Optimization of sputum-processing methods for the measurement of interleukin-5: effects of protease inhibition.
BACKGROUND
Interleukin (IL)-5 measurement in sputum samples has produced variable results that appear to be due to methodological problems. The aim of the present study was to investigate the effects of dithiothreitol (DTT), sputum protease inhibition and sample storage on IL-5 recovery in order to develop a method to accurately measure IL-5 in dispersed sputum supernatant.


METHODS
Measurement of IL-5 in sputum was performed in 22 subjects with airway disease. Interleukin-5 recovery was measured in samples spiked with recombinant human IL-5 using a commercial ELISA. A mix of four protease inhibitors (PI) was added to sputum processed using the selection method with dispersion using DTT and stored with and without inhibitors.


RESULTS
The addition of PI to sputum resulted in a 24% increase in IL-5 recovery. Recovery was not further increased with the addition of a blocking protein. Storage of IL-5-spiked sputum gave significantly less recovery. The addition of PI to sputum processed with DTT had no effect on total cell count, viability or cell differential.


CONCLUSIONS
Interleukin-5 recovery is increased by the addition of PI to samples processed using the selected portion method with DTT dispersion. A protease inhibitor cocktail should be added to sputum for IL-5 assay.
Simpson
Jodie L
JL
Airways Research Centre, Department of Respiratory and Sleep Medicine, John Hunter Hospital, Newcastle, New South Wales, Australia.
Gibson
Peter G
PG
Wark
Peter A B
PA
D000328:Adult; D000368:Aged; D004229:Dithiothreitol / Q000494:pharmacology*; D005260:Female; D006801:Humans; D015848:Interleukin-5 / Q000032:analysis*; D008297:Male; D008875:Middle Aged; D011480:Protease Inhibitors / Q000494:pharmacology*; D013183:Sputum / Q000737:chemistry*
D015848:Interleukin-5; D011480:Protease Inhibitors; D004229:Dithiothreitol