Nutritional biomarkers, including 25-hydroxyvitamin D, 25(OH)D; red blood cell (RBC) fatty acids; IL-1β; calcium; albumin; iron; and vitamin B12 were assayed from a non-fasted venous blood sample. The blood was protected from light and allowed to clot for 30 min and centrifuged for 10 min at 2000 rpm at 4 °C within 2 h of sampling. For detailed information regarding the assays and methods refer to the study protocol paper [49 (link)].
IL-1β was assayed using spare samples collected at baseline and stored at −80 °C. IL-1β was measured from thawed serum (at room temperature) by Abcam IL-1β ELISA kit according to the manufacturer’s instructions. In this assay, an antibody specific for IL-1β was coated onto the wells of the microtiter plates. Samples, including standards of known IL-1β concentrations and unknowns were pipetted into these wells and incubated at room temperature. The wells were then washed and a biotinylated antibody specific for IL-1β was added to the wells and incubated. After further washing, a streptavidin-peroxydase conjugate was added to each well and incubated. The wells were then washed to remove all unbound enzymes and TMB solution, which acts on the bound enzyme was added to induce a coloured reaction product. The intensity of this coloured product was directly proportional to the concentration of IL-1β present in the samples.
IL-1β was assayed using spare samples collected at baseline and stored at −80 °C. IL-1β was measured from thawed serum (at room temperature) by Abcam IL-1β ELISA kit according to the manufacturer’s instructions. In this assay, an antibody specific for IL-1β was coated onto the wells of the microtiter plates. Samples, including standards of known IL-1β concentrations and unknowns were pipetted into these wells and incubated at room temperature. The wells were then washed and a biotinylated antibody specific for IL-1β was added to the wells and incubated. After further washing, a streptavidin-peroxydase conjugate was added to each well and incubated. The wells were then washed to remove all unbound enzymes and TMB solution, which acts on the bound enzyme was added to induce a coloured reaction product. The intensity of this coloured product was directly proportional to the concentration of IL-1β present in the samples.
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