A simplified direct on-chip forward or reverse immunoassay for evaluating protein-protein interactions in the serum.

ABSTRACT

BACKGROUND: The identification of protein-protein interactions is a great challenge. In this study, we fabricated a gold surface-modified biochip with activated sophorolipids (SLs) in combination with 16-amino-1-hexadecanethiol hydrochloride to detect serum proteins. MAIN METHODS AND MAJOR RESULTS: The on-chip immunoassay reported here included a forward assay, in which a ligand is immobilized on the biochip surface and allowed to interact with its free specific receptor in liquid phase, and a reverse assay, in which a receptor is loaded on the biochip surface and combined with its free specific ligand in solution. The specificity of the molecular interactions on the biochip was evaluated using immunological blocking assays and chemiluminescent immunoassays (CLIA). Hemophagocytic lymphohistiocytosis (HLH) serum was used to test the potential utilization of the biochip. Reverse receptor CD25-based interleukin (IL)-2 and forward ligand IL-2-based CD25 assays revealed that the limit of detection of the target proteins was as low as 156 and 78 pg/ml, respectively. Using receptor- or ligand-based platforms, we found that the positive rates of free IL-2 and soluble CD25 (sCD25) monomers in the sera of HLH patients were 14.3% and 71.4%, respectively. In addition, the biochip showed good compatibility with CLIA for the measurement of sCD25 (r = 0.77, p < 0.01). CONCLUSIONS AND IMPLICATIONS Biochip platforms, such as on-chip immunoprecipitation (IP), can be used to evaluate the interactions between proteins, ligands, and receptors, or enzymes and substrates in serum.