KIR3DL1 allotype binding to HLA by Luminex (W632 nomalised values).

HLA-I recognition by KIR3DL1 allotypes was assessed through binding of KIR3DL1 tetramers to beads coated with a panel of 100 different HLA-A, -B, and -C molecules (LABScreen HLA Class I Single Antigen; One Lambda). PE-conjugated KIR3DL1 tetramer (5 µg per test) was incubated with beads for 30 min at room temperature in the dark in 300 mM of phosphate-buffered NaCl (PBS-300) with 5% fetal calf serum (AusGeneX). The beads were then washed three times in PBS-300 with 0.05% Tween 20 and resuspended in PBS-300. Binding was measured on a Luminex platform (LABScan 100; One Lambda) through identification of the individual HLA allotypes via unique bead labeling and detection of tetramer fluorescence intensity on each bead set. Normalized fluorescence values for analysis were obtained using the HLA Fusion software suite (One Lambda) that subtracted background values using the following formula:

(S#N − SNC bead) − (BG#N − BGNC bead), 

where S#N is the sample-specific fluorescence value (trimmed mean) for bead #N, SNC bead is the sample-specific fluorescence value for the negative control (nude) bead, BG#N is the background negative control fluorescence value for bead #N, and BGNC bead is the background negative control fluorescence value for negative control bead. To obtain binding values for each HLA-I, mean fluorescence intensity (MFI) values for an isotype control (a PE-conjugated IgG) were subtracted from the raw values obtained for each experiment, and recognition was assessed as being higher than that of the tetramer to beads without conjugated HLA-I. MFI values were normalized to the highest value for each experiment and also provided averaged over three experiments.