Preparation and characterization of immunochemical reagents for bioanalytical applications.

Persistent Link:
http://hdl.handle.net/10150/185457
Title:
Preparation and characterization of immunochemical reagents for bioanalytical applications.
Author:
Wimalasena, Rohan Lalith.
Issue Date:
1991
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
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.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Dissertations, Academic; Immunology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Chemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Wilson, George S.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titlePreparation and characterization of immunochemical reagents for bioanalytical applications.en_US
dc.creatorWimalasena, Rohan Lalith.en_US
dc.contributor.authorWimalasena, Rohan Lalith.en_US
dc.date.issued1991en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractImmunological 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.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectDissertations, Academicen_US
dc.subjectImmunology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorWilson, George S.en_US
dc.contributor.committeememberBurke, Michael F.en_US
dc.contributor.committeememberFernando, Quintusen_US
dc.contributor.committeememberMiller, Walteren_US
dc.contributor.committeememberByers, Jamesen_US
dc.identifier.proquest9124165en_US
dc.identifier.oclc710218984en_US
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