[Detection of the antigenic peptides in pHLA complexes by ultrafiltration-high performance liquid chromatography]

Recombinant HLA-Ⅰ molecules/antigenic peptide complexes (pHLA complexes) are applied in the research of human T cell-specific immune responses. The preparation of pHLA complex is based on genetic engineering and protein in vitro dilution and folding-refolding technology. In an in vitro refolding system, recombinant HLA-Ⅰ molecules correctly fold and bind with antigenic peptides to form complexes. In this study, ultrafiltration-high performance liquid chromatography (ultrafiltration-HPLC) was used for quantitative determination of the antigenic peptides in recombinant pHLA complexes, especially for those in a small amount of prepared products. By adding the recombinant HLA-Ⅰ molecules and antigenic peptides into the refolding buffer, the heavy chain (HC) and light chain (β2m) of recombinant HLA-Ⅰ molecules were refolded and bond with the VYF antigenic peptide containing anchor residues to form a pHLA complex. The unbound free antigenic peptide VYF was removed by ultrafiltration to retain the complex. Finally, the pHLA complex was treated by acid to destroy its interaction, thus releasing the antigenic peptide. The results showed that the prepared recombinant pHLA complex was recognized by HLA-Ⅰ molecule specific antibody W6/32, which indicated that the recombinant HLA-Ⅰ class molecule had correct folding and was identified as pHLA complex. The antigen peptide VYF contained in the pHLA complex was also detected by ultrafiltration-HPLC, so it is feasible to apply ultrafiltration-HPLC for determination of pHLA complex. Compared with Western blotting, the concentration of antigenic peptides detected by ultrafiltration-HPLC was 0-9 μg/mL. The binding conditions can be optimized according to the amount of antigenic peptides bound in the complex in order to improve the folding efficiency of HLA-Ⅰ molecules and promote the binding of HLA-Ⅰ molecules to antigenic peptides. The production rate of pHLA complexes in the refolding system can also be calculated according to the content of antigenic peptides bound by pHLA complexes. Therefore, ultrafiltration-HPLC in this study can be used for the quality control of the preparation process of pHLA complexes, and may facilitate the research of T cell-specific immunity, artificial antigen-presenting cells, and development of specific tetramer probe applications.