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Isolation and characterization of nucleic acid-binding antibody fragments from autoimmune mice-derived bacteriophage
M J Calcutt1, M T Kremer, M F Giblin
1Department of Biochemistry, University of Missouri, Columbia 65212.
The 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, including haptens, proteins and viral particles. We have examined the possibility of isolating nucleic acid-binding Fab by constructing a combinatorial library of phage displaying Fab derived from autoimmune (MRL/lpr) mice. Autoimmune mice were chosen because they contain antibodies (Ab) reactive against nuclear components, including DNA, RNA and protein complexes. The library was panned against single-stranded (ss) calf thymus (CT) DNA and the selected Fabs were analyzed further. Characterization of the nucleic acid-binding phage led to the identification of two kinds of Fab with quite different properties. One Fab bound with high affinity a variety of ssDNA molecules, as well as several model RNA substrates. This Fab has been affinity purified to greater than 95% and competition studies revealed a marked preference for binding to poly(dT). The second Fab showed a reduced binding to RNA ligands and a restricted number of ssDNA molecules. Analysis of the deduced amino acid (aa) sequences of the Fab variable (V) regions revealed that the heavy (H) chain V region from the strong nucleic acid-binding Fab was derived from a VH gene that is used recurrently in autoantibodies. This VH domain was most similar to an anti-ssDNA autoimmune monoclonal antibody (mAb) suggesting that antigen-binding specificities present in an autoimmune repertoire may be directly accessed by this approach.(ABSTRACT TRUNCATED AT 250 WORDS)
The 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, including haptens, proteins and viral particles. We have examined the possibility of isolating nucleic acid-binding Fab by constructing a combinatorial library of phage displaying Fab derived from autoimmune (MRL/lpr) mice. Autoimmune mice were chosen because they contain antibodies (Ab) reactive against nuclear components, including DNA, RNA and protein complexes. The library was panned against single-stranded (ss) calf thymus (CT) DNA and the selected Fabs were analyzed further. Characterization of the nucleic acid-binding phage led to the identification of two kinds of Fab with quite different properties. One Fab bound with high affinity a variety of ssDNA molecules, as well as several model RNA substrates. This Fab has been affinity purified to greater than 95% and competition studies revealed a marked preference for binding to poly(dT). The second Fab showed a reduced binding to RNA ligands and a restricted number of ssDNA molecules. Analysis of the deduced amino acid (aa) sequences of the Fab variable (V) regions revealed that the heavy (H) chain V region from the strong nucleic acid-binding Fab was derived from a VH gene that is used recurrently in autoantibodies. This VH domain was most similar to an anti-ssDNA autoimmune monoclonal antibody (mAb) suggesting that antigen-binding specificities present in an autoimmune repertoire may be directly accessed by this approach.(ABSTRACT TRUNCATED AT 250 WORDS)