Quantifying Soluble IL-6 Receptor Levels

Circulating sIL-6R concentrations were measured in BABYDIET and BABYDIAB serum samples using a highly sensitive non-isotopic time-resolved fluorescence ELISA assay based on the dissociation-enhanced lanthanide fluorescent immunoassay technology (DELFIA; PerkinElmer), as described45 (link). Test samples were diluted 1:20 in PBS+10% FBS and measured in duplicate on 384-well MaxiSorp microtiter plates (Nunc), coated with 1 μg ml⁻¹ monoclonal anti-human IL-6R antibody (clone 17506; RD Systems). Detection was performed using a biotinylated mouse anti-CD126 monoclonal antibody (clone M182, BD Biosciences) diluted to a final concentration of 100 ng ml⁻¹ in PBS+10% FBS and a Europium-Streptavidin detection solution (PerkinElmer), diluted in PBS+0.05% tween, 1% BSA, 7 μg ml⁻¹ DTPA to a final concentration of 0.05 μg ml⁻¹. Quantification of test samples was obtained by fitting the readings to a human recombinant IL-6Rα (RD systems) serial dilution standard curve plated in quadruplicate on each plate. Data for 782 unique individuals existed from 722 families.

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Dopico X.C., Evangelou M., Ferreira R.C., Guo H., Pekalski M.L., Smyth D.J., Cooper N., Burren O.S., Fulford A.J., Hennig B.J., Prentice A.M., Ziegler A.G., Bonifacio E., Wallace C, & Todd J.A. (2015). Widespread seasonal gene expression reveals annual differences in human immunity and physiology. Nature Communications, 6, 7000.

Publication 2015

MaxiSorp microtiter plates

Anti antibody Antibody Assay Cd126 Clone Dilution Dtpa Elisa Enhanced technology Europium Fluorescence Human Il 6r 1 Immunoassay Isotopic Lanthanide Monoclonal antibody Mouse Serum Streptavidin Tween

Corresponding Organization : Wellcome Trust

Other organizations : MRC Unit the Gambia, London School of Hygiene & Tropical Medicine, Technical University of Munich, Klinikum rechts der Isar, Helmholtz Zentrum München, Paul Langerhans Institute Dresden, TU Dresden, MRC Biostatistics Unit

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Circulating sIL-6R concentrations

control variables

Test samples were diluted 1:20 in PBS+10% FBS
Microtiter plates were coated with 1 μg ml^-1 monoclonal anti-human IL-6R antibody (clone 17506; RD Systems)
Detection was performed using a biotinylated mouse anti-CD126 monoclonal antibody (clone M182, BD Biosciences) diluted to a final concentration of 100 ng ml^-1 in PBS+10% FBS
Europium-Streptavidin detection solution (PerkinElmer) was diluted in PBS+0.05% tween, 1% BSA, 7 μg ml^-1 DTPA to a final concentration of 0.05 μg ml^-1

positive controls

Human recombinant IL-6Rα (RD systems) serial dilution standard curve plated in quadruplicate on each plate

negative controls

None explicitly mentioned

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