Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display.
Functional antibody fragments may be displayed on the surface of filamentous bacteriophage by introducing variable region genes into the viral genome at a gene encoding a viral coat protein. "Phage display" enables the isolation of antibody genes from large libraries according to the binding specificities they encode. We have constructed a new phage-display vector encoding a polyhistidine tag that has been used for rapid purification of soluble antibody fragments. An antibody library derived from immunized mice was cloned into this vector. This library was panned against the transition state analog RT3, and a high proportion of binders isolated after two rounds of panning. PCR analysis revealed that there were 24 different pattern groups. Sequencing of 15 clones within the major pattern group revealed 10 related clones with a range of point mutations. Thus, phage display can provide a large diverse repertoire of candidate catalytic antibodies based on TSA selection and screening.
McCafferty
J
J
Cambridge Antibody Technology, Melbourne.
Fitzgerald
K J
KJ
Earnshaw
J
J
Chiswell
D J
DJ
Link
J
J
Smith
R
R
Kenten
J
J
D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002846:Chromatography, Affinity; D004247:DNA; D004797:Enzyme-Linked Immunosorbent Assay; D006639:Histidine; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008670:Metals; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc; D016415:Sequence Alignment
D007128:Immunoglobulin Fragments; D008670:Metals; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; C033223:polyhistidine; D006639:Histidine; D004247:DNA