- 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 levels
were 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. The reaction was stopped by adding the
stop solution, 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.