In this study, we have investigated in-situ the process of antigen immobilization and subsequent antigen-antibody interaction on solid surfaces by using infrared absorption spectroscopy in the multiple internal reflection (MIR-IRAS) geometry. Three kinds of MIR substrates, Si, GaAs and SiO2-deposited GaAs, were investigated in terms of transparency to the IR light and immobilization performance of antigen peptides, which was evaluated by using MIR-IRAS and enhanced chemiluminescence (ECL) assay. The SiO2-deposited GaAs prism provides the highest peptide immobilization efficiency that is compatible with MIR-IRAS measurements in the mid-infrared region. By using Pluronic F-127 as a blocking agent to inhibit nonspecific protein binding to the surfaces, we observed an antiserum-induced increase in the peak intensity corresponding to the amide I and amide II bands, which was selective for the antigen-antibody interaction. The present results show that MIR-IRAS is a promising tool not only for the characterization of the peptide-immobilized surface but also for the label-free detection of antigen-antibody interactions on solid surfaces.
- Antigen-antibody interaction
- Infrared spectroscopy
ASJC Scopus subject areas
- Atomic and Molecular Physics, and Optics
- Electrical and Electronic Engineering