Investigations of enzyme-linked potentiometric immunoassay systems.

Abstract

Pseudo-homogeneous and heterogeneous immunosensors for proteins based upon the binding interaction between protein A and IgG are described. Enzyme electrodes utilizing immobilized alkaline phosphatase and asparaginase are presented. A homogeneous enzyme immunoassay using a synthetic macromolecular substrate is examined. The proposed homogeneous immunoassay relies upon steric hindrance induced when an antibody binds an enzyme-antigen conjugate, in this case adenosine deaminase-theophylline. Methods of synthesizing the required macromolecular dextran-adenosine substrate are described. In the final system, the enzyme conjugate was inhibited five to ten percent in the presence of anti-theophylline. Therefore, a configuration utilizing an asparaginase enzyme electrode detector was examined. Response characteristics of the enzyme electrode are reported. An enzyme channeling immunosensor is also described. This sensor operates on the principle that electrochemical detectors only sense the concentration of species in the diffusion layer adjacent to the detector surface. The enzyme channeling sensor comprises an ammonium-selective electrode with immobilized adenosine deaminase and IgG. When protein A-alkaline phosphatase conjugate binds to IgG, a two-enzyme catalytic layer is created where adenosine 5'-monophosphate is converted to ammonium. In the presence of sample IgG, which competes for protein A-alkaline phosphatase conjugate, the signal resulting from the addition of substrate is diminished. The sensor was used to determine human serum IgG. Based on principles similar to the enzyme channeling immunosensor, an immunosensor employing one enzyme is reported. This sensor consists of a p-nitrophenolate-sensitive membrane with immobilized IgG. Protein A-alkaline phosphatase binds to IgG in a competitive assay arrangement. Pseudo-homogeneous and heterogeneous modes of use are examined. Two different sensor membrane matrices are compared. Two immobilization methods for the preparation of alkaline phosphatase enzyme electrodes are also reported. Finally, the response characteristics and analytical performance of a new thiocyanate-selective electrode are described. The electrode utilizes a novel manganese (III) porphyrin as the membrane-active component. The porphyrin's sterically hindered axial coordination sites contribute to the electrode's enhanced selectivity for thiocyanate over chloride and other anions. Determination of thiocyanate in human saliva samples with the new electrode agrees with more traditional methods.