Preparation and characterization of immunochemical reagents for bioanalytical applications.

Abstract

Immunological reagents were prepared and characterized for the development of analytical methodology in bioanalytical research. Monoclonal antibodies to glucose oxidase (E.C. 1.1.3.4) from Aspergillus niger were prepared with apoenzyme as the antigen. Five of these antibodies, all of the IgG, subisotype, were further characterized. The carbohydrate moiety of the enzyme is not immunogenic. Binding of the five antibodies to the enzyme had no detectable effect on its catalytic properties. All the antibodies are shown to be directed towards segmental epitopes of the enzyme, not involving the carbohydrate moiety. Each enzyme subunit has more than one non-overlapping epitope. All five antibodies bound enzyme in a non-native conformation when coated on ELISA plates in preference to the native solution conformation. The importance of having a solution phase screening procedure for monoclonal antibodies is demonstrated. Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human IgG. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multisite attachment, multiple orientations, and steric hindrance imposed by crowding of antibody and the size of the antigen. With oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab') fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance. Preparation of fragments of immunoglobulin were studied. Within the same species different antibodies showed different sensitivities to proteolytic cleavage by pepsin. A rapid, simple, high performance size exclusion chromatographic method was developed to monitor the reaction progress. Conditions must be optimized for each antibody in the preparation of F(ab')₂. Preparation of F(ab') from F(ab')₂ shows that 10-15% of goat anti-mouse F(ab')₂ was resistant to reduction. The procedure causes reduction of disulfide bonds other than the inter-heavy chain disulfide bonds.