2.5. Luminex assay

Diluted sera (1:5, 1:100) of pediatric lung transplant recipients were used to measure the Ab to human HLA class I and class II using a LABScreen mixed Ag kit (One Lambda, Canoga Park, CA) by Luminex assay according to the manufacturer’s protocol. Briefly, 5 μl of beads coated with mixed HLA class I and Class II Ag was added to 20 μl of diluted serum samples (1:5, 1:100) in 96-well plates and incubated for 30 min at room temperature. The plates were washed and 100 μl of PE-conjugated goat anti-human Ab was added and incubated for 30 min. Plates were then washed and up to 100 beads were analyzed using the Bio-Plex 200 system (Bio-Rad Laboratories, Hercules, CA). Negative controls (One Lambda, Canoga Park, CA) used for normalization.