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Diamide does not improve the analysis of IL1b and TNFa in dithiothreitol-treated sputum supernatants

: Holz, Olaf; Lavae-Mokhtari, Bianca; Witte, Lena; Hohlfeld, Jens Michael

American Journal of Respiratory and Critical Care Medicine 189 (2014), Art. A5641
ISSN: 1073-449X
ISSN: 0003-0805
ISSN: 1535-4970
American Thoracic Society (ATS International Conference) <2014, San Diego/Calif.>
Fraunhofer ITEM ()

INTRODUCTION: There is one report (Milone ERS2012) that diamide can be used to re-oxidize disulfide bonds that were separated by DTT treatment during the homogenization of sputum samples, a process that can affect the analysis of mediators in sputum supernatants. It was the aim of this study to test this method for two selected cytokines in sputum samples of healthy subjects induced before and after inhalation of LPS.
METHODS: IL1b and TNFa ELISA standards (R&D DuoSet) were treated with DTT (final 0.05%) or PBS and frozen. After thawing diamide was added (3.75-60mM). DTT treated sputum supernatants from 8 healthy subjects at baseline and following LPS challenge (Holz et al. BMC Pul Med 2012) were thawed and incubated with diamide (7.5 or 60mM). Pooled DTT treated sputum supernatants were spiked with IL1b and diamide (2-120mM) was added before freezing or after thawing.
RESULTS: Treatment of assay standards with DTT resulted in a significant decline in the slope of the standard curves, which was reversed to the level of PBS treated standards by the addition of diamide. The effect was smaller for TNFa, which was already very sensitive to PBS treatment (>60% reduction). The median concentration of IL1b after LPS treatment was 56.3, 61.6 and 56.5 pg/ml (without, 7.5, 60mM diamide) and only 2/8, 4/8 and 2/8 samples had detectable IL1b levels at baseline. No positive effect of diamide was observed for TNFa. The median recovery of IL1b was 49 and 66% (125 and 15.6 pg/ml spike) when diamide was added before freezing and 47.6 and 61.7 % when diamide was added after thawing. The recovery did not differ from samples that were not treated with diamide.
CONCLUSION: In our hands the addition of diamide was not successful in reversing the negative effects of DTT on the analysis of IL1b and TNFa in sputum supernatants.