2.6. Measurement of IL-31 Serum Level
BLOOD
Blood samples were collected in EDTA tubes and centrifuged for 20 min at 2000–3000 rpm speed. The supernatant without sediment was then taken out. The plasma was stored at −80 °C for six months. About 50 μL standard solution was added to the standard well. Then, 40 μL of the sample was added into the sample well followed by 10 μL of antibody anti IL-31. A total of 50 μL streptavidin–HRV was added into the sample well and standard well and mixed. Both solutions were left for 30 min of incubation under a temperature of 37 °C. After 30 min, the plate was washed five times with buffer solution and the wells were left with 0.35 buffer solution for 30 s to 1 min. About 50 μL of A substrate solution and 50 μL of B substrate solution were added into each well and left for another 10 min at 37 °C temperature. Then, 50 μL of stop solution was added into each well, after which the bluish color changed to yellowish. Interleukin-31 serum levels were assessed using a microplate reader at 450 nm within 10 min after the addition of stop solution.