- Laboratory analysis
The samples were immediately centrifuged at 5,000 rpm and 4 °C for 10
min to remove cells and debris, and stored in aliquots at –70 °C until
analysis. The urinary N-methylhistamine, histamine and tryptase levelswere measured using test kits by the sandwich
enzyme-linked immunosorbent assay (Genzyme Corporation, Cambridge, MA,
USA). The specific biotinylated detection antibody and avidin
horseradish peroxidase conjugate were added to the wells and incubated.
After removing the non-washing parts, the blue color was created in the
boxes by adding a substrate solution. By adding the stop solution, the
reaction was stopped, and the blue color was observed to turn yellow.
Optical density (OD) was determined spectrophotometrically at a
wavelength of 450 ± 2 nm. Since the OD value was proportional to mast
cell mediator levels in the samples, the N-methylhistamine, histamine,
and tryptase levels concentrations were calculated using standard curve
graphics.