Soluble HLA Technology as a Strategy to Evaluate the Impact of HLA Mismatches

Figure 2

Generation of peptide sequences from sHLA molecules. This figure gives a schematic overview of the steps towards the generation of peptide sequences obtained from sHLA molecules. The HLA heavy chain is given in blue; β2m is shown in yellow; and the peptide is shown in red. Cell culture supernatants containing sHLA molecules are passed over an N-hydroxysuccimide- (NHS-) activated HiTrap column coupled to mAb w6/32. Trimeric complexes (class I heavy chain, β2m and peptide) are eluted using a pH 2.7 elution buffer. Here, peptides from sHLA complexes can be differentiated into low and high binding peptides. The trimeric elution fractions are filtered through a 10 kDa cut-off membrane and the peptides detected in the flow through are considered to be of low affinity. The retentate containing dimeric (heavy chain and β2m) as well as trimeric complexes is then treated with 0.1% trifluoroacetic acid (TFA) to elute high binding peptides that can finally be separated by filtration through an additional 10 kDa cut-off membrane. Flow through fractions containing the low or high affinity peptides are subjected to mass spectrometric analysis using an Eksigent NanoLC Ultra 2D HPLC coupled to an orbitrap ion trap. Database queries can finally be carried out using Mascot software [25] incorporating the IPI human and the respective decoy databases.