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

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 ha...

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Bibliographic Details
Published in:ACS sensors 2017-11, Vol.2 (11), p.1730-1736
Main Authors: Arts, Remco, Ludwig, Susann K. J., van Gerven, Benice C. B., Estirado, Eva Magdalena, Milroy, Lech-Gustav, Merkx, Maarten
Format: Article
Language:English
Subjects:
Alkynes - chemistry
Antibodies - immunology
Azides - chemistry
Creatinine - analysis
Creatinine - immunology
Dinitrophenols - analysis
Dinitrophenols - immunology
Epitopes - chemistry
Epitopes - immunology
Immunoassay - methods
Luminescent Proteins - chemistry
Phenylalanine - analogs & derivatives
Phenylalanine - chemistry
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Summary: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 detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.
ISSN:2379-3694
2379-3694
DOI:10.1021/acssensors.7b00695