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Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution.
- Source :
-
ACS sensors [ACS Sens] 2017 Nov 22; Vol. 2 (11), pp. 1730-1736. Date of Electronic Publication: 2017 Nov 02. - Publication Year :
- 2017
-
Abstract
- 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.
- Subjects :
- Alkynes chemistry
Azides chemistry
Creatinine immunology
Dinitrophenols immunology
Epitopes chemistry
Epitopes immunology
Phenylalanine analogs & derivatives
Phenylalanine chemistry
Solutions
Antibodies immunology
Creatinine analysis
Dinitrophenols analysis
Immunoassay methods
Luminescent Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 2379-3694
- Volume :
- 2
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- ACS sensors
- Publication Type :
- Academic Journal
- Accession number :
- 29037030
- Full Text :
- https://doi.org/10.1021/acssensors.7b00695