Anna Swietlikowska,Eva van Aalen,Max Bossink,Laura van Weesep,Maarten Merkx
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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.
期刊介绍:
ACS Sensors is a peer-reviewed research journal that focuses on the dissemination of new and original knowledge in the field of sensor science, particularly those that selectively sense chemical or biological species or processes. The journal covers a broad range of topics, including but not limited to biosensors, chemical sensors, gas sensors, intracellular sensors, single molecule sensors, cell chips, and microfluidic devices. It aims to publish articles that address conceptual advances in sensing technology applicable to various types of analytes or application papers that report on the use of existing sensing concepts in new ways or for new analytes.