Author Type

Graduate Student

Date of Award

2019

Document Type

Dissertation

Publication Status

Version of Record

Submission Date

October 2026

Thesis/Dissertation Advisor [Chair]

Hartmann, James X.

Abstract

Two novel methodologies were developed for purification and functional (DNA hydrolytic) assessment of anti-DNA antibodies of IgG isotype from patients with Systemic Lupus Erythematosus (SLE). Earlier protocols for purification and analysis of antibody hydrolytic abilities were lengthy, laborious, and potentially disruptive to antibody function. Purification protocols failed to capture all four IgG subclasses and produced multiple bands outside the range of IgG on electrophoretic separation. Hydrolysis assays were discontinuous increasing the likelihood of introducing error and making them better suited to analysis of endpoint kinetics rather than reaction kinetics. A two-step, affinity-based purification protocol was developed which utilized magnetic Dynabeads to capture serum components with binding affinity for a thymine 20mer followed by capture of the antibody components of this initial anti-T 20mer serum fraction using Protein G. A fluorescence-based method for real-time, continuous analysis of anti-DNA antibody hydrolytic activity utilizing hydrolysis probes was developed and used to characterize abzyme reaction kinetic parameters. Anti-DNA antibodies demonstrated significantly different Vmax and Km values in the hydrolysis assay (p <0.001) when compared with a DNAse I control.

Extent

218 p.

Comments

Includes bibliography.|Dissertation (Ph.D.)--Florida Atlantic University, 2019.|FAU Electronic Theses and Dissertations Collection|Includes bibliography.

Identifier

FA00013363

Format

pdf

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