Isolation and characterization of nucleic acid-binding antibody fragments from autoimmune mice-derived bacteriophage display libraries.
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)
Calcutt
M J
MJ
Department of Biochemistry, University of Missouri, Columbia 65212.
Kremer
M T
MT
Giblin
M F
MF
Quinn
T P
TP
Deutscher
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D000974:Antibodies, Antinuclear / Q000378:metabolism; D000918:Antibody Specificity; D015551:Autoimmunity; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D017912:Inovirus; D051379:Mice; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism*; D011993:Recombinant Fusion Proteins
D000974:Antibodies, Antinuclear; D004268:DNA-Binding Proteins; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D012313:RNA; D004247:DNA