title: Semi-continuous, label-free immunosensing approach for Ca2+-based conformation change of a calcium-binding protein.
abstract: A label-free immunosensing method based on the conformational change of calcium-binding protein (CBP) depending on analyte concentration was explored for semi-continuous analysis of free Ca(2+). Glucose-galactose-binding protein as a CBP and produced as a recombinant protein by Escherichia coli was used as the immunogen to produce monoclonal antibodies by hybridoma technology. We finally screened the 3-6F cell clone, which produced the desired antibody specific to a particular structural conformation of the protein that occurred only upon CBP-calcium complex formation. To construct an immunosensor, the antibody was immobilized via a secondary antibody on an Octet Red optical fiber-based label-free sensor. Calcium analysis was conducted on the sensor in combination with CBP previously added to the aqueous sample, which distinguished the sensor signal according to the analyte concentration. The immunosensor produced a signal in real time with a response time of approximately 15 min and could be reused for analyses of different samples in a semi-continuous manner. The minimum detection limit of the analyte under optimal conditions was 0.09 mM and the upper limit was about 5 mM (log-logit transformed standard curve linearity: R(2) > 98%). In sample tests with milk, the analytical performance of the sensor was highly correlated (R(2) > 99%) with that of the reference system based on the KMnO4 titration method (ISO 12081). Although the sensor showed cross-reactivity at high concentrations (>1 mM) of cations including zinc, iron, manganese, and copper, these ionic components were not traceable (<0.01 mM) in milk.
authors.affiliation: Department of Bio-Microsystem Technology, Korea University, 1, 5-ka, Anam-dong, Sungbuk-gu, Seoul 136-701, Korea. shpaek@korea.ac.kr.
authors.forename: Sung-Ho
authors.initials: SH
authors.lastname: Paek
authors.forename: Ji-Na
authors.initials: JN
authors.lastname: Park
authors.forename: Dong-Hyung
authors.initials: DH
authors.lastname: Kim
authors.forename: Hee-Soo
authors.initials: HS
authors.lastname: Kim
authors.forename: Un-Hwan
authors.initials: UH
authors.lastname: Ha
authors.forename: Sung-Kyu
authors.initials: SK
authors.lastname: Seo
authors.forename: Se-Hwan
authors.initials: SH
authors.lastname: Paek
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000276:immunology; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D005336:Fiber Optic Technology / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D051379:Mice; D008892:Milk / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D055100:Optical Fibers; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D055910:Antibodies, Immobilized; D002135:Calcium-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011994:Recombinant Proteins; C023550:galactose-binding protein; D002118:Calcium