Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.
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.
Mecklenburg
M
M
Chemical Center, University of Lund, Sweden.
Lindbladh
C
C
Li
H
H
Mosbach
K
K
Danielsson
B
B
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
D002021:Buffers; D007328:Insulin; D009243:NAD; D011384:Proinsulin; D006899:Mixed Function Oxygenases; D007770:L-Lactate Dehydrogenase; C021497:lactate 2-monooxygenase; D000469:Alkaline Phosphatase