Sunrise absorbance reader

A commercially available colorimetric Iron Assay Kit (MAK025; Sigma-Aldrich, St. Louis, MO, USA) was used to determine serum and hepatic iron concentrations according to the manufacturer’s instructions. Serum was used directly while liver tissue weighing approximately 30–50 mg were freshly-homogenised in 10 volumes (w/v) of iron assay buffer. Tissue homogenate was centrifuged at 13,000× g for 10 min, and resultant supernatant was collected for the assay. Serum or liver samples were loaded and an acidic assay buffer was added to all wells in a ratio of 1:1 (v/v), to separate iron from bound proteins and an iron reducer added for total iron measurement. Absorbance was measured at 593 nm with a 700 nm reference, using a Sunrise™ absorbance reader (Tecan Group Ltd., Männedorf, Switzerland). Data were corrected for background, normalised with corresponding protein content, and presented as μg/mL for serum iron and μg/mg for hepatic iron.

To measure the effects of febrile temperature and MPs, the ADAMTS13 activity was analyzed in 30 citrated plasma samples by CBA, as described with slight modifications [17 (link)]. Importantly, febrile plasma samples which were previously incubated at 38°C and 39°C in plasma preparation process would be incubated at 38°C and 39°C instead of 37°C in digestion step, respectively. The chromogenic reaction in the final process was performed by addition of 100 μl of substrate o-phenylene-diamine dihydrochloride (5 mg) (Med OPD P6912, Sigma Aldrich, USA). The ELISA reaction was stopped by addition of 100 μl H₂SO₄ (3%) and absorbance was read at 492 nm with reference length at 620 nm using Sunrise™ absorbance reader (Tecan Group Ltd. Switzerland). The ADAMTS13 activity in each sample was determined in duplicate.
Normal pooled plasma (NPP), which was collected from 30 healthy participants was used to generate a calibration curve. The NPP was diluted in to 7 dilutions, 1:5, 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320 in 1.5 M urea, 5 mM Tris, pH 8.0. The ADAMTS13 activity was read from the sigmoid-like calibration curve using point-to-point plot. The NPP dilution of 1:10 was defined as 107% of ADAMTS13 activity.

The commercially available colorimetric Haptoglobin Kit (Phase™ Range TP801; Tridelta Development Ltd., Maynooth, Ireland) was used to measure haptoglobin concentration in plasma, based on its preservation of haemoglobin’s peroxidase activity. Following the manufacturer’s instructions, samples were loaded into a 96-well microplate and incubated with haemoglobin and chromogen. After 5 min, absorbance was measured at 620 nm with a 700 nm reference, using the Sunrise™ absorbance reader (Tecan Group Ltd., Männedorf, Switzerland). Sample haptoglobin was determined from a 0.039–2.5 mg/mL standard curve. Data were corrected for background and presented as mg/mL.