Autoimmune derived combinatorial phage display libraries: methods in construction of and affinity selection for anti-RNA Fabs.
Display of antibody fragments (Fab) on the surface of filamentous bacteriophage and selection of phage that bind to a particular antigen has enabled the isolation of Fab with numerous specificities. We have examined the possibility of isolating RNA-binding Fab by constructing and screening combinatorial libraries of phage displaying Fab derived from the antibody repertoires of autoimmune humans. The genes and corresponding Fabs will allow for examination of the mechanism by which antibodies recognize RNA. Patients selected exhibit mixed connective tissue disease (MCTD), which is a subset of systemic lupus erythematosus (SLE), MCTD patients contain antibodies reactive against various nucleic acid, protein, and nucleoprotein complexes, most notably those involved in RNA processing. The cDNA libraries were constructed from total RNA derived from leukophoresed patient samples and the resulting Fab genes were expressed in E. oli using the pComb system1. Affinity selection procedures have been designed to isolate anti-RNA Fabs from these libraries. Results demonstrate the ability to enrich for anti-RNA Fabs using biotinylated RNA-streptavidin capture methodologies.
Marchbank
M T
MT
Department of Biochemistry, University of Missouri School of Medicine, Columbia 65212, USA.
Deutscher
S L
SL
D000974:Antibodies, Antinuclear / Q000235:genetics / Q000378:metabolism; D000915:Antibody Affinity; D000918:Antibody Specificity; D015551:Autoimmunity; D001435:Bacteriophages / Q000235:genetics / Q000276:immunology; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008947:Mixed Connective Tissue Disease / Q000276:immunology; D012313:RNA / Q000276:immunology*
D000974:Antibodies, Antinuclear; D007140:Immunoglobulin Fab Fragments; D012313:RNA