Detection of a large antigen through the masking and exposure of a fragment of split luciferase.

We developed PMBiT, an antibody-binding Protein M (PM)-based bioluminescent probe that detects large antigens via luciferase reconstitution by exposing a luciferase fragment. Detection is achieved by exploiting the principle that the antibody, large antigen, and PM cannot form a complex simultaneously. PMBiT was prepared by conjugating PM with a HiBiT-based peptide from split NanoLuc luciferase through an Azide-DBCO click reaction. It retained its binding activity to the antibody and showed bioluminescence upon reconstitution of the luciferase with LgBiT, the other fragment of the 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, a dose-dependent increase in bioluminescence was obtained. Molecular dynamics simulations of PM showed that two regions in the C-terminus contribute to steric clashes with antigens owing to their relatively rigid structures. Furthermore, in silico analysis of the structure suggested that the antigen size was the primary factor blocking the binding of PMBiT to IgG for antigen detection. An 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.