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PublisherThe University of Arizona.
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AbstractThe components involved in an immunoassay were investigated in order to improve the detection limits of the ELISA and to make the assay adaptable to a flow injection analysis (FIA) configuration. The goal being the total automation of the ELISA procedure which is long, tedious and has high standard deviation. The antibody purification and cleavage methods were studied with special emphasis on obtaining products with highest immunological activity. The antibody-enzyme coupling reactions using homobifunctional reagents and heterobifunctional reagents were studied in order to attempt the preparation of highly characterized reagents. The fragments of IgG were coupled to polymeric supports via the hinge thiol groups to retain the maximum immunological activity. This method was found to be superior to those methods involving coupling via amino group. These reagents were used in the development of a sandwich ELISA for bovine IgG. The range of assay was in the 20-1000 femtomole range with a linear dynamic range of 2 orders of magnitude and an accuracy of 2-5%. A competitive ELISA based on the use of immobilized anti-human IgG Fab' fragments was developed. The linear dynamic range for this assay was found to be less than one order of magnitude. The detection limit was in the low picomole range with an accuracy of 2-5%. Based on the principle used in the two assays an enzyme immobilization scheme was developed for the reversible immobilization of these enzymes. Which was subsequently utilized in the determination of substrate in the picomole range in a reagent less FIA technique. The goals of this research project were realized in that the FIA system utilized in this work was capable of carrying out totally automated ELISA assays with an accuracy far surpassing the conventional plate ELISA assays.