title: Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.
abstract: A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.
authors.affiliation: Chemical Center, University of Lund, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Mecklenburg
authors.forename: C
authors.initials: C
authors.lastname: Lindbladh
authors.forename: H
authors.initials: H
authors.lastname: Li
authors.forename: K
authors.initials: K
authors.lastname: Mosbach
authors.forename: B
authors.initials: B
authors.lastname: Danielsson
mesh_terms: D000469:Alkaline Phosphatase; D002021:Buffers; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology; D017022:Flow Injection Analysis; D006801:Humans; D007328:Insulin / Q000032:analysis*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D006899:Mixed Function Oxygenases; D009243:NAD; D011384:Proinsulin / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013821:Thermometers
chemical_list: D002021:Buffers; D007328:Insulin; D009243:NAD; D011384:Proinsulin; D006899:Mixed Function Oxygenases; D007770:L-Lactate Dehydrogenase; C021497:lactate 2-monooxygenase; D000469:Alkaline Phosphatase