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.
These data support the notion that DNA hydrolytic ability is an intrinsic characteristic of some lupus anti-DNA antibodies rather than a result of contamination of antibody preparations with blood DNAses as has been suggested based on the output from prior purification protocols.
|Advisor:||Hartmann, James X.|
|Commitee:||Iragavarapu-Charyulu, Vijaya, Binninger, David, Milton, Sarah L., Dawson-Scully, Ken|
|School:||Florida Atlantic University|
|School Location:||United States -- Florida|
|Source:||DAI-B 81/6(E), Dissertation Abstracts International|
|Subjects:||Biology, Immunology, Molecular biology|
|Keywords:||Anti-DNA antibody, Anti-ssDNA antibody, Hydrolysis probe, Hydrolytic activity, Real-time fluorescence-based, Systemic lupus erythematosus|
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