Detection of a Large Antigen through the Masking and Exposure of Fragment of Split Luciferase

Abstract

We developed PMBiT, an antibody-binding Protein M (PM)-based bioluminescent probe, which detects large antigens through luciferase reconstitution by exposing the luciferase fragment. The detection is achieved by exploiting the principle that the antibody, the large antigen, and PM are not able to form a complex at the same time. The PMBiT is prepared by conjugating PM with HiBiT-based peptide from the split NanoLuc luciferase through click reaction. It retained its binding activity to antibody, and showed bioluminescence upon reconstitution of the luciferase, by assembling with LgBiT, the other fragment of split NanoLuc. Mixing PMBiT with various IgG antibodies resulted in decreased bioluminescence. In contrast, when PMBiT was mixed with IgG bound to its large antigen, such as human C-reactive protein, the decreased bioluminescence was lessened, leading to bioluminescence increase in a dose dependent manner. Molecular dynamics simulations of PM showed that two regions in the C-terminus contribute to steric clashes with antigens due to their relatively rigid structures. Furthermore, in silico analysis of the structure suggested that antigen size is the primary factor blocking PMBiT binding to IgG for antigen detection. The immunoassay utilizing PMBiT does not require genetic manipulation of antibodies, allowing for seamless and scalable antibody replacement, and will advance the future of on-site detection and rapid diagnostics.