Semisynthetic Bioluminescent Sensor Proteins for DirectDetection of Antibodies and Small Molecules in Solution

摘要

Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detectionof small molecules using a competitive assay format, which is demonstratedhere for the detection of creatinine. Using a preassembled complexof CR-LUMABS and an anti-creatinine antibody, the detection of highmicromolar concentrations of creatinine was possible both in bufferand in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensorssignificantly expands the range of antibody targets and enables theapplication of LUMABS sensors for the ratiometric bioluminescent detectionof small molecules using a competitive immunoassay format.