Preparation and characterization of immunological reagents for analytical applications.

Persistent Link:
http://hdl.handle.net/10150/184395
Title:
Preparation and characterization of immunological reagents for analytical applications.
Author:
Nielsen, Randall Gunnar.
Issue Date:
1988
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:
Immunochemical reagents were characterized under carefully controlled laboratory conditions using conventional high performance liquid chromatography instrumentation. The stationary phase was prepared by attaching antigen molecules to an insoluble support through a covalent linkage. Experiments were carried out by introducing antibody molecules into the mobile phase and monitoring their interaction with the stationary phase. Monoclonal antibodies were employed because of their more homogeneous properties compared to polyclonal antisera. Radioisotopes were employed to study low level adsorption on the stationary phase. Recovery experiments were carried out in which it was possible to account for all of the material introduced into the mobile phase. Antibodies were purified over a preparative scale antigen affinity column following labeling to insure high immunoreactivity. Studied under normally dissociating conditions, irreversible adsorption of picomole amounts of protein on the antigen stationary phase was greater than on other ligand modified stationary phases. This accumulation decreased with repeated use of the affinity column. The present study provides a framework for evaluation of other immunoaffinity systems and demonstrates that reproducible recovery of immunologically active material in high yield is possible. Monoclonal antibodies labeled with fluorescein were different from unlabeled molecules in binding and physical characteristics. Computer simulations were used to describe binding behavior. Although fluorescein labels improve detection sensitivity over native protein absorbance, their use in this case decreased binding affinity significantly. Heterogeneity of affinity purified fluorescein labeled and unlabeled monoclonal antibodies was examined with two dimensional gel electrophoresis. In addition to increased charge heterogeneity in the labeled antibody fragments, both light and heavy chains possessed more negative character. These results agree with each other. Fluorescein contains a carboxylic acid group, and modification of antibody light chains may interfere with binding affinity. The number and location of labels covalently attached to antibodies must be carefully controlled to obtain maximum detection sensitivity and preserve immunoreactivity.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Chemical tests and reagents.; Monoclonal antibodies -- Reactivity.; Biological reagents.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Chemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Wilson, George

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titlePreparation and characterization of immunological reagents for analytical applications.en_US
dc.creatorNielsen, Randall Gunnar.en_US
dc.contributor.authorNielsen, Randall Gunnar.en_US
dc.date.issued1988en_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.abstractImmunochemical reagents were characterized under carefully controlled laboratory conditions using conventional high performance liquid chromatography instrumentation. The stationary phase was prepared by attaching antigen molecules to an insoluble support through a covalent linkage. Experiments were carried out by introducing antibody molecules into the mobile phase and monitoring their interaction with the stationary phase. Monoclonal antibodies were employed because of their more homogeneous properties compared to polyclonal antisera. Radioisotopes were employed to study low level adsorption on the stationary phase. Recovery experiments were carried out in which it was possible to account for all of the material introduced into the mobile phase. Antibodies were purified over a preparative scale antigen affinity column following labeling to insure high immunoreactivity. Studied under normally dissociating conditions, irreversible adsorption of picomole amounts of protein on the antigen stationary phase was greater than on other ligand modified stationary phases. This accumulation decreased with repeated use of the affinity column. The present study provides a framework for evaluation of other immunoaffinity systems and demonstrates that reproducible recovery of immunologically active material in high yield is possible. Monoclonal antibodies labeled with fluorescein were different from unlabeled molecules in binding and physical characteristics. Computer simulations were used to describe binding behavior. Although fluorescein labels improve detection sensitivity over native protein absorbance, their use in this case decreased binding affinity significantly. Heterogeneity of affinity purified fluorescein labeled and unlabeled monoclonal antibodies was examined with two dimensional gel electrophoresis. In addition to increased charge heterogeneity in the labeled antibody fragments, both light and heavy chains possessed more negative character. These results agree with each other. Fluorescein contains a carboxylic acid group, and modification of antibody light chains may interfere with binding affinity. The number and location of labels covalently attached to antibodies must be carefully controlled to obtain maximum detection sensitivity and preserve immunoreactivity.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectChemical tests and reagents.en_US
dc.subjectMonoclonal antibodies -- Reactivity.en_US
dc.subjectBiological reagents.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, Georgeen_US
dc.identifier.proquest8814263en_US
dc.identifier.oclc701246526en_US
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