Flow injection enzyme-based immunoassay systems for measurement of biomolecules.

This work describes the development of a fast, non-equilibrium, universal enzyme-linked flow-injection immunoassay (FIIA) system using an immobilized secondary-antibody reactor and the development of a rapid, sensitive, non-equilibrium flow-injection sandwich type EIA system suitable for the direct determination of proteins in biological samples. The flow-through secondary-antibody reactor technique using a reusable immunoreactor is shown to be simpler, faster, and more precise than conventional heterogeneous, double antibody, solid phase EIA techniques. By using non-equilibrium conditions, a single assay takes less than 15 min including regeneration of the immunoreactor. To illustrate the application of this system, theophylline and insulin are chosen as model hapten and macromolecule analytes, respectively. Results show that this new FIIA system is suitable for determining theophylline in serum samples with high selectivity (xanthine analogs) and high precision (RSD = 3.79%). The sandwich type EIA proposed system utilizes highly active adenosine deaminase-antibody conjugates in conjunction with a flow-through immunoreactor and an ammonium ion-selective potentiometric detector. By using non-equilibrium flow-rates of sample and reagent slugs, a single protein assay takes less than 12 min, including regeneration of the reactor. The proposed assay method is shown to be precise (RSD = 4.25%) and capable of determining accurately the model protein (human IgG) at sub-$\mu$g/ml concentrations. In addition, the development of a post-column HPLC biodetection system to monitor toxic substances (e.g., heavy metal ions) is also examined. The detection scheme is based on the potent inhibition of enzyme urease activity by toxic heavy metal ions. Continuous flow analysis in used to enlarge the inhibition of enzyme by toxic metal ions and amplify the enzymatic reaction. The inhibition of urease activity is then detected downstream with a flow-through ammonium ion selective electrode. In order to overcome the dispersion problems in the post-column continuous flow detector, a hydrophobic organic solvent segmentation technique is described. This segmentation technique can be used to significantly reduce band broadening and the adsorption of metal ions onto the glass coils of the flow-through post-column system.Ph.D.Analytical chemistryBiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128371/2/9001665.pd