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|Title:||Characterization of Homogeneous and Heterogeneous Anti-Nucleic Acid Autoantibodies in Systemic Lupus Erythematosus|
|Author(s):||Rezaei Poor Kardost, Robabeh|
|Department / Program:||Microbiology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Three murine monoclonal anti-ssDNA autoantibodies were generated by fusion of spleen cells from autoimmune diseased NZB/NZW F1 hybrid mice with Sp 2/0-Ag 14 murine myeloma cell line. These monoclonal antibodies were purifed by affinity chromatography, and their immunochemical properties were studied using different methods. Specificity studies characterizing monoclonal anti-ssDNA autoantibodies revealed specificity for nucleotides. Clone E-4-1 showed preferential binding with mono, di, and polyguanosine. Clones E-4-4 and E-11-1 specifically bound mono, di, and polythymidine. Results of inhibition studies indicated the following order of reactivity for each monoclonal antibody: Polynucleotide > dinucleotide > mononucleotide. In a fluorescence immune assay, using aminonaphthyl derivatives of nucleotides, all three monoclonal antibodies exhibited low affinity for the homologous mononucleotides. The affinity of clone E-4-1 increased 10 folds when a dinucleotide derivative was used relative to a mononucleotide fluorescent probe.
To determine the relative role of nucleotides in autoanti-nucleic acid antibodies produced spontaneously in systemic lupus erythematosus, the relative role of uridine and thymidine were analyzed as a prelude to determine if both RNA and DNA are important in autoimmune disease SLE. To achieve this, anti-uracil, anti-thymidine, and anti-ssDNA were induced experimentally in rabbits and mice and characterized extensively using different methods and approaches. These antibodies were used as model systems and were compared with heterogeneous and homogeneous SLE antibody populations.
To study binding specificity in a heterogeneous SLE antibody population, human SLE sera were studied in terms of their binding specificities. The SLE sera also served as a comparative control with the homogeneous murine monoclonal autoantibody studies. In the modified Farr assay, all three human SLE sera showed preferential binding for ssDNA. The specificity of the interaction of the SLE autoantibodies with nucleic acids was further verified in inhibition assays, in which ssDNA gave complete inhibition, while, nucleotides only partially inhibited the reaction. No anti-nucleotide activity was detected in the human SLE sera using the fluorescence immune assay.
Problems encountered in many classical assays (i.e. lack of reproducibility, high background or nonspecific binding, low sensitivity, etc.) have imposed general restrictions and limitations on such assays. Therefore, weighing the various factors and the potential fluorescence-based assays provide (e.g. simplicity, sensitivity, precision, speed, etc.) a fluorescence immune assay was designed and developed to measure the primary intrinsic interaction between nucleotides and antibodies. The fluorescence immune assay as devised in these studies provided many advantages over other conventional methods.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1981.
|Date Available in IDEALS:||2014-12-16|