab X-Pand (Gyros Protein Technologies, ref. number P0020520). Cleaning cycles were performed in 20% ethanol.
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4

Biotinylated rhIFN-γ for Gyrolab Assay

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Recombinant human (rh) IFN-γ (R&D Systems, 285-IF-100/CF or 10067-IF-100) was first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG [Thermo Fisher Scientific, A21445]) diluted in Rexxip F (Gyros Protein Technologies, P0004825; 1:500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Buffer phosphate-buffered saline, 0.01% Tween 20 (PBS-T) and Gyros Wash buffer (Gyros Protein Technologies, P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1:100 in 0.01% PBS-T and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, P0004253) and the Gyrolab xPand (Gyros Protein Technologies, P0020520). Cleaning cycles were performed in 20% ethanol. Threshold for positivity was determined by the average of the positive controls run on the day of the experiment in each run.
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5

Biotin-labeled rIFN detection assay

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Recombinant human (rh) IFNα2 (Miltenyi Biotec, reference number 130-108-984) or rhIFNω (Merck, reference number SRP3061), was first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, catalog number A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody [Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, reference number A21445)] diluted in Rexxip F (Gyros Protein Technologies, reference number P0004825; 1:500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Buffer phosphate-buffered saline, 0.01% Tween 20 (PBS-T), and Gyros Wash buffer (Gyros Protein Technologies, reference number P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1:100 in PBS-T and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, reference number P0004253) and the Gyrolab xPand (Gyros Protein Technologies, reference number P0020520).
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6

Detecting Cytokine Levels via Gyros Bioaffinity

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Cytokines, recombinant human (rh)IFN-α2 (Milteny Biotec, ref. number 130-108-984) or rhIFN-ω (Merck, ref. number SRP3061), were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, cat. number A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, ref. number A21445) diluted in Rexip F (Gyros Protein Technologies, ref. number P0004825; 1/500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Buffer PBS-T 0.01% and Gyros Wash buffer (Gyros Protein Technologies, ref. number P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1/100 in PBS-T 0.01% and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, ref. number P0004253), and the Gyrolab X-Pand (Gyros Protein Technologies, ref. number P0020520). Cleaning cycles were performed in 20% ethanol.
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7

Screening for Auto-Antibodies in CCP and FFP Samples

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CCP and FFP samples were screened for auto-Abs by Gyros: cytokines, recombinant human (rh)IFN-α2 (Milteny Biotec, Paris, France) or rhIFN-ω (Merck, St. Quentin Fallavier, France), were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, Illkirch, France), according to the manufacturer's instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific Cat# A-21445, RRID:AB_2535862) diluted in Rexip F (Gyros Protein Technologies, ref. number P0004825; 1/500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). 0.01% PBS-T buffer and Gyros Wash buffer (Gyros Protein Technologies, Uppsala, Sweden) were prepared according to the manufacturer's instructions. Plasma or serum samples were then diluted 1/100 in 0.01% PBS-T and tested with Bioaffy 1000 CD (Gyros Protein Technologies) and Gyrolab X-Pand (Gyros Protein Technologies). Cleaning cycles were performed in 20% ethanol.12
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8

Quantification of Biotinylated Cytokines

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Cytokines, recombinant human (rh)IFN-α2 (Miltenyi Biotec, ref. number 130-108-984) or rhIFN-ω (Merck, ref. number SRP3061), were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, cat. number A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, ref. number A21445) diluted in Rexxip F (Gyros Protein Technologies, ref. number P0004825; 1/500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Buffer PBS-T 0.01% and Gyros Wash buffer (Gyros Protein Technologies, ref. number P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1/100 in PBS-T 0.01% and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, ref. number P0004253), and the Gyrolab xPand (Gyros Protein Technologies, ref. number P0020520). Cleaning cycles were performed in 20% ethanol.
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9

Quantification of Anti-Type I IFN Auto-Antibodies Using Gyros Platform

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Gyros was used for the detection of anti-type I IFN auto-Abs, as described by Bastard et al. [8 (link)]. Cytokines, recombinant human (rh)IFN-α2 (Miltenyi Biotec, reference number 130–108-984) and rhIFN-ω (Merck, reference number SRP3061) were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, catalog number A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody [Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, reference number A21445)] diluted in Rexxip F (Gyros Protein Technologies, reference number P0004825; 1:500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Phosphate-buffered saline, 0.01% Tween 20 (PBS-T) and Gyros Wash buffer (Gyros Protein Technologies, reference number P0020087) were prepared according to the manufacturer’s instructions. BAL or plasma samples were then diluted 1:100 in 0.01% PBS-T and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, reference number P0004253) and the Gyrolab xPand (Gyros Protein Technologies, reference number P0020520). Cleaning cycles were performed in 20% ethanol.
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10

Cytokine Quantification by Gyrolab

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Cytokines, recombinant human (rh)IFN-α2 (ref. number 130-108-984; Miltenyi Biotec) or rhIFN-ω (ref. number SRP3061; Merck), were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (cat. number A39257; Thermo Fisher Scientific), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained an Alexa Fluor 647 goat anti-human IgG Ab (ref. number A21445; Thermo Fisher Scientific) diluted in Rexip F (ref. number P0004825; 1/500 dilution of the 2 mg/ml stock to yield a final concentration of 4 µg/ml; Gyros Protein Technologies). PBS-Tween (PBS-T) 0.01% buffer and Gyros Wash buffer (ref. number P0020087; Gyros Protein Technologies) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1/100 in PBS-T 0.01% and tested with Bioaffy 1000 CD (ref. number P0004253; Gyros Protein Technologies) and Gyrolab X-Pand (ref. number P0020520; Gyros Protein Technologies). Cleaning cycles were performed in 20% ethanol.
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