Isolation and characterization of nucleic acid-binding antibody fragments from autoimmune mice-derived bacteriophage display libraries

Abstract

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/1pr) 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 v h 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. A light (L) chain shuffling experiment was performed to investigate how indiscriminate a H chain fragment from a nucleic acid-binding Fab could be and still retain antigen binding. In contrast to previous reports employing haptens or proteins, only a few L chains were isolated that enabled DNA and RNA binding. Furthermore, the deduced aa sequences of these L chains were almost identical to that of the original Fab. The few aa changes present resulted in a shift in antigen specificity, suggesting that fine tuning of substrate recognition may be possible. This report demonstrates that phage Fab display libraries will be a powerful method for the isolation of nucleic acid-binding Ab in vitro and may be applicable to the study of immune recognition, especially as it occurs in autoimmune disease.