Reference - Detail
| RRC ID | 56348 |
|---|---|
| Author | Su J, Dong J, Kitaguchi T, Ohmuro-Matsuyama Y, Ueda H. |
| Title | Noncompetitive homogeneous immunodetection of small molecules based on beta-glucuronidase complementation. |
| Journal | Analyst |
| Abstract |
In this study, a novel noncompetitive homogeneous immunoassay for antigen detection was developed. We utilized β-glucuronidase (GUS), a homotetrameric enzyme, the assembly of all of whose subunits is necessary to attain its activity. By using a mutant GUS (GUSm), wherein the dimerization of dimers, which is a rate-limiting step, can be effectively inhibited by a set of interface mutations, we attempted to create a biosensor for detecting various molecules. Usually, the affinity between the two variable region domains (VH and VL) of an antibody, especially for a small molecule, is relatively low. However, in the presence of an antigen, the affinity increases so that they bind tighter to each other. A pair of fusion proteins, comprising the VH and VL regions of the antibody as the detector tethered to a GUSm subunit as the reporter, was constructed to detect antigen 4-hydroxy-3-nitrophenylacetyl (NP) and bone Gla protein (BGP) through GUS activity measurement. Colorimetric and fluorescence assays could detect NP, 5-iodo-NP, and BGP within 1 h without separation steps and with a higher signal/background ratio than conventional ELISA. The instantaneous response after simple mixing of the components makes this system convenient and high-throughput. The system could be effective for the analyses of various small molecules in environmental and clinical settings. |
| Volume | 143(9) |
| Pages | 2096-2101 |
| Published | 2018-4-30 |
| DOI | 10.1039/c8an00074c |
| PMID | 29634056 |
| MeSH | Antibodies Antigens / analysis* Biosensing Techniques* Enzyme-Linked Immunosorbent Assay Glucuronidase / chemistry* Humans Immunoassay* Nitrophenols / analysis Osteocalcin / analysis Phenylacetates / analysis |
| IF | 3.978 |
| Times Cited | 5 |
| Altmetric score |
オルトメトリクス指標項目
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| The most frequently cited source | Patent(IFI CLAIMS) |
| Total number of mentions | 1 |
| Altmetric score changes over past 6months | 0.0 |
| Resource | |
| Prokaryotes E. coli | ASKA(-) pCA24N ASKA(-) JW1609 |