Homogeneous enzyme-linked assays mediated by enzyme antibodies; a new approach to electrode-based immunoassays

A new homogeneous enzyme--immunoassay system is described. The assay employs an ammonia-liberating enzyme covalenty coupled to protein antigens along with two antibodies. An anti-enzyme antibody inhibits the enzyme. However, an antibody selective for the antigen reverses the inhibition process. When samples containing free antigen are present in the assay mixture, there is competition for anti-antigen antibody sites and protection against the anti-enzyme antibodies is diminished. The extent of the enzymatic reaction is monitored with an ammonium ion-selective electrode. Preliminary data demonstrating the feasibility of this approach for human serum albumin are presented.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25005/1/0000432.pd

Development and Application of Electrode-Based Enzyme-Labeled Competitive Binding Assays for the Determination of Haptens and Proteins (Ion-Selective Electrodes, Immuno).

Research concerning the feasibility of adapting various enzyme-labeled competitive binding assay arrangements to ion-selective electrode based detection systems is described. Assays for haptens and proteins were investigated by applying conventional methodologies for hapten analysis and novel protocols for protein determinations. A flow-through nonactin electrode detection system was used to determine the final enzymatic activity in the majority of these investigations. A heterogeneous assay for the determination of adenosine 3':5'-cyclic monophosphate (cyclic AMP) is described. This assay uses various enzyme-cyclic nucleotide conjugates in a double antibody separation scheme. Calibration curves demonstrate the sensitivity and selectivity of the assay. Real sample application was shown by assaying cyclic AMP in urine. Attempts to develop an enzyme-labeled competitive binding assay for cyclic AMP using its specific binding protein instead of an antibody are discussed. Progress was hindered due to difficulties associated with the reduced ability of the conjugated cyclic AMP-dependent protein kinase to bind cyclic AMP. Various assay schemes and enzymes were investigated, but failed to produce satisfactory results. A new homogeneous enzyme-labeled competitive binding assay which incorporates potentiometric detection is described. This assay uses two antibodies; one with specificity towards the lig and of interest, and one with specificity towards the enzyme label. The enzyme antibody reduces the enzyme's activity and is used to indicate the extent of binding of the lig and antibody to the enzyme-labeled lig and . Development of enzyme antibodies is discussed. Specific assay criteria, characteristics, and protocol for optimizing and conducting the assay are also described. Application of this assay for the selective determination of the protein human serum albumin is presented. The development of an immunoelectrode tube is detailed. This tube combines an enzyme-labeled competitive binding assay with an ion-selective electrode detection system in one disposable tube. Electrode membranes were cast on the bottom of polystyrene and polypropylene tubes. Antibodies were immobilized in the interior of the tubes. Various methods for accomplishing both of these tasks were investigated. Preliminary calibration curves for the determination of human serum albumin using the immunoelectrode tubes are shown.Ph.D.Analytical chemistryUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/160098/1/8422200.pd