RAPPID-M: A Mix-and-Measure Bioluminescent Sandwich Immunoassay Based on Generic Antibody-Binding Protein M

Homogeneous immunoassays that allow direct in-sample detection of biomarkers provide an attractive alternative to traditional heterogeneous immunoassays that require multiple washing and incubation steps. We previously reported the development of RAPPID, a bioluminescent sensor platform that uses split luciferase complementation to enable sandwich immunoassays directly in solution. Although RAPPID provides many benefits over traditional heterogeneous immunoassays, it requires protein G-mediated photoconjugation of split luciferase fragments to the Fc part of IgG monoclonal antibodies. Here, we report a new generation of RAPPID sensors (RAPPID-M) that do not rely on photoconjugation of protein G, but instead use a generic antibody-binding domain derived from protein M. The use of protein M substantially expands the application of RAPPID to include protein G-incompatible but analytically important mouse IgG1 antibodies, as well as scFv and Fab antibody fragments. Moreover, binding of protein M is found to be essentially irreversible, abolishing the need for photo-cross-linking. RAPPID-M thus improves upon the original RAPPID platform by the mix-and-measure-type assembly of antibody-split-luciferase conjugates and its compatibility with a broader scope of antibody types without compromising and even improving the analytical performance.